A MANUAL OF BACTEBIOLOGIY BY GEOEGE M. STERNBEEG, M.D. DEPUTY SURGEON-GENERAL U. S. ARMY Director of the Hoagland Laboratory (Brooklyn, N. Y.); Honorary Member of the Epidemiological Society of London, of the Royal Academy of Medicine of Rome, of the Academy of Medicine of Rio de Janeiro, of the American Academy of Medicine, etc., etc. ILLUSTRATED BY IIELIOTYPE AND CHROMO-LITHOGRAPIIIC PLATES AND TWO HUNDRED AND SIXTY-EIGHT ENGRAVINGS -v X NEW YORK WILLIAM WOOD & COMPANY 1893 OUPLIC/JE BIOLOGY LIBRARY G COPYRIGHT BY WILLIAM WOOD & COMPANY 1802. PREFACE. THE progress of our knowledge relating to the bacteria has been so rapid and the literature of the subject is now so extensive that it is no small task to keep pace with this progress, even when one has the literature at hand and devotes a large share of his time to bac- teriological studies. Fortunately, recent researches in this depart- ment of science have been largely made by exact methods and by trained investigators, and the results can be accepted as well estab- lished. 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 manual, and at the same time a text book of bacteriology for stu- dents 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 depart- ment 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. In the preparation of this man- ual various text books in foreign languages have been consulted, and I am especially indebted to the works of Fliigge, of Baumgarten, and of Eisenberg. But the descriptions of species and experimental data have been very largely taken from original memoirs. ' The illustrations also are to a considerable extent reproductions from the original papars of those engaged in research work. NKW YORK, April 1st, 1892. 268472 TA.BLE OF CONTESTS. 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, CO 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, Ill II. CONDITIONS OF GROWTH, 118 III. MODIFICATIONS OF BIOLOGICAL CHARACTERS, . . . .122 IV. PRODUCTS OF VITAL ACTIVITY, 126 V. PTOMAINES AND TOXALBUMINS, .... . 139 VI. INFLUENCE OF PHYSICAL AGENTS, . . . . . .145 VII. ANTISEPTICS AND DISINFECTANTS (GENERAL ACCOUNT OF THE ACTION OF), 156 VIII. ACTION OF GASES AND OF THE HALOID ELEMENTS UPON BAC- TERIA, 164 IX. ACTION OF ACIDS AND ALKALIES 172 X. ACTION OF VARIOUS SALTS, . . . * 178 XI. ACTION OF COAL-TAR PRODUCTS, ESSENTIAL OILS, ETC., . 189 XII. ACTION OF BLOOD SERUM AND OTHER ORGANIC LIQUIDS, . 198 XIII. PRACTICAL DIRECTIONS FOR DISINFECTION, . . . . 201 VI TABLE OP CONTENTS. PART THIRD. PATHOGENIC BACTERIA. PAGB I. MODES OF ACTION, , . 215 II. CHANNELS OF INFECTION 22.3 III. SUSCEPTIBILITY AND IMMUNITY, . . . . . 226 IV. PYOGENIC BACTERIA, 263 V. BACTERIA IN CROUPOUS PNEUMONIA, . . . . 288 VI. PATHOGENIC MICROOOCCI NOT DESCRIBED IN SECTIONS IV. ANDV., 310 VII. THE BACILLUS OF ANTHRAX, .... . 327 VIII. THS BACILLUS OF TYPHOID F VER . 337 IX. BACTERIA IN DIPHTHERIA, . . .- . . . . 356 X. BACTERIA IN INFLUENZA. , . . 370 XI. BACILLI IN CHRONIC INFECTIOUS Di EASES, . . . . 374 XII. BACILLI WHICH PRODUCE SEPTICAEMIA ix SUSCEPTIBLE ANI- MALS, . . . . .. . . . . . , . . 407 XIII. PATHOGENIC AEROBIC BACILLI NOT DESCRIBED IN PREVIOUS SECTIONS, . . .>-,'. 438 XIV. PATHOGENIC ANAEROBIC BACILLI, . . . ... .482 XV. PATHOGENIC SPIRILLA, . . . . „ , . . . 497 XVI. BACTERIA IN INFECTIOUS DISEASES NOT PROVED TO BE DUE TO SPECIFIC MICROORGANISMS, . . . . . . . 514 XVII. CLASSIFICATION OF PATHOGENIC BACTERIA, ... . 533 PART FOURTH. SAPROPHYTES. I. BACTERIA IN THE AIR, . 541 II. BACTERIA IN WATER, 553 III. BACTERIA IN THE SOIL, 567 IV. BACTERIA OF THE SURFACE OF THE BODY AND OF EXPOSED Mucous MEMBRANES, 573 V. BACTERIA OF THE STOMACH AND INTESTINES, .... 580 VI. BACTERIA OF CADAVERS AND OF PUTREFYING MATERIAL FROM VARIOUS SOURCES, 585 VII. BACTERIA IN ARTICLES OF FOOD, 588 VIII. NON-PATHOGENIC MICROCOCCI, 593 IX. NON- PATHOGENIC BACILLI, 620 X. NON-PATHOGENIC SPIRILLA 694 XI. LEPTOTRICHE.E AND CLADOTRICHE^E, 703 XII. ADDITIONAL SPECIES OF BACTERIA, NOT CLASSIFIED, . . 709 XIII. BACTERIOLOGICAL DIAGNOSIS, . 735 BIBLIOGRAPHY, . 769 INDEX, 877 LIST OF ILLUSTRATIONS. FIG. PAGE 1. Staphylococci, ........... 21 2. Zoogla-a 21 3. Ascococcus, ............ 21 4. Streptococci, 21 5 Tetrads, . . 22 6. Packets — sarciiia, ........... 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. Karliuski'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. ..... 56 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 61 34. Flask used by Pasteur, 61 35. Platinum wire loop, 62 36. Platinum needle 63 37. Sternberg's bulb, 64 38. Method of making stick culture, 67 Vlll LIST OP ILLUSTRATIONS. FIG. PAGE 39. Sloping surface of culture medium, ....... 68 40. Growth of non-liquefying bacteria in gelatin stick cultures, . . C8 41. Growth of same along line of puncture, ....... 69 42. Growth of liquefying bacilli, 70 43. Colonies of bacteria, 71 44. Apparatus for gelatin plates, 73 45. Esmarch roll tube, . 74 46. (See Fig. 15). 47. Mode of development of a facultative anaerobic bacillus, . . . 78 48. Mode of development of strict anaerobic in long stick culture, . . 78 49. Exhausted-air flask for liquid media , . . . 80 50. Method of displacing air with hydrogen, ...... 80 51. Salomonson's tube, HO 53. Friinkel's method of cultivation, 81 53. Sternberg's method of cultivation, . 81 54. Sternberg's method of cultivation 82 55. Buchner's method of cultivation, . . . . . . . . 83 56. Hydrogen generator, 83 57. Hydrogen apparatus for plate cultures, ....... 85 58. Incubating oven, . . ' '. \ .' 87 59. Thermo-regulator for gas, . . . . . . . . 88 60. Moitessier's pressure regulator, . .... -. . . 88 61. Mica screen for flame, . . . . . .' .- . . . 89 62. Koch's device for cutting off flame, -..''"-•« • • > " • • 89 63. Reichert's thermo regulator, . . . . . ' . . . 89 64. Bohr's thermo-regulator, . . . • * • • «'''.'. 89 65. Munckc's thermo-regulator 4 . 90 66. Sternberg's thermo-regulator, 90 67. Gas valve for the same, . 91 68. D'Arsonval's incubating apparatus, 91 69. Roux's incubating oven and thermo-regulator, ..... 91 70. Roux's thermo-regulator, -. .91 71. Koch's syringe, ........... 95 72. Sternberg's glass syringe, .- . 9f 73. Pringle's photomicrographic apparatus 106 74. Sternberg's photomicrographic apparatus for gas, ..... 107 75. Spores of bacilli, 115 76. Method of germination of spores, . . . . . , . . 116 77. Apparatus for cultivating anaerobic bacilli 131 78. Bacillus of mouse septicaemia in leucocytes from blood of mouse, . 245 79. Staphylococcus pyogenes aureus, . . . . . . . . 266 80. Gelatin culture of Staphylococcus pyogenes aureus 267 81. Vertical section through a subcutaneous abscess caused by inoculation with staphylococci in the rabbit, ....... 269 82. Pus containing streptococci, 275 83. Streptococcus of erysipelas in nutrient gelatin 276 84. Section from margin of an erysipelatous inflammation, showing strepto- cocci in lymph spaces, 277 85. Gouococci, 283 86. Gonococcus in gonorrhrcal pus, ........ 284 87. Gonorrhooal conjunctivitis, second day of sickness, . . . 286 LIST OF ILLUSTRATIONS. ix no. PAGE 88. FriedlSnder's bacillus, 296 89. Friedlander's bacillus; stick culture in gelatin, 297 90. Micrococcus pneumoniae crouposae, 301 91. Micrococcus pneumoniae crouposae, 301 92. Micrococcus pneumoniae crouposae, ....... 301 93. Micrococcus pneumoniae crouposae, showing capsule 302 94. Single colony of Micrococcus pneumonise crouposae upon agar plate, . 303 95. Micrococcus pneumoniae crouposae in blood of rabbit inoculated with sputum, 307 96. Micrococcus of progressive tissue necrosis in mice, . . . . 311 97. Micrococcus of pyaemia in rabbits, 312 98. Micrococcus tetragenus, 314 99. Streptococcus of mastitis in cows, 321 100. Bacillus anthracis, showing development of long threads in convoluted bundles, 328 101. Bacillus anthracis, showing formation of spores, . . ... . 329 102. Culture of Bacillus anthracis in nutrient gelatin 330 103. Colonies of Bacillus anthracis upon gelatin plates, . . . . 331 104. Bacillus anthracis in liver of mouse, ....... 334 105. Bacillus anthracis in kidney of rabbit, 335 106. Bacillus of typhoid fever; colonies in stained sections of spleen, . . 340 107. Bacillus of typhoid fever; colonies in stained sections of spleen, . . 340 108. Bacillus typhi abdominalis, 346 109. Bacillus typhi abdominalis, 346 110. Bacillus typhi abdominalis, showing flagella, 347 111. Single colony of Bacillus typhi abdominalis in nutrient gelatin, . . 348 112. Bacillus typhi abdominalis; stick culture in nutrient gelatin, . . 348 113. Section through wall of intestine, showing invasion by typhoid bacilli, 352 114. Bacillus diphtheriae, 360 115. Colonies of Bacillus diphtheriae in nutrient agar, 361 116. Bacillus tuberculosis, . 376 117. Bacillus tuberculosis in sputum, . . .' » . . .... 377 118. Section through a tuberculous nodule in the lung of a cow, showing two giant cells, 379 119. Tubercle bacilli from surface of culture upon blood serum, . . . 382 120. Culture of tubercle bacillus upon glycerin-agar, 384 121. Limited epithelioid-celled tubercle of the iris, . . . . . 390 122. Section of a recent lepra nodule of the skin, 395 123. Bacillus mallei 397 124. Section of a glanders nodule, . . , 397 125. Section through a glanders nodule in liver of field mouse, . . . . 400 126. Migrating cell containing syphilis bacilli, ...... 402 127. Pus from hard chancre containing syphilis bacilli, .... 402 128. Bacillus of rhinoscleroma in lymphatic vessels of the superficial part of tumor, 405 129. Bacillus septicaemias haemorrhagicae in blood of a rabbit, . . . 408 130. Bacillus septicaemiae haemorrhagicae; stick culture in nutrient gelatin, . 410 131. Bacillus of Schweineseuche, 410 132. Colonies of bacillus of swine plague, 410 133. Bacillus of Schweineseuche in blood of rabbit, . . . . . 412 134. Bacillus of hog cholera, ... ... . 414 X LIST OF ILLUSTRATIONS. PIG. PAGE 135. Bacillus of mouse septictemia in leucocytes from blood of mouse, . 420 136. Bacillus of rouget, 421 137. Bacillus of mouse septicaemia ; culture in nutrient gelatin, . . . 4'31 138. Bacillus of mouse septicaemia; single colony in nutrient gelatin, . . 422 139. Section of diaphragm of a mouse dead from mouse septicaemia, . . 423 140. Bacillus cavicida Havaniensis, " 425 141. Bacillus crassus sputigenus, . . . . -. . . . . 426 142. Proteus hominis capsulatus, .* . . . . . , . . . 428 143. Bacillus capsulatus, . . . . . • . » . . . 432 144. Bacillus hydrophilus fuscus, . . , . . ... . 433 145. Culture of Bacillus hydrophilus fuscus in nutrient gelatin, . • . . 433 14C. Bacillus coli communis, . . » ; . . . . . . 440 147. Bacillus coli communis in nutrient gelatin, . . . . 442 148. A portion of the growth shown in Fig. 147, 442 149. Bacillus lactis aerogenes, . .•-..-. . . - . . . 447 150. Bacillus acidiformans, . . ... . . . . . . 449 151. Culture of Bacillus acidiformans in nutrient gelatin, . . . '- . 449 152. Bacillus cuniculicida Havaniensis, 450 153. Colonies of Bacillus cuniculicida Havaniensis, . . . . - '•'•. 451 154. Colonies of Bacillus cuniculicida Havaniensis, , 451 155. Bacillus pyocyanus, . . • . .• . ' . . . . . 454 156. Proteus vulgaris, . . . . . . .'.".. 457 157. " Swarming islands" from a culture of Proteus mirabilis, . .' . 461 158. Spiral zooglcea from a culture of Proteus mirabilis, . . . . 461 159. Tetanus bacillus, ' . '. . . . -V . 483 160. Tetanus bacillus, . . . . . •*"'"• ' • ; ' "* * '• 483 161. Culture of Bacillus tetani in nutrient gelatin, ..... 484 162. Bacillus cedematis maligni, . . 489 163. Bacillus redematis maligni, . .. '.• . . . . . 489 164. Cultures of Bacillus oedematis maligni in nutrient gelatin, . . . 490 165. Bacillus cadaveris, - . . . '•••'. . 492 166. Bacillus cadaveris, . . .492 167. Bacillus of symptomatic anthrax, _ . 493 168. Bacillus of symptomatic anthrax, . • . . . . . . . 493 169. Culture of bacillus of symptomatic anthrax, ...... 494 170. Spirillum Obermeieri 498 171. Spirillum Obermeieri, .......... 498 172. Spirillum cholerae Asiaticae, 500 173. Spirillum cholerae Asiaticse, 500 174. Colonies of Spirillum cholerae Asiaticae. . . . . . . ; 501 175. Spirillum cholerae Asiaticae, 501 176. Cultures of Spirillum choleras Asiaticae in nutrient gelatin, . . . 502 177. Spirillum cholerae Asiaticae, 502 178. Colonies in nutrient gelatin of Spirillum cholerae Asiaticae, Spirillum tyrogenum, and Spirillum of Finkler and Prior, .... 503 179. Section through mucous membrane of intestine from cholera cadaver, 507 180. Spirillum of Finkler and Prior, . 510 181. Colonies of Spirillum of Finkler and Prior, 510 182. Spirillum of Finkler and Prior ; culture in gelatin, .... 510 183. Spirillum tyrogenum 511 184. Colonies of Spirillum tyrogenum, 511 LIST OF ILLUSTRATIONS. xj FIG. PAGE 185. Spirillum Metschnikovi, 512 186. Penicillum glaucum, 542 187. Miquel's aeroscope, 543 188. Hesse's aeroscope 545 189. Miquel's flask ' 547 190. Straus and Wurtz's soluble filter, 547 191. Petri's sand filter 548 192. Sugar filter, 549 193. Sedgwick and Tuckers apparatus, 549 194. Sternberg's vacuum tube, 554 195. Lepsius' apparatus for collecting water at various depths, . . . 555 196. Koch's plate method, 556 197. Smear preparation from liver of yellow-fever cadaver, .... 586 198. Bacillus cadaveris grandis, 586 199. Micrococcus of Freire, from a gelatin culture, ' G09 200. Culture of Freire's micrococcus in nutrient gelatin, .... 609 201. Streptococcus coli gracilis, . 610 202. Streptococcus cadaveris, 612 203 Streptococcus Ha vaniensis, „ 612 204. Streptococcus liquefaciens, from anaerobic culture in nutrient gelatin, . 613 205. Streptococcus liquefaciens ; culture in nutrient gelatin, . . . 613 206. Micrococcus tetragenus versatilis, from a single colony in nutrient gelatin, 614 207. Micrococcus tetragenus ; culture in nutrient gelatin, .... 614 208. Sarcina lutea, 616 209. Ascococcus Billrothii, 618 210. Bacillus cyanogenus, 627 211. Bacillus arborescens, from a gelatin culture, 633 212. Colony of Bacillus arborescens 633 213. Bacterium termo of Vignal, from a bouillon culture 642 214. The same from a culture fifteen days old 642 215. Bacillus ubiquitus 644 216. Bacterium Zopfii, 655 217. Bacillus scissus, 657 218. Bacillus scissus; superficial colony on gelatin plate, .... 657 219. Bacillus circulans, 664 220. Bacillus diffusus, from a gelatin culture 667 221. Bacillus diffusus; superficial colony in nutrient gelatin, . . . 667 222. Bacillus vermicularis, 668 223. Bacillus mesentericus vulgatus, from a culture in bouillon, . . . 673 224. Bacillus mesenteriqus vulgatus, from an agar culture 673 225. Bacillus megatherium, 674 226. Bacillus subtilis, 678 227. Bacillus ulna of Vignal, 681 228. Bacillus buccalis maximus 683 229. Colonies of Bacillus muscoides, 687 230. Colonies of Bacillus polypiformis, 688 231. Bacillus butyricus, . 689 232. Colonies of Bacillus liquefaciens magnus, 690 233. Colonies of Bacillus liquefaciens parvus, 691 234. Colony of Bacillus radiatus, 691 Xii LIST OF ILLUSTRATIONS. FIG PAGE 235. Culture of Bacillus liquefacieus magnus 692 236. Culture of Bacillus radiatus, 692 237. Culture of Bacillus spinosus, 692 238. Culture of Bacillus liquefaciens parvus, 692 239. Culture of Clostridium fcetidum, . 4 . . . . . . . 692 240. Colony of Bacillus spiuosus 693 241. " Spirochoete dentium," . 694 242. " Spirochaete plicatilis, vibrio rugula, and other bacteria," . . . 694 243. " Spirillum dentium," 694 244. Vibrio rugula, 695 245. Spirillum volutans, . . ' . . . . . . . . . 696 246. Spirillum sanguineum, . . . . , . . . . . 696 247. Spirillum serpens, 696 248. Spirillum tenue, . 696 249. Spirillum undula, . . ..... . . . . . 696 250. Spirillum linguae, . j ... 697 251. Spirillum a of Weibel, . . . 698 252. Spirillum ft of Weibel, .'.... 698 253. Spirillum y of Weibel, . . . . ., .... 699 254. Spirillum aureum, 699 255. Crenothrix Kuhniana, . . - . . 704 256. Beggiatoa alba, 704 257. Cladothrix dichotoma 707 258. Nitrifying bacillus of Winogradsky, 710 259. Bacillus thalassophilus, . . . . . . . . . 712 260. Bacillus granulosus, . . . . . . . . . • . 712 261. Bacillus limosus, 713 262. Spirillum marinum, , . 713 263. Bacillus litoralis, 715 264. Bacillus balophilus, 715 265. Bacillus of Dantec, 718 266. Bacillus Havaniensis 718 267. Bacillus gracilis cadaveris, 733 268. Colonies of Bacillus gracilis cadaveris, 73C 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. PART FIRST. 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 (0. F. Miiller, 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 algSB ; and Davaine (1859) insisted that the vibrioniens are vegetable organ- 4 HISTORICAL. isms, nearly allied to the algae. His classification will be found in the " Dictionnaire Encyclop. des Sciences Medicales," art. " Bac- teries " (18G8). This view is also sustained by the German botanist Cohn and is now generally accepted. Spallanzani, in 1776, 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 not 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- tained in it. Schwann (1839) demonstrated the same fact by another method. Air was freely admitted to his boiled liquids through a tube which was 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 — Saccharomyces cerevisice — 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- duce fermentation of any kind. It was objected to these experiments that the air, having been subjected to a high temperature, had perhaps undergone some chem- ical change which prevented it from inaugurating processes of fer- mentation. This objection was met by Schroder and Von Dusch (1854) by a very simple device which has since proved to be of inestimable value in bacteriological researches. These observers showed that a loose plug of cotton, through which free communication with the external air is maintained, excludes all suspended microorganisms, and that HISTORICAL. 5 air passed through such a filter does not cause the fermentation of boiled organic liquids. The experiments of Pasteur and of Hoffman, made a few years later, showed that even without a cotton filter, when the neck of the flask containing the boiled liquid is long drawn out and turned down- ward, the contents may be preserved indefinitely without change. In this case suspended particles do not reach the interior of the flask, as there is no current of air to carry them upward through its long- drawn-out neck, and they are prevented by the force of gravity from ascending. Tyndall showed at a later date that in a closed chamber, in which the air is not disturbed by currents, all suspended particles settle to the floor of the chamber, leaving the air optically pure, as is proved by passing a beam of light through such a chamber. Notwithstanding the fact that the experimenters mentioned had succeeded in keeping boiled organic liquids sterile in flasks to which the oxygen of the air had free access, the question of the possibility of spontaneous generation — heterogenesis — still remained unsettled, inasmuch as occasionally a development of bacterial organisms did occur in such boiled liquids. This fact was explained by Pasteur (1860), who showed that the generally received idea that the temperature of boiling water must destroy all living organisms was a mistaken one, and that, especially in alkaline liquids, a higher temperature was required to insure ster- ilization. His experiments showed that a temperature of 110° to 112° C. (230° to 233.6° F.), which he obtained by boiling under a pressure of one and a half atmospheres, was sufficient in every case. These experiments, which have been repeated by numerous investi- gators since, settled the spontaneous-generation controversy ; and it is now generally admitted that no development of microorganisms occurs in organic liquids, and no processes of putrefaction or fermen- tation occur in such liquids, when they are completely sterilized and guarded against the entrance of living germs from without. Pasteur, at a later date (1865) showed that the atmospheric or- ganisms which resist the boiling temperature are in fact reproduc- tive bodies, or spores, which he described under the name of " corpus- cles ovoides " or " corpuscles brillants." Spores had been previously seen by Perty (1852) and Robin (1853), but it was not until 1876 that the development of these reproductive bodies was studied with care by Cohn and by Koch. The last-named observer determined the conditions under which spores are formed by the anthrax bacillus. Five years later (1881) Koch published his valuable researches relat- ing to the resisting power of anthrax spores to heat and to chemical agents. 6 HISTORICAL. The development of our knowledge relating to the bacteria,, stimulated by the controversy relating to spontaneous generation and by the demonstration that various processes of fermentation and putrefaction are due to microorganisms of this class, has depended largely upon improvements in methods of research. Among the most important points in the development of bacterio- logical technique we may mention, first, the use of a cotton air filter (Schroder and Von Dusch, 1854) ; second, the sterilization of culture fluids by heat (methods perfected by Pasteur, Koch, and others) ; third, the use of the aniline dyes as staining agents (first recommended by Weigert in 1877) ; fourth, the introduction of solid culture media, and the " plate method " for obtaining pure cul- tures, by Koch in 1881. The various improvements in methods of research, and espe- cially the introduction of solid culture media and Koch's "plate method " for isolating bacteria from mixed cultures, have placed bacteriology upon a scientific basis, and have shown that many of the observations and inferences of the earlier investigators were erroneous owing to the imperfection of the methods employed. Since it has been demonstrated that certain infectious diseases of man and the lower animals are due to organisms of this class, phy- sicians have been especially interested in bacteriological researches, and the progress made during the past fifteen years has been largely due to their investigations. It was a distinguished French physi- cian, Davaine, who first demonstrated the etiological relation of a microorganism of this class to a specific infectious disease. The an- thrax bacillus had been seen in the blood of animals dying from this disease by Pollender in 1849 and by Davaine in 1850, but it was sev- eral years later (1863) before the last-named observer claimed to have demonstrated by inoculation experiments the causal relation of the bacillus to the disease in question. The experiments of Davaine were not generally accepted as con- clusive, because in inoculating an animal with blood containing the bacillus, from an infected animal which had succumbed to the disease, the living microorganism was associated with material from the body of the diseased animal. This objection was subse- quently removed by the experiments of Pasteur, Koch, and many others 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- croorganism to an infectious malady was made by Pasteur, who de- voted several years to the study of an infectious disease of silkworms which 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- tneieri — 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 gonorrhceal 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 pneumonia}," but described in the present volume under the name of Micrococcus pneumonias 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 (1885) Koch published his discovery of the tubercle bacillus. The same year Pasteur published his researches upon the disease of swine, known in France as ronget. The same investigator (Pasteur) also published in 1883 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 — * ' co*ima 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- 8 HISTORICAL. nitely the characters of the various microorganisms found in pus from acute abscesses, etc. The tetanus bacillus was discovered in 1884 by Nicolaier, a stu- dent in the laboratory of Prof. Flugge, 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 has been discovered since the year 1884 until the present year (1892). After numerous unsuccessful researches by competent bacteriologists, a bacillus has finally been discovered by Pfeiffer, of Berlin, and independently by Canon, which is believed to be the specific cause of influenza. Having briefly passed in review some of the principal events in the progress of our knowledge in this department of scientific inves- tigation, 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 Ger- many the leading countries in this line of investigation. The very great advantages of Koch's methods of research, introduced at the commencement of the last decade, have attracted many students from various parts of the world to Berlin, and to other cities of Ger- many 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 Germany to obtain such instruction. The literature of the sub- ject is, however, largely in the German and French languages. We can only refer here to such periodicals as are principally devoted to bacteriological research work. The Zeitschrift fur Hygiene has been published since 188(3, and contains numerous valuable papers, contributed for the most part by the pupils of Koch and of Flugge, who are the editors of the journal. Nine volumes, each containing three numbers, have thus far been published (1891). The Annales de I'Institut 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 hydrophobia. The Annales de Micrography is a monthly journal, published in Paris. It is now (1892) in its fourth year. The principal editor is Miquel. HISTORICAL. 9 The Centralblatt fiir 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. 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. Dujardin (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 Davaiue (18G8) provides for the motionless, fila- mentous 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, i Moving spontane- \ 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 bacteridie. Hoffman 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, as it was not constant in the same species and depended to some ex- tent upon temperature conditions, etc. CLASSIFICATION. 11 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 Oscillator iacece 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 them 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^OGENES — cells free or united into glairy families by an intercel- lular substance. 2. NEMA.TOGENES— cells disposed in filaments. In the first tribe he has placed the genera Micrococcus (Hallier), Bacte- rium (Dujardin), Merismopedia (Meyer), Sarciita (Goodsir),and^lscococcM6f (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. Cyanophyceae (Oscillatoria- A. Schizomycetes (Bacteria), ceae, etc.). B. Palmellaceae. B. Saccharomycetes. 12 CLASSIFICATION. Zopf, who insists upon the polymorphism of these low organisms, divides the bacteria into four groups : Genera. Streptococcus, Merismopedia, 1. COCCKCE^E. — Up to the pre- sent time, only known in the form of cocci. Sarcina, Micrococcus, Ascococcus. 2. BACTERIACE.E. — Have for the 1 Bacterium, most part spherical, rod-like, and j Spirillum, filamentous forms ; the first (cocci) • vibrio, may be wanting ; the last are not j Leuconostoc, different at the two extremities; fila- Bacillus, ments straight or spiral. Clostridium. 3. LEPTOTRICHE^. — Spherical, ] Crmnnriv rod-shaped, and filamentous forms; Beaaiatoa the last show a difference between the \ p*S™ ,v//o/7i w-r two extremities ; filaments straight | LevSrix orspiral; spore formation not known. J ^ 4. CLADOTRICHE.E. — Spherical, 1 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?riacese — which has only been established for a few species, and which appears not to be general among the rod- shaped and spiral bacteria. De Bary divides 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. The first group includes the micrococci, the bacilli, and the spirilla; the second group the spirulina of Hueppe, leptotrichece (Zopf), and cladotrichece. The pleomorphous species described by Hauser under the generic name Proteus are included in the second group among the spirulina. In the present volume we have described these pleomorphous species among the bacilli. The Cocci, in the classification of Baumgarten, constitute a single genus with the following subgenera : 1, Diplococcus ; 2, Strepto- coccus; 3, Merismopedia (Zopf) — "Merista" (Hueppe); 4, Sar- cina (Goodsir) ; 5, Micrococcus (" staphylococci"). The BACILLI are included in a single genus embracing all of CLASSIFICATION. 13 those species which only form rod-shaped cells, and filaments com- posed of red-like segments ; or straight filaments not distinctly seg- mented, which may be rigid or flexible. The SPIRILLA are also included in a single genus, embracing all of those species in which the filaments are spiral in form and the segments more or less spiral or "comma-shaped" — filaments either rigid or flexible. This simple morphological classification of the monomorphous group of Baumgarten corresponds with the nomenclature now gene- rally in use among bacteriologists. We speak of the spherical bac- teria as cocci or as micrococci, of the rod-shaped bacteria as bacilli, and of the spiral bacteria as spirilla, It is true, however, that we are sometimes embarrassed to decide whether a particular microorganism belongs to one or the other of these morphological groups or so-called genera. Among the bacilli, for example, we may have, in the same pure culture, rods of very different lengths, some being so short that if alone they might be taken for cocci, while others may have grown out into long fila- ments. But if we are assured that the culture is pure the presence of rod forms establishes the diagnosis, and usually the cocci-like elements, when carefully observed, will be seen to be somewhat longer in one diameter than in the other. The German bacterio- logists generally insist upon placing among the bacilli all straight bac- teria in which, as a rule, one diameter is perceptibly greater than that transverse to it ; and several species of well-known bacteria which were formerly classed as micrococci are now called bacilli — e. g. , Friedlander's bacillus ("pneumococcus"), Bacillus prodigiosus. The distinction made by Cohn and others between the genus Bacterium (Duj.) and the genus Bacillus (Cohn) cannot be main- tained, inasmuch as we may have short rods and quite long fila- ments in the same pure culture of a single species ; and, again, the character upon which the genus Vibrio (Ehr.) was established — viz., the fact that the filaments are flexible and the movements sinuous — is not a sufficient generic or even specific character, for in a pure culture there may be short rods which are rigid, and long filaments which are flexible and have a sinuous movement. We therefore to-day speak of all the elongated forms as bacilli, unless they are spiral and have a corkscrew-like motion, in which case they are known as spirilla. The bacteria are also classified according to their biological char- acters, and it will be necessary to consider the various modes of grouping them from different points of view other than that which relates to their form. This is the more important inasmuch as we are not able to differentiate species by morphological characters 14 CLASSIFICATION. alone. Thus, for example, there are among the spherical bacteria, or micrococci, 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 "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 is a 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 of the zymogenes. These characters must therefore be considered separate^ as regards each species, and in studying its life history and distinguishing characters we determine whether it is chromogenic or noii-chromogenic ; whether it produces special fermentations ; and whether it is or is not pathogenic when inoculated into the lower animals. In making the distinction between pathogenic and 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 septica3mia 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- 10 CLASSIFICATION. mals, and that, consequently, the immense destruction of human life which 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- lished 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 strict anaerobics, may be 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- aerobics. 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- tempted a classification based upon the mode of reproduction, in which he makes two groups, or " tribes," one in which reproduction occurs by the formation of endogenous spores — " endospores " — the other in which it occurs by the formation of "• ar thro spores." ' 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- mon to all. In the 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, zooglcea masses by a rather firm intercellular substance. LEUCONOSTOC. — Cocci, solitary or in chains, surrounded by a thick, gelatinous envelope arid forming zooglcea 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 18 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 denned 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 zoogloea (differing from the zoogloea of spherical bacteria by a more abundant aud 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 desci-ibed by some authors under the generic name Vibrio, e.g., the so-called ''comma bacillus" of Koch—" Spirillum cholerae Asiatic*"; the spirillum of Finkler and Prior — "Vibrio proteus" ; the spirillum described by Gameleia — " Vibrio Metschnikovi,"etc. These microorganisms have not the characters which distinguished 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. SPIRULINA (Hueppe). — The vegetative cells are sometimes rod- shaped 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. " LEPTOTRiCHEyE (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 the 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 yw. One >M (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 0.1 >w, while some of the larger species are from one to two yw 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 >w, 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 as 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- stances may be of a short or long oval. When division has taken place, if the two members of a pair remain associated they are often more or less flattened at the point of contact (Fig. 1, a). MORPHOLOGY. 21 The staphylococci are characterized by the fact that, for the most part, the individual cocci in a culture are solitary (Fig. 1, 6). 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). 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. Frequently after staining 00 OQO 00 o a 8° b &83 8Soo C ficbQ} FIG. 1. and mounting a preparation we find that the cocci are associated in irregular groups, although we may have endeavored to distribute them in a drop of distilled water. This results from the fact that they are surrounded by a glutinous material which causes them to FIG. 2. FIG. 3. FIG. 4. adhere to each other (Fig. 1, e). A mass of cocci held together in this way by a transparent, ;glutinous, intercellular substance is spoken of as a zoogloea (Fig. 2). In the genus Ascococcus the in- tercellular substance is quite firm and the zooglcea are in the form of spherical or irregularly lobulated masses surrounded by a resist- ant 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, b. 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 88 00 S e Fia. 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 suspended 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 be remembered that we may have 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 CO CTXZD CO 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 FIG. 9. chains in which the elements remain associated may be formed (Fig. 0) ; 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 zooglcea 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- MORPHOLOGY. merits, 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 dichotoma, 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 FIG. 10. FIG. 11. FIG. 12. of this class are provided with similar appendages and that these are organs of locomotion. Recently, by improvements in methods of staining, Loffler 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, 6); 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 drops 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 stained granules, which are not spores, may be seen at the extremi- ties of the rods — end-staining. The morphological characters de- pending upon the formation of endogenous spores will be referred to hereafter. IV. STAINING METHODS. THE rapid development of our knowledge with reference to the minute microorganisms under consideration depends very largely upon the discovery that they may be stained by various dyes, and es- pecially by the aniline colors. Weigert (1876) was the first to employ these colors in studying the bacteria, and Koch at once recognized the value of the method and made use of it in his researches. The basic aniline colors are those employed, and among these the most useful are fuchsin, methylene blue, gentian violet, Bismarck brown, and vesuvin. Staining upon the Cover Crlass or Slide. — By a " cover-glass preparation " we mean that material supposed to contain bacteria has been spread out upon a thin glass cover, dried, and stained for microscopical examination. A small drop of a liquid culture may, for FIG 13. example, be spread upon a perfectly clean cover glass by means of a platinum wire held in a glass handle (Fig. 13). Or we may place a drop of water in the centre of the thin glass cover, and by means of the same instrument take a little material from a culture made upon the surface of a solid medium and distribute it through the drop. In this case we must be careful to take very little of the material, as the smallest quantity will contain an immense number of bacteria, and for a satisfactory view of the individual cells it is necessary that they be well separated from each other, in some parts of the prepa- ration at least, and not massed together. Where the object is to make a cabinet preparation for permanent preservation, special care should be taken to distribute the bacteria unif ormly through the drop of water. The next step consists in eva- porating the liquid so that the bacteria may remain attached to the surface of the glass cover. This may be done by simple exposure to the air or by the application of gentle heat. When the bacteria are 20 STAINING METHODS. suspended in an albuminous medium it will be necessary, after the film is dry, to heat the preparation sufficiently to coagulate the albu- men, in order that it may not ba 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 be 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 flame 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 neoessary 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 is especially 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 upon 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 the cover 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. Staining 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, ...... lf) 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. C arbol-Fuchsin (Ziehl's solution). Fuchsin, . . . . . ... . . 1 cc. 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 methylene 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 cover 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, potassio iodide two parts, water 30 STAINING METHODS. three hundred parts) ; then washed in alcohol, dried, and, if for per- manent preservation, mounted in balsam. METHODS OF 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 containing 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 bs 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-fuchsiii 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 as a 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. 4. Gabbett's Method. — This is a slight modification only of a very useful method recommended by B. Frankel in 1884. The con- trast stain is added to the decolorizing solution. After staining with STAINING METHODS. 31 carbol-f uchsin solution (No. 3) the cover glass is placed for one or two minutes in a solution containing: Sulphuric acid (twenty-five-per-cent solution), . . 100 cc. Methylene blue, ...... 2 cc. Wash, dry, and mount in cedar oil or balsam. METHODS OF STAINING SPORES. — When preparations containing the spores of bacilli are stained by any of the methods above given, these remain unstained and appear as highly refractive bodies in the interior of the rods or filaments in which they have been formed, or scattered about in the field if they have been set free. Owing to the contrast with the stained protoplasm of the rod or spore-bearing filament, they are especially well seen in recent cultures ; while in older cultures the bacilli often do not stain well, or are entirely dis- integrated and spores only are to be seen. The discovery was made at about the same time by Buchner (1884) and by Hueppe that spores may be stained if they are first exposed to an elevated tem- perature for some time. This may be accomplished by placing the slide or cover glass, upon which the spore-containing culture has been dried, in a hot-air oven at a temperature of 120° C. for an hour; or a higher temperature (180° C.) may be employed for a shorter time (fifteen minutes) ; or the cover glass may be passed through the flame of an alcohol lamp or Bunsen burner eight or ten times, instead of three times as is customary when the object in view is simply to coagulate the albumen and fix the film upon the cover glass. After such treatment the spores may be stained with an aqueous solution of one of the basic aniline colors — fuchsin, methyl violet, etc. — but the bacilli no longer take the stain so well. To obtain satisfactory double-stained preparations, showing both spores and bacilli, a different method is employed. The film upon the cover glass is passed through the flame three times, as heretofore directed ; it is then floated upon aniline-f uchsin solution in a watch glass, and this is heated to near the boiling point for an hour — Neisser's method. The aniline-f uchsin solution is prepared by shaking an excess of aniline oil in a test tube with dis- tilled water, filtering the saturated solution into a watch glass, and then adding a few drops of a saturated alcoholic solution of fuchsin. After this prolonged action of the hot staining fluid the spores of some bacilli are deeply stained, while others do not take the stain so well. The cover glass is next washed in water and then placed in a decolorizing solution containing twenty-five parts of hydrochloric acid to seventy-five parts of alcohol. This removes the stain from the bacilli, but, if not allowed to act too long, leaves the spores still stained. The preparation is next stained in a saturated aqueous 32 STAINING METHODS. solution of methylene blue ; and if the operation has been successfully carried out the spores will be stained red and the protoplasm of the bacilli in which they are present will be blue. Moller has recently (1891) published the following new method of staining spores : The cover-glass preparation, dried in the air, is passed three times through a flame or placed for two minutes in absolute alcohol ; it is then placed in chloroform for two minutes and washed in water ; it is now immersed in a five-per-cent solution of chromic acid for from half a minute to two minutes and again thoroughly washed in water ; next a solution of carbol-fuchsin is poured upon it and it is heated over a flame until it commences to boil, for sixty seconds ; the carbol-fuchsin solution is then poured off and the cover glass is immersed in a five-per-cent solution of sulphuric acid until the film is decolorized, after which it is again thoroughly washed in water. It is then placed for thirty seconds in an aqueous solution of methylene blue or of malachite green, and again washed in water, after which the preparation should be dried and mounted in balsam. As a result of this procedure the spores are stained dark red and the protoplasm of the bacilli blue or green. METHODS OF STAINING FLAGELLA. — Koch first succeeded in de- monstrating the flagella of certain bacilli and spirilla by staining them with an aqueous solution of hsematoxylon, and dilute chromic acid as a mordant. Recently Loffler (1889) has succeeded in demonstrat- ing, by an improved staining method, the presence of flagella in a con- siderable number of species in which they had not previously been seen, although generally suspected to be present. His method is as follows : Loffler 's Method. — The following solution is used as a mordant : No. 1. Solution of tannin of twenty per cent, . . . 10 cc. Saturated (cold) solution of ferrous sulphate, . . 5 cc. Aqueous or alcoholic solution of fuchsin, . . 1 cc. (Or one cubic centimetre alcoholic solution of methyl violet.) No. 2. A one per-eent solution of caustic soda. No. 3. A solution of sulphuric acid of such strength that one cubic centi- metre 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 STAINING METHODS. 33 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. By carefully conducted experiments Loffler has found that suc- cess in staining the flagella depends upon adding just the right quan- tity of acid or alkali, a very slight variation from the proper quantity being sufficient to give an imperfect or negative result. In general, those bacilli which produce an acid reaction in a neutral culture medium require the addition of the alkaline solution, and those which cause an alkaline reaction require the addition of an acid — acetic or sulphuric. Loffler gives the following detailed account of his method : A small quantity of a pure culture is suspended in a few drops of distilled water. Upon perfectly clean glass covers small drops of water are distributed by means of a platinum wire loop ; these are sowed with bacilli from the first drop. These little drops are spread out upon the cover with a platinum wire and allowed to dry in the air, after which they are passed through the flame in the usual way to fix the bacteria to the cover glass. Great care must be taken not to heat the preparation too much, as this prevents the flagella from taking the stain. Loffler recommends that the cover glass be held between the thumb and forefinger in passing it through the flame, instead of in forceps, as this insures it from being overheated. The mordant (solution No. 1) is now placed upon the cover glass so as to completely cover it as an arched drop. The cover glass is then carefully heated over a flame until steam commences to be given off ; too much heat causes a precipitate which cannot be washed away. The mordant is left upon the cover glass for from half a minute to a minute, and during this time it is gently moved to and fro. The cover glass is then washed by means of a stream of dis- tilled water. All remnants of the mordant attached to the margins of the cover glass should then be washed away with absolute alco- hol. The staining solution is now dropped upon the surface of the glass cover so as to completely cover it, and heat is applied as before for about a minute until steam commences to be given off. The staining solution recommended is a neutral, saturated aniline- water-fuchsin solution. 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 3 34 STAINING METHODS. 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 for 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 ma}T 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 Lofflers alkaline solution of methylene blue (No. 4). A freshly-prepared so- lution will stain sections in four or five minutes. Superfluous color is removed by immersing the sections in diluted alcohol or in a one- half-per-cent solution of acetic acid for a few seconds. The sectiofis 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. Kiihne'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 STAINING METHODS. 35 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 solu- tion 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 re- main 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 dehy- drated 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 of 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 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 following method of staining sections for the purpose of demonstrating bacteria present in the tissues is recommended by Pregl (1891) as a substitute for the method of Kiihne. 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 em- bedded 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 36 STAINING METHODS. 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 pre- pared by adding celloidin in small, dry pieces to aceton until a con- centrated 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 having 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 Kiihne's carbol-methylene-blue solution, which is dropped 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 de- sirable. They are then washed in water and immediately placed in fifty-per-cent alcohol, where they remain until the sections have a pale-blue color with a greenish tinge. They are now completely dehydrated in absolute alcohol and subsequently cleared up in xylol. STAINING SECTIONS OP GELATIN STICK CULTURES. — Fischl, Wei- gert, 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 direc- tions : 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, ren- dering it insoluble. The gelatin cylinder is thoroughly washed and then hardened in alcohol, first of seventy per cent and then of ninety- six per cent. It is then cut into suitable pieces, and these are attached to a cork in the usual manner and placed for twenty-four hours in ab- solute alcohol. Thin sections may now be made with a microtome, and these are attached to a glass slide and stained by Gram's or Weigert's method or by the use of Loffler's solution (No. 4). The decolorization should be effected by the use of alcohol and not with an acid solution. 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 38 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 FIG. 14. FIG. 15. FIG. 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 medium in the little flask, or it may be transferred to a test tube and solidified by heat whenever a solid blood-serum medium is re- quired. The advantage of preserving blood serum and other liquid media in these little flasks is in the fact that they may be preserved indefinitely without becoming contaminated or drying up, and that they 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, Milk 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 six nuts averaged 339.1 grammes. The specific gravity averaged 1.02285. The amount of water averaged 95 percent; 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 per cent. 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- strate certain facts relating to their physiology. Infusions of meat, or " flesh water," are made by chopping fine lean beef or mutton (one 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. Flesli-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. Ten-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 FIG. 17. next step consists in clarifying the nutrient medium. It is allowed to cool to about 50° C., and an egg, previously broken into one hundred grammes of water, is gradually added while stirring the liquid with a glass rod. A whole egg is used for a litre of the solu- tion. 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 apd 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 solution is then filtered, A hot- water funnel (Fig. 17) is usually employed, as the gelatin 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 by 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 nitration 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- FIQ. 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 made by adding one to two per cent of agar-agar to the standard flesh-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 Fio. 19. 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 46 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-ceiit agar will pass through it in about two hours. For special purposes various substances are added to the above- described solid and liquid media. A favorable addition for the growth of a considerable number of bacteria is from one to three per cent of glucose. The phosphorescent bacteria grow best in a medium containing 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 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. Quite recently 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 Sleskin (1891) have made experiments which indicate that this medium has considerable value. CULTURE MEDIA. 47 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 jequirity seeds has recently been recommended by Kaufmann (1891), who has found, by experimenting upon various bacteria, that such a medium is useful in differentiating species. 'The jequirity 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 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 48 CULTURE MEDIA. 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 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). FIG. 20. FIG. 21. FIG. 22. 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. CULTURE MEDIA. 40 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. 4 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 52 STERILIZATION OF 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. FIG. 23. (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 Loftier 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. FIB. 55 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 sli-p 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 yalve. A single sterilization in this ap- paratus, at a temperature of 115° C., for half an hour, will usually FIG. 26. Fro. 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 temperature of 100° C. for ten minutes, at intervals of twenty-four hours, four days in succession. Bouillon, flesh infusions, and agar-agar jelly 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. Sterilized ion 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 GO0 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 00° 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 08° 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 of the most important pathogenic bacteria produce toxic substances during their growth which may cause the death of susceptible ani- mals independently 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- lain and baked at a high temperature. 58 STERILIZATION OF CULTURE MEDIA. In Fig. 30 the Pasteur-Chamberlain 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 Chamberlain 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 the filtrate when it overflows from the interior of the filter. Of course all the necessary precautions must be taken with reference 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-Chamberlain 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 Chamberlain 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. to the introduction of gelatinous media by Koch in 1881, 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- full}' 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 the other hand, liquid media are more convenient than solid when it is the intention to isolate by filtration the soluble products of bacterial 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. 31. 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 32 CULTURES IN 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 Math 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 FIG. 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 studied ; also their power to break up various organic substances, as shown by the evolution of 'gas or other volatile products which may be collected, or by substances which remain in solution and can be 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 slide, is very useful. Such a drop culture may be left under the microscope and kept under observation for hours or days. In making these drop cultures it is necessary to sterilize the glass slides and thin glass covers by heat, and to take every precaution to prevent the inoculation of the drop of culture liquid with any other bacteria than those which are to be studied. The simplest form of moist chamber for drop cultures consists of an ordinaiy glass slide having a concave depression, about fifteen millimetres in diameter, ground out in its centre. This and the thin glass cover, having been sterilized by exposure in the hot-air oven at 150° C. for an hour 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 platinum loop heretofore described. It is best to spread the drop out as thin as possible, and it may be inoculated, from a pure cul- FIG. 86. ture, with a platinum needle (Fig. 36) after it has been placed upon the cover. This is then inverted over the hollow place in the glass slide, and it is customary to prevent the entrance of air and attach the cover by spreading a little vaseline around the margin of the " excavation. Another form of moist chamber is made by attaching a glass ring, having parallel, ground surfaces, to the centre of a glass slide by a suitable cement. In Ranvier's moist chamber there is a central eminence sur- rounded by a groove ground into the glass slide, and the drop of culture fluid is in contact with a polished glass surface below as well as above. This affords a more satisfactory view under the micro- scope. The Author's Culture Method. — In a paper read at the meeting of the American Association for the Advancement of Science, in August, 1881, the writer described a method of conducting culture experiments which he has since used extensively and with very satis- factory results. The liquid culture medium is preserved in little flasks having a long neck which is hermetically sealed. The principal ad- vantages connected with the use of these little flasks, or " Stern- 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 FIG. 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 :' 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.2 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. 9 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. 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 io 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. 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 2-t° 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. 38. 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 FIQ. 39. sloping surface, as shown in Fig. 39. 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. 40; 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. 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 maybe translucent or opaque ; or we may have little tufts, like moss, projecting from the line of puncture (b, 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. 70 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, L'J 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 fungi than of bacteria, but the principle is the same, viz., that a colony developed from a single germ 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 psrtains 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, having 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 FIG. 48.— Colonies of Bacteria. outlines may be irregular, giving rise to amoeba-like forms, or to a fringed or plaited margin, or the form may ba 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 bast 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 develops! 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- D3red 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 I the glass plate is adjusted to a hori- zontal position. The sterilized glass plate is placed in a 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 FIG. 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 other 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 Esinarch'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 aside for the development of colonies from the bacteria which had been introduced. When roll tubes are made from the agar jelly it is best 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. 4G) 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. 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 anae'robics'' grow FIG. 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- quire 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 oedema, and the bacillus 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 tuba 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 skill is required in the successful perform- ance of the last step in this procedure, and it will be easier for those FIG. 49. Fia. 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 gas is flowing, and then the horizontal tube at b. Frdnkel's Method. — Instead of these tubes specially made for the purpose, an ordinary test tube may be used, as recommended by Frankel. This is closed by a soft rubber cork through which two glass tubes 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. 52. ^sV hJji -t 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 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. FIG. 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 aiiaerobics. Method of Esmarcli. — The 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 OF 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 Ihbe, and sections are 'f a FIG. P6. 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. Buchners 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 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, form- FIG. 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 of 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 gas 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- tion extending downward as a tube, g, makes an enclosed space in INCUBATING OVENS AND THERMO-REGULATORS. 87 the lower part of the instrument, and this, when immersed in water, acts as a thermometer bulb. This space contains mercury below and air or the vapor of ether above. When the air is expanded by heat the mercury is forced up the tube g until it meets the end of the inlet tube for gas at h, and by shutting off the flow of gas pre- vents the temperature from going any higher. A small opening in the inlet tube at e permits a small amount of gas to flow, so that the flame under the brood oven ( Fig. 58, /) may not be entirely extin- guished. The lower end of the bent tube a is bevelled, so that a tri- angular opening is formed, which is closed gradually by the rising FIG. 58. mercury, instead of abruptly as would be the case if the lower end of the tube a were cut off square. To adjust the temperature in the air space of the incubating oven when the thermo-regulator is in position, a full flow of gas is admitted to the burner until the ther- mometer (Fig. 58, b) shows the desired temperature ; then the bent tube a is pushed down through the cork until its lower extremity meets the mercury and the flame / is somewhat reduced. The ap- paratus is then left for a time, to see whether the flame runs too high or too low, and a further adjustment is made. When the changes in the exterior temperature are slight and the gas pressure regular the temperature in the air chamber is controlled with great precision. But this is not the case under the reverse conditions. Changes in INCUBATING OVENS AND THERMO-REGULATORS. the pressure of gas, especially, interfere with the maintenance of a constant temperature, and for this reason a pressure regulator will be required when great precision is desired. That of Moitessier is commonly used in bacteriological laboratories (Fig. 60). But for most purposes variations of temperature of 1° to 2° C. are not of great importance. For ordinary use a brood oven should be regu- lated to about 35° to 37° C. It is best to have a little cylindrical screen of mica around the gas jet beneath the incubating oven, for the purpose of preventing the flame from being extinguished by cur- rents of air (Fig. 61). Koch's ingenious automatic device for shutting off the gas if the flame is accidentally extinguished is shown in Fig. 62. Fia. 59. Another form of thermo-regulator, which answers very well, is that of Reichert (Fig. 63). In this the gas enters at a and escapes at c. The mercury, which fills the bulb, shuts off the gas at the point for which the instrument is regulated. By means of the screw d the height of the mercury in the tube may be very accu- rately adjusted for any desired temperature. The regulator of Bohr, shown in Fig. 64, is more sensitive than that of Reichert, and rather simpler in construction than the usual form shown in Fig. 59. The thermometer bulb a contains only air, and the gas which passes through the tube / is shut off at the proper temperature by the mercury in the U-shaped tube c. The stopcock b is left open when the bulb a is immersed in the water INCUBATING OVENS AND THERMO-REGULATORS. 89 bath, and when the proper temperature is reached is closed so as to confine the air in the bulb. An increase of temperature now causes FIG. 61. FIG. 63. ihe air in the bulb to expand, the mercury in the U-tube is forced up FIG. 63. FIG. 64. and shuts off the gas flowing through the tube /at its lower ex- tremity, d. A small opening, e, permits sufficient gas to pass to 90 INCUBATING OVENS AND THERMO-REGULATORS. maintain a small flame which must not be sufficient by itself to keep up the desired temperature in the water bath. Altmann has recently (1891) described a thermo-regulator which is made by Miincke, of Berlin, and which is shown in Fig. 65. This is said to act with great precision. It is a modification of Reichert's FIG. 65. FIG. G6. regulator. Its mode of action will be readily understood by a refe- rence to the figure. A thermo-regulatQr which gives very accurate results, which are not influenced by differences in pressure, is that invented by the FIG. 67. writer over twenty years ago. The regulating thermometer may contain mercury only, or air and mercury, as shown in the thermo- regulator for gas (Fig. 59). In the simplest form a large bulb con- taining mercury is used, and a platinum wire is hermetically sealed in the glass so as to have contact with the mercury (Fig. 60, a). INCUBATING OVENS AND THERMO-REGULATORS. 9L 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, b, having an out- FIG. 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. 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 FIG. 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- ral use in connection 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' Arson val is shown in Fig. 68. It is a cylindrical vessel of copper having double walls, and is provided with the thermo-regulator of D' Arson val, 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 gives 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. 9& of this simple and convenient incubating oven are manufactured by Rohrbeck and by Miincke, of Berlin. The apparatus of D'Arson- val, and other forms in favor at the French capital, may be obtained from Wiesnegg, of Paris. The last-named manufacturer also sup- plies the incubating oven and thermo-regulator recently described by Roux (1891). This is shown in Fig. G9. 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. 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 caniivora, 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. As a rule, the specific infectious diseases 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 power of bacteria are the mouse, the guinea-pig, and the rabbit. Domestic fowls and pigeons are 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 Fia. 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 Fm. 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. 9(5 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 the 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 penetrated by the point of a hypodermic syringe without any pre- vious dissection. The ear is first washed with a solution of bichloride of mercury or simply with warm water. The animal had better be carefully wrapped in a towel to control its movements. The veins are distended by compressing them near the base of the ear. When the point of the needle has not been properly introduced, and the fluid to be injected escapes in the surrounding connective tissue, it will commonly be best to withdraw the syringe and make the attempt upon another vein. As pointed out by Abbott, the needle of the syringe should be ground flat at the point, and not curved as is commonly the case. EXPERIMENTS UPON ANIMALS. 97 Large quantities of fluid may be injected into the cavity of the abdomen or into the circulation by slowly forcing the fluid through a slender canula, properly introduced, which is coupled with a large syringe by means of rubber tubing, or with a glass receptacle from which the fluid is forced by the pressure of air pumped in with a rubber hand ball. Mice are usually injected subcutaneously near the tail. The little animal is first' seized by a long pair of forceps, or "mouse tongs, and the hair is clipped away on the back just above the tail. If solid material is to be introduced a little pocket is made with scis- sors or with a lancet, into which the infectious material is carried by means of a platinum needle or slender forceps. Liquids may be in- jected by the little glass syringe heretofore described, the point of which is easily forced through the skin. Pasteur's method of inoculating rabbits with the virus of hydro- phobia consists in trephining the skull and injecting the material beneath the dura mater. An incision through the skin is first made to one side of the median line a short distance back of the eyes. The edges of the wound are separated, and a small trephine (five or six millimetres in diameter) is used to remove a button of bone. The emulsion of spinal cord from a hydrophobic animal is then carefully injected beneath the dura mater — two or three drops will be sufficient. The wound is washed out with a two-per-cent solution of carbolic acid and closed with a couple of sutures. Injections into the intestine are made by carefully opening the abdomen with antiseptic precautions, gently seizing a loop of the in- testine, and passing the point of the syringe through its walls ; the loop is then returned and the incision in the walls of the abdomen carefully closed with sutures and dressed antiseptically. Inoculations into the anterior chamber of the eye of rabbits and other animals have frequently been practised, and offer certain ad- vantages in the study of the local effects of pathogenic microorgan- isms. The animal should be fastened to an operating board, belly down, and its head held by an assistant, who at the same time holds the eyelids apart. The conjunctiva is seized with forceps to steady the eye, and an incision about two millimetres long is made through the cornea with a cataract knife. Through this opening a small quantity of a liquid culture may be injected, or a bit of solid material introduced with slender curved forceps. Ordinary injections give but little pain and do not call for the use of an anaesthetic. When anaesthesia is required ether will usually be preferable to chloroform. Rabbits, especially, are very apt to die from chloroform, no matter how carefully it may be administered. Dog3, rats, and mice stand ether very well. The smaller animals 7 98 EXPERIMENTS UPON ANIMALS. may be brought under the anaesthetic by placing them in a covered jar into which a pledget of cotton wet with ether has been dropped. Before making injections into the anterior chamber of the eye it is well to use a two-per-cent solution of cocaine as a local anaesthetic. Mice which have been inoculated are usually kept in a glass jar having a wire-gauze cover. A quantity of cotton is put into the jar to serve as a shelter for the little animal, and it is well to partly fill the jar with dry sawdust. Larger animals are kept in suitable cages of wire or wood, and, as a rule, each one should be kept in a separate cage while under observation after an inoculation experiment. In experimenting upon animals the following points should be kept in view and noted : (a) The age and weight of the animal. Young animals are, as a rule, more susceptible than older ones, and with many pathogenic bacteria the lethal dose of a culture bears some relation to the size of the animal. (b) The point of inoculation. Injections into the circulation are generally more promptly fatal and require a smaller dose than those into a serous cavity or into the connective tissue. Pathogenic bacteria introduced into the abdominal cavity reach the circulation more promptly than those injected subcutaneously. But certain microorganisms owe their pathogenic power to the local effect about the point of inoculation and the absorption of toxic products formed in the limited area invaded, and do not enter the general circulation, or at least do nqt multiply in the circulating fluid, and quickly dis- appear from it. (b) The age of the culture injected. Old cultures sometimes have greater and sometimes less pathogenic potency than recent cul- tures. Some kinds of virus become "attenuated" when kept. But when the pathogenic power depends chiefly upon toxic products formed during the growth of the bacteria, old cultures are, as a rule, more potent than those recently made. (d) The medium in which the pathogenic bacteria are sus- pended. Cultures in albuminous media, like blood serum, are in some cases more potent than bouillon cultures ; and the virulence of several pathogenic bacteria is greatly intensified by successive cul- tures— by inoculation — in the bodies of susceptible animals. Ogston found that pus cocci cultivated in the interior of eggs had an in- creased virulence. According to Arloing, Cornevin, and Thomas, the activity of a culture of the bacillus of symptomatic anthrax is doubled by adding one-five-hundredth part of lactic acid to the cul- ture fluid. (e) The quantity injected is evidently an essential point when the result depends largely upon the toxic products formed in the cul- EXPERIMENTS UPON ANIMALS. 99 ture medium. It is also an essential point when pathogenic bacteria are injected which kill susceptible animals in very minute doses, for it has been shown by the experiments of Watson Cheyne and others that in the case of some of these, at least, there is a limit below which infection does not occur. Inoculated animals should be carefully observed, and a note made of every symptom indicating a departure from the usual con- dition of health, such as fever, less of activity, loss of appetite, weakness, emaciation, diarrhoea, convulsions, dilated pupils, the for- mation of an abscess or a diffuse cellulitis extending from the point of inoculation, etc. The temperature is usually taken in the rectum. The temperature of small animals, like rabbits and guinea-pigs, va- ries considerably as a result of external conditions. In the rabbit the normal temperature may be given as about 102° to 103° F. ; in the guinea-pig it is a little lower. In making a post-mortem examination of an inoculated animal it is best to stretch it out on a board, belly up, by tying its legs to nails or screws fastened in the margin of the board. When the abdomen is dirty, as is usually the case, it should be carefully washed with a disinfecting solution. An incision through the skin is then made in the median line the full length of the body, and the skin is dis- sected back so as to expose the anterior walls of the abdomen and thorax. These cavities are then carefully opened with a sterilized knife or scissors, and the various organs and viscera examined. At- tention should also be given to the appearances at the point of in- oculation. To ascertain whether the microorganism injected has invaded the blood, smear preparations should be made with blood obtained from a vein or from one of the cavities of the heart. It will be well also to make a smear preparation from a cut surface of the liver and spleen. In the various forms of acute septicaemia the spleen is usually found to be enlarged. If but few microorganisms are present in the blood and tissues they may escape observation in stained smear preparations, and it will be necessary to make cultures to demonstrate their presence. A little blood from a vein or from one of the cavities of the heart is transferred, by means of a plati- num loop (ose) or a sterilized collecting tube (see page 38), to a test tube containing liquefied nutrient gelatin or agar-agar, and an Esmarch roll tube is made. This is put aside for the development of colonies from any scattered bacteria which may be present. As a rule, it will be best to make agar cultures, as these can be placed in the incubating oven at 35° to 38° C. Stick cultures may also be made and will serve to show the presence of microorganisms, but will not give information as to how numerous they may be. The roll tube also has the advantage of showing whether there is a 100 EXPERIMENTS UPON ANIMALS. mixed infection or whether a pure culture of a single microorganism is obtained from the blood. In the same way cultures may be made from material obtained from the liver or spleen, and it may happen that one or both of these organs contain bacteria when none are found in the blood. Before passing the platinum needle or collect- ing tube into the organ, the surface, which has been more or less ex- posed to contamination, should be sterilized by applying to it a hot spatula ; then at the moment of lifting the spatula the sterilized needle is introduced into the interior of the organ, and the blood and crushed tissue adhering to it at once carried over to the culture me- dium. Or blood obtained with proper precautions from a vein, a cavity of the heart, or the interior of the spleen or liver, may be used to inoculate another animal. Animals are also sometimes inoculated by excoriating the cutis as in vaccination. They may also, in rare cases, be infected by in- troducing cultures into the stomach, either mixed with the food in- gested or by injection through a tube. Infection by inhalation is accomplished by causing the animal to respire an atmosphere, in a properly enclosed space, in which the pathogenic organism is sus- pended, by the use of a spray apparatus for liquid cultures, or some form of powder blower for powders containing the bacteria in a desiccated condition. One method of obtaining a pure culture of pathogenic bacteria consists in the inoculation of susceptible animals with material con- taining a pathogenic species in association with others which are not. When the blood is invaded by the pathogenic species and the animal dies from an acute septicaemia, we may usually obtain a pure cul- ture by inoculating a suitable culture medium with a minute drop of blood taken from a vein or from one of the cavities of the heart. Sometimes, however, a mixed infection occurs and some other mi- croorganism is associated in the blood with that one which was the immediate cause of the death of the animal. XII. PHOTOGRAPHING BACTERIA. WELL-MADE photomicrographs are unquestionably superior to drawings made by hand as a permanent record of morphological characters. This being the case, bacteriologists would no doubt re- sort to this method more generally but for the technical difficulties and the time and patience required in overcoming these. Koch, in his earlier studies, gave much time to photographing bacteria, and with very remarkable success. In his work on "Traumatic Infec- tive Diseases " (1878) he says : "With respect to the illustrations accompanying this work, I must here make a remark. In a former paper ' on the examination and photographing of bacteria I expressed the wish that observers would photograph pathogenic bacteria in order that their representa- tions of them might be as true to nature as possible. I thus felt bound to photograph the bacteria discovered in the animal tissues in traumatic infective diseases, and I have not spared trouble in the attempt. The smallest, and in fact the most interesting bacteria, however, can only be made visible in animal tissues by staining them and by thus gaining the advantage of color. But in this case the photographer has to deal with the same difficulties as are expe- rienced in photographing colored objects — e.g., colored tapestry. These have, as is well known, been overcome by the use of colored collodion. This led me to use the same method for photographing stained bacteria, and I have, in fact, succeeded, by the use of eosin- collodion, and by shutting off portions of the spectrum by colored glasses, in obtaining photographs of bacteria which had been stained with blue and red aniline dyes. Nevertheless, from the long ex- posure required and the unavoidable vibrations of the apparatus, the picture does not have sharpness of outline sufficient to enable it to be of use as a substitute for a drawing, or, indeed, even as evidence of what one sees. For the present, therefore, I must abstain from pub- lishing photographic representations ; but I hope, at a subsequent period when improved methods allow a shorter exposure, to be able to remedy this defect." 1 The paper referred to is published in Colm's "Beitrage zur Biologie d. Pflanzen." 102 PHOTOGRAPHING BACTERIA. Since the above was written considerable progress has been made in removing the technical difficulties, and a few bacteriologists have succeeded in making very satisfactory photomicrographs. As speci- mens of what may be done with the best apparatus and the highest degree of skill, we may call attention to the photomicrographs in the Atlas der Bakterienkunde of Frankel and Pfeiffer, and those of Roux in the Annales of the Pasteur Institute. The writer, also, has devoted much time to making photomicrographs which have served as illustrations for several of his published works. Those who have had no practical experience in making photo- micrographs are apt to expect too much and to underestimate the technical difficulties. Objects which under the microscope give a beautiful picture, which we desire to reproduce by photography, may be entirely unsuited for the purpose. In photographing with high powers it is necessary that the objects to be photographed be in a single plane and not crowded together or overlying each other. For this reason photographing bacteria in sections presents special difficulties, and satisfactory results can only be obtained when the sections are extremely thin and the bacteria well stained. Even with the best preparations of this kind much care must be taken in selecting a field for photography. It must be remembered that the expert microscopist, in examining a section with high powers, has his finger on the fine adjustment screw and focuses up and down to bring different planes into view. He is in the habit of fixing his at- tention on that part of the field which is in the focus and disregard- ing the rest. But in a photograph the part of the field not in focus appears in a prominent way which mars the beauty of the picture. In a cover-glass preparation made from a pure culture, when the bacteria are well distributed, this difficulty does not present itself, as the bacteria are all lying in a single plane; but the portion of the field which can be shown at one time is limited by the spherical aberra- tion of the objective, which the makers do not seem able to overcome in high-power lenses of wide angle, at least not without loss of de- fining power. Usually preparations of bacteria are stained for photography, but with some of the larger forms, such as the anthrax bacillus, very satisfactory photomicrographs may be made from unstained preparations. In this case a small quantity of a recent culture is put upon a slide, covered with a thin cover glass, and placed at once upon the stage of the microscope. The main difficulty to be encoun- tered results from the change of location of the suspended bacteria resulting from the pressure of the objective in focussing. Motile bacteria, of course, cannot be photographed in this way without first arresting their movements by means of some germicidal agent ; PHOTOGRAPHING BACTERIA. 103 and in general it will be found more satisfactory to fix the micro- organisms to be photographed to a slide or cover glass by desiccation and heat, and to stain them with one of the aniline colors. Objects which are opaque cannot be photographed by transmitted light, and objects which have a deep orange or red color are practi- cally opaque for the actinic rays which are at the violet end of the spectrum. Such objects simply intercept the light, but this gives the outlines, and, where there are no details of structure, is all that is required to illustrate the form and mode of grouping. Softer and more satisfactory photomicrographs of bacteria are made when the staining is not such as to entirely arrest the actinic rays. Among the aniline colors Bismarck brown and vesuvin are the most suitable, care being taken, with the larger bacteria especially, not to make the staining too intense. Objects which are transparent for the ac- tinic rays, or nearly so, give a very feeble photographic image, or none at all, on account of the want of contrast in the impression made upon the sensitive plate. This is the case when we attempt to photograph, by ordinary white light, objects which are stained violet or blue. But this want of contrast in the negative can be overcome by the use of specially prepared plates and colored screens of glass interposed between the object and the source of light. The so-called orthochromatic plates are more sensitive to the rays toward the red end of the spectrum than ordinary plates. They are prepared by treating the plates with a solution of eosin, of erythrosin, or of rose bengal (Vogel), and may now be purchased in this country from dealers in dry plates. If we shut off the violet rays by the use of a yellow screen, objects having a yellow or orange color may be pho- tographed upon orthochromatic plates, although the time of exposure will be quite long owing to the comparatively feeble actinic power of the yellow rays. We may also make photomicrographs of objects stained with methylene blue or with fuchsin, because objects stained with these colors are opaque for the rays from the red end of the spectrum, and sufficiently so with yellow light to give a good photographic con- trast. Frankel and Pfeiffer recommend the use of a green light-fil- ter (green glass screen) for all preparations stained with methyl vio- let, fuchsin, or methylene blue; and for brown-stained preparations a pure blue light. The writer has been in the habit of using a yellow glass screen for fuchsin-stained preparations, and has had excellent results, but the time of exposure is necessarily long. A yellow glass screen may be prepared by dissolving tropjeolin in negative varnish, and pouring this upon a clean glass slide, where it is permitted to dry. To show bacteria in photographs in a satisfactory manner we 104: PHOTOGRAPHING BACTERIA. require an amplification of five hundred to one thousand diameters ; and as it is often desirable to make comparisons as to the dimen- sions of microorganisms which resemble each other in form, it is best to adopt a standard amplification. The writer has himself adopted, and would recommend to others, a standard amplification of one thousand diameters. This is about as high a magnifying power as we can get with satisfactory definition, or as we require, and it is a convenient number when measurements are made from the photograph. The beginner, after having put his apparatus in position, should focus the lines of a stage micrometer upon the screen with the optical apparatus which he proposes to use ; then by moving the screen forward or back as required, and carefully focus- sing the lines, he will ascertain what is the position of the screen for exactly one thousand diameters. If the stage micrometer is ruled with lines which are one one-thousandth of an inch apart, it is evi- dent that when projected upon the screen they should be one inch apart to make the amplification one thousand diameters. But it must be remembered that any change in the position of the optical combination will change the amplification. If, therefore, the cover correction of the objective is changed, or the position of the eyepiece — if one is used — it will be necessary to again adjust the distance of the screen. Apparatus required. — A first-class immersion objective of one- twelfth of an inch or higher power, a substantial stand which can be placed in a horizontal position, and a camera which can be coupled with the microscope tube, are the essential pieces of apparatus. If sunlight is to be used a heliostat will also be required. The oil-immersion objectives of any good maker may be used, but the apochromatic objectives and projection eyepieces of Carl Zeiss, of Jena, are especially to be recommended. Indeed, those who can afford it will do well to get Zeiss' complete apparatus, which includes a stand having a mechanical stage and a camera mounted upon a metal frame conveniently provided with focussing appliances, etc. However, good work may be done with less expensive appa- ratus. The stand should be substantial and well made, with a delicate, fine adjustment. A mechanical stage is not essential, but is a great convenience in finding and adjusting to the centre of the screen a satisfactory field to photograph. The substage should be provided with a good apochromatic condenser, and with appliances for moving the condensing lens forward and back and for centring it, with dia- phragms, etc. By the use of a high-power objective, like the one-eighteenth-inch oil-immersion of Zeiss, the desired amplification may be obtained PHOTOGRAPHING BACTERIA. 105 without the use of an eyepiece ; and, as a rule, it is best not to use an ordinary eyepiece to secure increased amplification, as this is ob- tained at the expense of definition. But an amplifier may be used in the tube of the microscope, as first recommended by Woodward. In this case the amplifier must be carefully adjusted with reference to the distance of the screen, to secure the best possible definition. The projection eyepieces of Zeiss are constructed especially for photography and possess a decided advantage. By the use of his three-millimetre apochromatic oil-immersion objective and projec- tion eyepiece No. 3 we may obtain an amplification of one thousand diameters with excellent definition. Light. — Sunlight is in many respects the most satisfactory for photography, but has the disadvantage that it is not always available. In some sections of the country weeks may pass without a single clear day suitable for making photomicrographs. In addition to the uncertainty arising from cloudy weather, we have to contend with the fact that the sun is only available for use with a heliostat for a limited time during each day, and that this time is greatly restricted in Northern latitudes during the winter months. When sunlight is to be employed the microscope and camera must be set up in a room having a southern exposure on a line corresponding with the true meridian of the place. The heliostat is placed outside the window in such a position that when properly adjusted the light of the sun will fall upon the condenser attached to the substage of the microscope. The condenser must be carefully centred, so that the circle of light falling upon the screen shall be uniform in intensity and outline. The calcium, magnesium, or electric light may be used as a sub- stitute for sunlight, but they are all rather expensive, unless, in the case of the electric light, a suitable current is available without the expense of generating it for the special purpose in view. The writer has obtained very good results with the calcium light, but has no ex- perience in the use of the electric light. Woodward, as a result of extended experiments, arrived at the conclusion that "the electric light is by far the best of all artificial lights for the production of photomicrographs." He used a Grove battery of fifty elements to generate the current, and a Duboscq lamp. The current from a dynamo would no doubt be much cheaper and more conveniently used, if an electric-lighting plant was in the vicinity. The apparatus shown in Fig. 73 was designed by Mr. Pringle for the use of the calcium light. It will serve to illustrate the arrange- ment of the microscope and camera in connection with any other light as well. An oil lamp may be placed in the position of the oxy- hydrogen burner ; or, if sunlight is to be employed, a heliostat will be placed in the same position. 106 PHOTOGRAPHING BACTERIA. When a colored screen is used this may be placed either before or behind the condensing lens — we prefer to place it behind, although Neuhauss has shown that it makes no difference in the length of the exposure. We cannot in the present volume give full details with reference 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 oxyhydrogeu lime light, the magnesium light, and the electric arc light 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 fuchsin 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 tropteolin 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 fuchsin-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 condenser. 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 the 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 FIG. 74. above the camera by arms depending from the ceiling, or by upright arms attached to the baseboard. " I 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 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 From Johns Hopkins University Circulars, vol. ix., No. 81, p. 72. PLATE I. STERNBERG'S BACTERIOLOGY. V :^v ^ V r / % N* ' /**- . ** 7 •• jl * < :$fafi\ "=*% T "" v* • jf/' ^;.f * N/''^ \A ^ . v ' x y»'*', >Sa ^ ^ I**?* ^» • - T\ PHOTOMICROGRAPHS BY GAS LIGHT. PLATE II. STERNBERG'S BACTERIOLOGY. COLONIES ANI PLATE I. PHOTOMICROGRAPHS OF BACTERIA MADE BY GASLIGHT. FIG. 1. — Streptococcus cadaveris, from a culture in agua coco; stained with fuchsin. x 1,000. (Steruberg.) 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 fuchsin. x 1,000. (Steruberg.) FlG. 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. x5. (Sternberg.) FIG. 2. —Colonies of Bacillus coli similis in gelatin roll tube, end of twenty-four hours at 22° C. xlO. (Sternberg.) FlG. 3. — Stick culture of Bacillus coli similis in nutrient gelatin, end of seven days at 20° C. (Sternberg.) FlG. 4. — Stick culture of Bacillus intestinus motilis in nutrient gelatin, end of four days at 22° C. (Sternberg.) FlG. 5. — Stick culture of Bacillus leporis lethalis in nutrient gelatin, end of eight days at 22° C. (Sternberg.) FlG. 6. — Stick culture of Micrococcus tetragenus versatilis in nutrient gelatin, end of two weeks at 22° C. (Sternberg.) FlG. 7. — Colonies of Bacillus cuniculicida Ha vaniensis in gelatin roll tube, end of forty-eight hours at 21° C. x 10. (Sternberg.) FlG. 8. — Colonies of Bacillus coli commuhis in gelatin roll tube, end of forty-eight hours at 22° C. x 10. (Sternberg.) PART SECOND. GENERAL BIOLOGICAL CHARACTERS: INCLUDING AN 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 other hand, we have ample evidence that widely distributed species exist having very definite morphological and biological characters which 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 has recently (1892) made an investigation for the pur- pose of ascertaining the structure of bacterial cells. Various meth- ods were employed, but the most satisfactory results were obtained by fixing with nitric acid, with or without alcohol, and without pre- 112 STRUCTURE, MOTIONS, REPRODUCTION. vious drying ; the preparations were then stained with carbol-meth- ylene-blue or carbol-fuchsin 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-fuchsin 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 o£ 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, as 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- ening of the outer layers of the cell wall. This jelly-like material causes the cells to adhere to each other, forming zooglcea masses. In 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 gelatinous 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 are provided with slender, whip-like appendages called flagella. The micrococci in general are not endowed with the power of executing spontaneous movements, and they are not provided with flagella. STRUCTURE, MOTIONS, REPRODUCTION. 113 But recently two motile species have been described, and in one of these — Micrococcus agilis of Ali-Cohen — the presence of flagella has been demonstrated. Many of the bacilli and spirilla are actively motile, and the pre- sence of flagella, which has long been suspected, has recently been demonstrated 'for a considerable number of species by Loffler and others. It must be remembered that the molecular movement which is common to all minute particles suspended in a fluid is a vibratory motion?'?*- si hi, which does not change the .relative position of the moving particles. This so-called Brownian movement has frequently been mistaken for a vital motion, as has also the movement due to currents in the liquid in which non-motile organisms are suspended. The latter is to be distinguished by the fact that the microorganisms are all carried in one direction. This movement due to a current, in connection with the vibratory Brownian movement, is very deceptive, and it is often hard for a beginner in bacteriological study to con- vince himself that what 'he sees is not a vital movement. But in true vital movements we have progression in different directions, and the individual microorganisms approach and pass each other, often in a most vigorous and active manner, passing entirely across the field of view or changing direction in an abrupt way. Sometimes the motion is slow and deliberate, the bacillus progressing with a to- and-fro motion, as if propelled by a trailing flagellum ; or it may be serpentine when the moving filament is flexible ; or again it is a darting forward motion which is so rapid that the eye can scarcely follow the moving body. The spirilla have a rotary movement as well as a progressive one, and this is often extremely rapid. Some- times bacilli spin around with a rotatory motion, as if they were an- chored fast to a fixed point, as they may be by the flagellum being attached to the slide or cover glass. Frequently, in a pure culture, the individual bacilli may be seen to come to rest, and, after an inter- val of repose, to dart forward again in the most active way. Or we may find, on examining the same culture at different times, that upon one occasion there is no evidence of vital movements, and on another all of the bacilli are actively motile. These differences de- pend upon the age of the culture, temperature conditions, etc. Reproduction by binary division is common to all of the bacte- ria, and in many species this is the only mode of reproduction known. When circumstances are favorable for rapid multiplication the indi- vidual cells grow in length, and a constriction occurs in the middle transverse to the long diameter. This becomes deeper, and after a time the cell is completely divided into two equal portions, which again divide in the same way. Separation may be complete, or the 8 114 STRUCTURE, MOTIONS, REPRODUCTION. cells may remain attached to each other, forming chains (strepto- cocci) or articulated filaments (scheinfdden of the Germans). The bacilli and spirilla divide only in a direction transverse to the long diameter of the cells, but among the micrococci division may occur either in one direction, forming chains ; or in two directions, forming tetrads ; or in three directions, forming ' ' packets " of eight or more elements. The staphylococci, in which the cells do not re- main associated, divide indifferently in any direction. The rapidity of multiplication by binary division varies greatly in different species, and in the same species depends upon conditions re- lating to the culture medium, age of the culture, temperature, etc. Under favorable conditions bacilli have been observed to divide in twenty minutes, and it is a matter of common laboratory experience that colonies of considerable size and containing millions of bacilli may be developed from a single cell in twenty-four to forty-eight hours. A simple calculation will show Avhat an immense number of cells may be produced in this time as a result of binary division oc- curring, for example, every hour. The progeny of a single cell would be at the end of twenty-four hours 16,777,220, and at the end of forty-eight hours the number would be 281,500,000,000. Some of the earlier observers have noted the presence of oval or spherical refractive bodies in cultures containing bacilli ; but that these were reproductive elements, although suspected, was not de- monstrated until a comparatively recent date. Pasteur was one of the first to point out the fact that certain bacteria have two modes of reproduction — by fission and by the formation of endogenous spores ; but the first careful study of the last-mentioned method was made by Koch in his classical study of the anthrax bacillus (1878), and by Cohn, who studied the formation of spores in Bacillus subtilis. These reproductive bodies serve the same purpose in the preserva- tion of species as the seeds of higher plants. They resist desiccation and may retain their vitality for months or years until circumstances are favorable to their development, when, under the influence of heat and moisture, they reproduce the vegetative form — bacillus or spiril- lum— with all of its biological and morphological characters. They are composed of condensed protoplasm which retains the vital char- acters of the soft protoplasm of the mother cell from which it has been separated ; and it is evident that whether reproduction occurs by fission or by the formation of endogenous spores, the protoplasm of 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 before the forma- tion of spores occurs ; and these filaments may be associated in bun- dles or intertwined in irregular masses. At first the protoplasm of the STRUCTURE, MOTIONS, REPRODUCTION. 115 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 -a. 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 ^ 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- 116 STRUCTURE, MOTIONS, REPRODUCTION. ity of the oval spore, as shown at a, Fig. 70, and in other species the exosporium is ruptured and the bacillus emerges from the side, as seen at 6. 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 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 temperatiire has an important in- fluence upon spore formation. Thus the anthrax bacillus does not form spores at temperatures below 20° 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. 117 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 zooglcea 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, 52.32 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 and Bacillus erythrosporus) in the following manner : Ten cubic centimetres of distilled water in a test tube were infected with a small 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. 3 was now ascertained by counting, and it was put aside for two or three days, at the end of which time the number was again estimated by counting. In every case there was an enormous increase in the number of bacteria. In order to be sure that the dis- CONDITIONS OF GROWTH. 119 tilled water was pure, it was distilled a second time in a clean glass retort, but the result was the same. Bolton remarks, with reference to these results: "If we seek to explain this remarkable fact we must remember, in the first place, what an extremely small abso- lute mass is represented by an enormous number of bacteria, and what a minute amount of material is required for the formation of this mass. In ten cubic centimetres of distilled water, in the experi- ment last referred to, there were about twenty million bacteria (two million per cubic centimetre). If we estimate the diameter of each at one j.i, with a specific weight of 1, the absolute weight would be for the entire number one-one-hundredth of a milligramme — that is to say, a quantity which cannot be determined by any of our methods of weighing." Bolton supposes that the small amount of organic pabulum re- quired fell into the water in the shape of dust, or was attached to the walls of the test tube in spite of all the precautions taken. Nitrogen is chiefly obtained from albuminoid substances, but Pasteur has shown that it may also be obtained from ammonia. This is shown by cultivating bacteria in a medium containing an ammonia salt, as in the following : PASTEUR'S SOLUTION. Distilled water, ...... 100 Cane sugar, ...... 10 Tartrate of ammonia. . . ... . 1 Ashes of one gramme of yeast, .... 0.075 CORN'S SOLUTION. Distilled water, . . . . . . 100 Tartrate of ammonia, ..... 1 Ashes of yeast, ....... 1 Many bacteria multiply abundantly in these solutions. Carbon is obtained from the various organic substances contain- ing it ; among others, from starch, sugars, glycerin, organic acids and their salts, etc. Temperature. — There are certain limits of temperature within which development may take place, but these differ greatly with different species. As a rule, growth is arrested when the tempera- ture falls below 10° C. (50° F.), but some species multiply at a still lower temperature. Thus Bolton observed a very decided increase in certain water bacteria kept in an ice chest at 0° C., and other ob- servers have witnessed development at the freezing temperature. Most saprophytic bacteria grow within rather wide temperature limits, but the rapidity of development is greatest at a certain favor- able temperature, which is usually between 25° and 30° C. The 120 CONDITIONS OF GROWTH. parasitic species have a more restricted range, which approaches the normal temperature of the animals in which they habitually de- velop. At 40° C. (104° F.) growth, as a rule, ceases, but there are some notable exceptions to this rule. Miquel some years ago found a bacillus in the water of the Seine which grew at a temperature of 69° to 70° C. ; Van Tieghem reports having observed species in thermal waters capable of growth at a still higher temperature (74° C.) ; and Globig has more recently ob- tained from garden earth several species which multiplied at 65° C. Some of the species found by the last-named observer were even found to require a temperature of about 60° for their development ; and yet this temperature is quickly fatal to a large number of the best known species. Low temperatures, while arresting the growth of bacteria, do not destroy their vitality. This has been demonstrated by numerous ex- periments, in which they have been exposed for hours in a refrigerat- ing mixture at —18° C. Frisch has even subjected them to a tempe- rature of — 87° C. by the evaporation of liquid carbon dioxide, and found that they still grew when placed in favorable conditions. Parasitism. — The strict parasites grow only in the bodies of liv- ing animals, or in artificial media kept at a suitable temperature. As examples we ma}' mention the bacillus of tuberculosis, the bacil- lus of leprosy, the micrococcus of gonorrhoea, the spirillum of re- lapsing fever. There is also a large class of facultative para- sites which, when introduced into the body of a susceptible animal, multiply in it, and may continue to live as parasites so long as they are transferred from one animal to another, but which are also able to live as saprophytes independently of a living host. To this class belong the pus cocci, the bacillus of typhoid fever, the spirillum of cholera, and many others. It seems extremely probable that the strict parasites were at one time capable of living a saprophytic existence, and that their restric- tion to a parasitic mode of life has been effected in course of time in accordance with the laws of natural selection. This view is sup- ported by the fact that the tubercle bacillus, which has been regarded as a strict parasite, which can only be cultivated artificially under very special conditions, has been shown to 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). Reaction of Medium. — Some bacteria grow readily in a medium having an acid reaction, while the slightest trace of acidity prevents the development of others. As a rule, the pathogenic species require a neutral or slightly alkaline culture medium. CONDITIONS OF GROWTH. 121 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 Freudenreich. 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 («) by exhausting the pabulum necessary for its growth or (b) by producing substances which inhibit the development of an- otlier species or destroy its vitality. Freudenreich 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 pyocyanus, Bacillus cyanogenus, Bacterium phosphorescens, Bacillus prodigiosus, Spi- rillum cholerse Asiaticse. 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 abdominalis, Bacillus anthracis, Bacillus septi- caemia? hsemorrhagicEe, Spirillum tyrogenum. The following have a decided antagonism : Bacillus pyogenes foatidus prevents the growth of Spirillum choleras Asiaticae ; Micrococcus roseus prevents the growth of Micrococcus tetragenus. The cholera spirillum will not grow in sterilized cultures of Bacillus pyocyanus, 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 iion-chromogenic varieties of some of the best known chromogenic species may be produced by special methods of cultivation. Thus Wasserzug obtained a non-chromogenic variety of the bacillus of green pus (Bacillus pyocyanus) by the action of time added to that of antiseptics. He says : ' ' 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 Havaniensis), 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. 123 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 p}~ogenes 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 124 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 1880, ob- tained evidence that attenuation of virulence may result from ex- posure 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 septica3mia induced by the subcutaneous injection of my own saliva, and due to the presence of a micrococcus (Micrococcus pneumoniEB crouposae), 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. 125 "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 recently 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. '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 of bacterial growth. Pigment Production. — A considerable number of species are 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- EPNBERG'ft BACTERIOLOGY. Plate III. Fig.l. Fig.Z. Fig3. Fig.l . Sarcinalutea'agar culture Fi^.2. Bacillus prodigiosus, a^ar culture. Fig. 3. Bacillus pyocyanus.agar culture. Fig.4-. Bacillus Havaniensis, potato culture, PRODUCTS OP VITAL ACTIVITY. 127 rent culture medium, coloring especially the upper portion, in stick cultures in nutrient gelatin or agar. This is the case with Bacillus pyocyanus, 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 pyocyanus (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 cul- tures 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 pig- ment, and cultures in bouillon to give both this and pyocyanin. In milk the fluorescent-green color is first seen, but subsequently, when the casein has been peptom'zed by a diastase produced in the culture, pyocyanin 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 pyocyanus in addition to pyocyanin : one, soluble in alco- hol, has by transmitted light a chlorophyll-green color, by reflected light it is blue ; the other, insoluble in alcohol and chloroform, by transmitted light is of a dark orange-red, by reflected light a green- ish-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 pyocyanus. 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. 128 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. 267), Bacillus fluorescens liquefaciens (No. 277), Bacillus indicus (No. 283), Bacillus pyocyanus (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 (1886) 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 the culture added to nutrient gelatin which had been liquefied by heat prevented it from subsequently forming a solid jelly when cold. In a recent study of the ferments produced by bacteria which PRODUCTS OF VITAL ACTIVITY. 129 "cause liquefaction of gelatin — " tryptic enzymes " — made by Fermir in the laboratory of the Hygienic Institute of Munich (1891), the fol- lowing 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 00° C. — Bacillus subtilis, Bacillus pyocyanus, Bacil- lus fluorescens liquefaciens, Sarcina aurantiaca ; some by 65° to 70° C. — Bacillus anthracis, Spirillum cholerae 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 pyocyanus (No. 95), Bacillus subtilis (No. 379), Bacillus indicus (No. 283), Ba- cillus prodigiosus (No. 284), Spirillum cholerse Asiaticae (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. 9 130 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 process 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 : CjH^O,, = 2(HC3H6O3). 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 previous^ 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 CHS.CH9.OH + O8 = CH3.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 starch, dextrin, sugar, or salts of lactic acid, when oxygen is ex- cluded it produces butyric acid in considerable quantity, and at the same 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. 131 This is introduced i-nto 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 pyocyanus produces butyric acid in addition to ethyl alco- hol 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. 132 PRODUCTS OF VITAL ACTIVITY. Botkin has recently (1892) described a "Bacillus butyricus" (No. 466) which he has not been able to identify positively with the butyric-acid ferment described by Prazmowski (No. 404). 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 anaerobic cultures, in a suitable medium, while all other bac- teria and spores present were destroyed. The writer has described a bacillus which causes active acid fermentation in culture solutions containing glycerin. The acid formed is volatile and is probably propionic acid — see Bacillus acidi- formans (No. 93). 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 bours until they swell; they are then carefully washed and 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 one 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 ready for use a portion (one- fifth to one- third) is left in the flask as ferment for a fresh quantity of milk. The temperature should be maintained at about 18° C. ; but at the commencement a higher temperature is desirable. The korner should be carefully cleaned from time to time and broken up to the size of peas. 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 urine is PRODUCTS OP VITAL ACTIVITY. 133 effected by various microorganisms, but chiefly by the Micrococcus urese, 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 : COH4N2 + 2H2O = CO, + 2NH9 + H,0 = (NH4),C03. According to Van Tieghem, Micrococcus urese 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 urese 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 urese liquef aciens — nas also been studied in Flugge'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 urese 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 (No. 467), Urobacillus Duclauxi (No. 468), Urobacillus Freu- denreichi (No. 469), Urobacillus Maddoxi (No. 470), Urobacillus Schutzenbergi (No. 471). 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 (No. 195). The fermented juices become very viscous, owing to the formation of a gum-like product resem- bling dextrin ; at the same time mannite and CO, are produced. The gum-like substance, called viscose by Bechamp, is soluble in cold water and is precipitated by alcohol. Recently (1892) Guille- beau has described a micrococcus and a bacillus which produce vis- cous fermentation in milk — Micrococcus Freudenreichi ' (No. 475) and 134 PRODUCTS OF VITAL ACTIVITY. Bacillus Hessi (No. 47G). A micrococcus producing viscous fermen- tation in milk has also been described by Schmidt-Miihlheim, and a bacillus by Loffler. Bacillus mesentericus vulgatus also produces a similar change in milk. Marsh gas, 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 CO2 and CH4 and small quantities of H2S. The second fermentation occurs when an alkaline solution of flesh extract containing cellulose in suspension is used. The gases formed are CO3 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 Lindenborn, 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 on 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 Fliigge, 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, hydrosulphuric acid (H2S), phosphoretted hydrogen (PH3), 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, ty rosin, neuridin, cadaverin, putrescin, cholin, neurin, peptotoxiii, 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 OP VITAL ACTIVITY. 135 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 saprophy tic 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 — COS, 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 anaerobics — 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 136 PRODUCTS OF VITAL ACTIVITY. doubt, according to whether they are or are not supplied with atmo- spheric oxygen. The anaerobic bacteria concerned in putrefaction have as yet re- ceived comparatively little attention. Among the aerobics and fac- ultative anaerobics the following are best known : Micrococcus fcetidus (No. 189), Bacillus saprogenes I., II., and III., Bacillus coprogenes fcetidus (No. 116), Bacillus putrificus coli (No. 324), Pro- teus vulgaris (No. 97), Proteus Zenkeri (No. 100), Proteus mirabilis (No. 99), Bacillus pyogenes foetidus (No. 72), Bacillus fluorescens liquefaciens (No. 277), Bacillus pyocyanus (No. 95), Bacillus coli communis (No. 89), Bacillus janthinus (No. 267). Soluble Ferments. — 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 from the human intestine a species which dis- solves starch. Marcano, by filtering cultures of species capable of this ferment action through porcelain, was 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 and 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 albumin a peptonizing ferment was formed ; in its ab- sence, 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- sirable. 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 peptonizing ferment produced by certain species of bac- teria. Some authors also speak of a soluble ferment capable of inverting cane sugar or milk sugar. According to Hueppe, such a ferment is produced by the Bacillus acidi lactici. A soluble ferment for cel- lulose is supposed by Fliigge to be produced by several species — among others by Bacillus butyricus and by Vibrio rugula. Several bacilli produce a soluble ferment capable of coagulating the casein of milk. Reduction of Nitrates, and Nitrification. — The researches of Gayon, Dupettit, and others show that certain bacteria are able to reduce nitrates with liberation of ammonia and free nitrogen. This is effected in the absence of oxygen by anaerobic bacteria, and, PRODUCTS OF VITAL ACTIVITY. 137 among others, by 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 Herseus, 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 recent researches of Winogradsky, of the Franklands, and of Jordan show that the failure of earlier investigators to obtain the nitrifying 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 isolating 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 (see Nos. 439, 440, 441). In his latest communication (September, 1891) Win- ogradsky arrives at the conclusion that the ferments which cause the oxidation of ammonia and production 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 formation of nitrates so long as the specific ferment was ab- sent to which this second oxidation is ascribed (nitrifying bacillus of Winogradsky, No. 451). Phosphorescence. — Recently several different bacteria have been studied which, in pure cultures, give rise to phosphorescence in the medium in which they are cultivated. In gelatin cultures the light is sufficient in some instances to enable one to tell the time by a 138 PRODUCTS OP VITAL ACTIVITY. watch in a perfectly 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- cens from sea water in the vicinity of the West Indies gives the most striking results, especially when planted upon the surface 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 harbor at Kiel, and the other a widely distributed species called by Fischer Bacterium phosphorescens. Katz (1891) has re- cently described several new species obtained by him from sea water and from phosphorescent fish in the markets at Sydney, New South Wales — Bacillus smaragdino- phosphorescens, Bacillus argenteo-phos- phorescens, Bacillus cyaneo-phosphorescens, Bacillus argenteo-phos- phorescens liquefaciens (Nos. 337, 338, 341, and 342). 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 dotfbtless 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, C5HMN2. — 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, C&H,4NS. — Isomeric with neuridin ; 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 140 PTOMAINES AND TOXALBUMINS. forms a crystalline mass. Is produced in cultures of the cholera spirillum and of the spirillum of Finkler and Prior which have been kept for a month or more at 37° C. Putrescin, CjH^N,. — A base resembling cadaverin and com- monly associated with it. Obtained by Brieger from various sources, most abundantly from substances containing gelatin and in the more advanced stages of putrefaction. It is obtained in the form of a hydrate, which is a transparent liquid having a boiling point of about 135°. With acids it forms crystalline salts. Saprin, C5AI6N2. — Resembles cadaverin and is commonly as- sociated with it in putrefying material. Isolated by Brieger. Methylamine, CH3.NH.,. — Obtained by Brieger from putrefying fish and from old cultures of the cholera spirillum. Dimethylamine, (CHJ.^.NH. — Obtained by Brieger from putre- fying gelatin and by Bocklisch from decomposing fish. Trimethylamine, (CH3)3N. — Obtained from various sources, and by Brieger from cultures of the cholera spirillum and of the strepto- coccus of pus. TOXIC PTOMAINES. Neurin, C5H13NO. — First obtained by Liebreich in 1865 as a decomposition 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 base is toxic in small doses. In frogs the injection of a few milli- grammes produces paralysis of the extremities. Respiration is first arrested 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. Cholin, C5H15NOa. — First obtained from hog's bile by Strecker in 1862. Has been obtained by Brieger from various sources, in- cluding cultures of the cholera spirillum. It is also found widely distributed in the vegetable kingdom. May be prepared from the yolk of eggs by the method of Diakonow. Cholin is obtained in the form of a syrupy, alkaline liquid which combines with acids to form deliquescent salts. At first this base was not supposed to have toxic properties, but more recent researches have shown that in compara- tively large doses it produces symptoms resembling those caused by minute doses of neurin. Muscarin, C5H15NO3. — This toxic principle of poisonous mush- rooms has also been obtained by Brieger from putrefying fish. It may be produced artificially by the oxidation of cholin. In small doses it kills rabbits and frogs. In the rabbit it produces lacrymation and PTOMAINES AND TOXALBUMINS. 141 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 Vaughan obtained 0. o 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, C2H7N3. — 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 fseces, 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. Typhotoxin, C7HnNO2. — 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 144 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. Quite recently (1891) G. and F. Klemperer have announced their success in obtaining a toxalbumin from cultures of Micrococcus pneumonisB crouposse (" diplococcus pneumonise "); this they propose to call pneumotoxin. 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 recently (1891) reported his success in isolating a toxic substance from tubercle cultures. The contents of tubes con- taining 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. Mullein. — 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 Preusse by treating old potato cultures of the glanders bacillus with glycerin and water. The extract was filtered several times and then sterilized in a steam sterilizer. This lymph injected into horses in- fected with glanders gives rise to a very decided elevation of tempe- rature, while in 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 vtilgaris 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. 10 146 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 Wolff hugel (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 this 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- organisms 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 diseases do not form spores. It is probable also that it may be safely used to disinfect the clothing of small-pox patients, for we have ex- perimental evidence that a lower temperature destroys the virulence of vaccine virus (90°-95° C. — Baxter). In practical disinfection by means of dry heat it will be necessary to remember that it has but little penetrating power. In the experi- ments of Koch and Wolffhiigel it was found that registering ther- mometers placed in the interior of folded blankets and packages of various kinds did not show a temperature capable of killing bacteria after three hours' exposure in a hot-air oven at 133° C. and above. Moist Heat. — The thermal death-point of bacteria, in the ab- sence of spores, is comparatively low when they are exposed to moist heat. The results of the writer's experiments are given below: " In my temperature experiments I have taken great pains to insure the exposure of the test organisms to a uniform temperature, and have adopted ten minutes as the standard time of exposure. The method employed throughout has been as follows: From glass tubing having a diameter of about three-sixteenths of an inch I draw out in the flame of a Burfsen burner a number of capillary tubes, with an expanded extremity which serves as INFLUENCE OF PHYSICAL AGENTS. 147 an air chamber. A little material from a pure culture of the test organ- ism is drawn into each of these capillary tubes by immersing the open extremity in the culture, after having gently heated the expandea end. The end of the tube is then hermetically sealed by heat These tubes are im- mersed in a water bath maintained at the desired temperature for the stan- dard time. The bath is kept at a uniform temperature by personal supervi- sion. At the bottom of the vessel is a thick glass plate which prevents the thermometer bulb and capillary tubes, which rest upon it, from being ex- posed to heat transmitted directly from the bottom of the vessel To further guard against thj,s I am in the habit of applying the flame to the sides of the vessel, and a uniform temperature throughout the bath is maintained by frequent stirring with a glass rod. It is impossible to avoid slight variations, but by keeping my eye upon the thermometer throughout the experiment I have kept these within very narrow limits. . . . No attempt has been made to fix the thermal death-point within narrower limits than 2° C., and in the table the lowest temperature is given which has been found, in the experi- ments made, to destroy all of the microorganisms in the material subjected to the test. No doubt more extended experiments would result, in some in- stances, in a reduction of the temperature given as the thermal death-point for a degree or more. But the results as stated are sufficiently accurate for all practical purposes." ' The results obtained in these experiments, for non-sporebearing bacteria, are given in the following table. The time of exposure was ten minutes, except for the cholera spirillum and the cheese spi- rillum of Deneke. THERMAL DEATH-POINT OF BACTERIA. Spirillum choler* Asiatic* 52° Spirillum tyrogenum (cheese spirillum) 52 Spirillum Finkler- Prior 50 Bacillus typhi abdominalis 56 Bacillus of schweiue-rothlauf (rouget) 58 Bacillus murisepticus 58 Bacillus Neapolitanus (Emmerich's bacillus) 62 Bacillus cavicida 62 Bacillus pneumonine (Friedlander's) 56 Bacillus crassus sputigenus 54 Bacillus pyocyanus . 56 Bacillus indicus 58 Bacillus prodigiosus 58 Bacillus cyanogenus 54 Bacillus fluorescens . . 54 Bacillus acidi lactici 56 Staphylococcus pyogenes aureus 58 Staphylococcus pyogenes citreus 62 Staphylococcus pyogenes albus 62 Streptococcus pyogenes 54 Micrococcus tetragenus ... .- . . 58 Micrococcus Pasteuri 52 Sarcina lutea . . 64 Sarcina aurantiaca . . 62 Centigrade. Fahrenheit. 125.6° 125.6 122. 138.8 136.4 136.4 143.6 143.6 132.8 129.2 132.8 136.4 136.4 129.2 129.2 132.8 136.4 143.6 143.6 129.2 136.4 125.6 147.2 143.6 (4m.) (4m.) The following determinations of the thermal death-point of path- 1 Quoted from the Report of the Committee on Disinfectants of the American Pub- lic Health Association, pages 136 and 152. 148 INFLUENCE OF PHYSICAL AGENTS. ogenic organisms have been made by the authors named : Bacillus anthracis (Chauveau), 5-4° C. ; Bacillus mallei — the bacillus of glan- ders— (Loffler), 55° C., Bacillus gallinarum — micrococcus of fowl cholera— (Salmon), 56° C. ; Bacillus of diphtheria (Loffler), 60° C. In the writer's experiments the micrococcus of gonorrhosa was apparently killed by exposure for ten minutes to a temperature of 60° C. % ' ' Some gonorrhceal pus from a recent case which had not undergone treatment was collected for me by my friend Dr. Rohe in the capillary glass tubes heretofore described. A microscopical examination of stained cover-glass preparations showed that this pus contained numerous ' gono- cocci ' in the interior of the cells. Two of the capillary tubes were placed in a water bath maintained at 60° C. for ten minutes. The pus was then forced out upon two pledgets of cotton wet with distilled water. Two healthy men had consented to submit to the experiment, and one of these bits of cotton was introduced into the urethra of each and left in situ for half an hour. As anticipated, the result was entirely negative. For obvi- ous reasons no control experiment was made to fix the thermal death-point within narrower limits. " In connection with these experiments upon the thermal death-point of known pathogenic organisms, it is of interest to inquire whether the viru- lence of infectious material, in which it has not been demonstrated that this virulence is due to a microorganism, is destroyed by a correspondingly low temperature. Evidently, if this proves to be the case, it will be a strong argument in favor of the view that we have to deal with a microorganism in these diseases also. We have experimental proof that a large number of pathogenic organisms are killed by exposure for ten minutes to a tempera- ture of 55° to t>0° C. But, so far as I am aware, this low temperature would not be likely to destroy any of the poisonous chemical products which might be supposed to be the cause of infective virulence, leaving aside the fact that such chemical products have no power of self-multiplication, and, there- fore, could not be the independent cause of an infectious disease. ' " Vaccine Virus. — Carstens and Coert have experimented upon the tem- perature required to destroy the potency of vaccine virus. In a paper read at the International Medical Congress in 1879 they report, as a result of their experiments, that the maximum degree of heat to which fresh vaccine virus can be exposed without losing its virulence probably varies between 52° and 54° C. Fresh animal vaccine heated to 52° C. for thirty minutes does not lose its virulence. Fresh animal vaccine heated to 54. 5 = for thirty minutes loses its virulence. "Rinderpest. — According to Semmer and Raupach, exposure for ten minutes to a temperature of 55° C. destroys the virulence of the infectious material in this disease. <% Sheep-pox. — The authors last mentioned have also found that the same temperature — 55° C. for ten minutes — destroys the virulence of the blood of an animal dead from sheep-pox. " Hydrophobia. — Desiring to fix the thermal death -point of the virus of hydrophobia, I obtained, through the kindness of Dr. H. C. Ernst, a rabbit which had been inoculated, by the method of trephining, with material which came originally from Pasteur's laboratory. The rabbit sent me showed the first symptom of paralytic rabies on the eighth day after inocu- lation. It died on the eleventh day (March 2d, 1887), and I at once pro- ceeded to make the following experiment : "A portion of the medulla was removed and thoroughly mixed with 1 Since this was 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. 149 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 crouposae, 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 minutes' 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, 04° 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 05° 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. 150 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 Globig 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 steam under pressure at from 109° to 113° C. They were de- stroyed, however, by exposure for twenty-five minutes in steam at 113° to 116°, 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. 151 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 pipes 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 Loftier 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 152 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 hsematoglobin, 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 bacilli, but that in fact they commenced to vegetate before they were killed. Strauss placed anthrax spores in sterilized distilled water and in bouillon, 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- sition 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. 153 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 Moment (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. Duclaux, in 1885, experimented upon several different species of micrococci and bacilli, with the following results: Recent cultures of micrococci in bouillon, which preserved their vitality for more than a year in a dark place, were sterilized in July by exposure in the sun for fifteen days, and two species even within two or three days (micrococcus of biskra and micrococcus of pemphigus). Bacilli showed greater resisting power. Still more recently Gaillard has conducted a series of experiments, under the direction of- Arloing, which confirm the results previously obtained by observers heretofore named as to the germicidal power of sunlight in the presence of oxygen. 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, from 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. 154 INFLUENCE OF PHYSICAL AGENTS. 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 fifty 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 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 INFLUENCE OF PHYSICAL AGENTS. 155 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. VII. ANTISEPTICS AND DISINFECTANTS. GENERAL ACCOUNT OF THE ACTION OF. THE term antiseptic is used by some authors to designate an agent 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 Ave 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 not destroy the vitality of the germs of putrefaction. In a certain degree of concentration they are antiseptics and are .largely used for 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. 157 Restrains. Kills. 1 . 2100 1 -300 1 . 1550 1 • 500 1 : 50000 1 • 4000 Sodium arseniate 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 158 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 urea3, 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, I : 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. 159 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 160 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-peptoiie-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 1880 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 of 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- tion of an 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. 101 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 -.1850 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. 11 162 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: GO, 000, while in blood serum 1: 800 was required to destroy it in the same time. (e) The time of exposure 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 the anthrax bacillus in one minute (Nissen). On the other hand, ANTISEPTICS AND DISINFECTANTS. 163 quicklime (milk of lime) requires a contact of several hours to in- sure the destruction of pathogenic bacteria. (/) The temperature at ivhich the exposure is made has a material influence upon the result. This is shown by the experi- ments of Henle and of Nocht. (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- ganate and the hypochlorite 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 spirillum in a moist 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. 165 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 Nis- 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 H2O., 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 H.,Oa 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 H2O.,). Anthrax spores were killed in the same time by a twenty-per-cent solution (0.8 per cent HaO,). Tested upon a pure culture of pus cocci, it was active in the proportion of ten per cent (0.4 per cent of 166 ACTION OF GASES AND OF THE H2O2); a solution containing 0.24 per cent of H3O., 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 H2O2, 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 H2O2 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 bacteria, 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 H.2O2 in one thousand of water. Carbon Dioxide. — The experiments of Frankel show that certain bacteria grow in an atmosphere of C0a 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 CO2, 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 C02. 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 abundantly when a little atmospheric oxygen was ad- mitted. In the experiments of Frankland the cholera spirillum and the Finkler-Prior spirillum failed to develop in an atmosphere of CO2, and at the end of eight days were no longer capable of growth when 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 Finkler-Prior spirillum, although it did not entirely HALOID ELEMENTS UPON BACTERIA. 167 prevent development, and after seven days' exposure the spirilla were not all killed, although a comparatively small number of colonies developed. Bacillus pyocyaiius 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 may be 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), O.G1 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 cholerse Asiaticse 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 pyocyanus 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 pyocyanus, Spirillum cholerse Asiaticse, Spirillum Finkler-Prior). Hydrosulphuric Acid, H2S. — In the experiments of Frankland this gas proved to be quickly fatal to the bacteria tested (Bacillus pyocyanus, Spirillum cholerse Asiaticse, Spirillum Finkler-Prior). On the other hand, Grauer found that this gas did not exercise any in- jurious 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 1G8 ACTION OF GASES AND OF THE others that certain species of bacteria cause an abundant evolution of H2S as a result of their development in an albuminous medium (Bacillus sulfureus and Proteus sulfureus). Sulphur Dioxide, S02. — 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 SO2, 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 (H2S03). 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 SO2 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 S02 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 pyocyanus. 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 SO2 developed by the combustion of sixty grains of sulphur per cubic metre. This amount corresponds closely with that fixed by the Committee on Dis- infectants of the American Public Health Association on the experi- mental evidence obtained by the writer in 1885. But the committee insisted upon the presence of moisture and made the time of exposure twelve hours — "exposure for twelve hours to an atmosphere con- HALOID ELEMENTS UPON BACTERIA. 169 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 170 ACTION OF ACIDS AND OF THE pneumonias croupossc in the proportion of 1 : 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-ceiit 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 Behririg, 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. Iodoform. — Numerous experiments have been made with this agent, which show 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. 171 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. Riedliii found it without any action, even upon the cholera spirillum. Hydrofluoric Acid, HFL — 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." IX. ACTION OF ACIDS AND ALKALIES. Sulphuric Acid, H2SO4. — 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. Anthrax bacillus 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, mixed 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 five minutes by five per cent. Sulphurous Acid, H,SO3. — In the writers experiments (1885) micrococci were destroyed in two hours by 1 : 2,000 by weight of SOa added to water. Kitasato found that solutions of sulphurous acid in the proportion of 0. 28 per cent killed the typhoid bacillus, and 0.148 per cent the cholera spirillum. De la Croix found that one ACTION OF ACIDS AND ALKALIES. 173 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 SO,2 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. Xitrous 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 0.3 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 :6,6G6 (Miquel). Phosphoric Acid. — Exposure for four or five hours to a solution 174 ACTION OF ACIDS AND ALKALIES. containing 0.3 per cent destroys the typhoid bacillus, and 0.183 per €ent the cholera spirillum (Kitasato). The acid used contained 0.152 gramme H3P04 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 0.30 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 per cent, the cholera spirillum 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- lution 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 percent kills the tubercle bacillus in six hours ( Yersin). In the proportion of 1 : 300 it destroys the cholera spirillum in half an hour (Van Ermengem). ACTION OF ACIDS AND ALKALIES. 175 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 tweiity-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). Thyinic 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 176 ACTION OF ACIDS AND ALKALIES. 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 bacillus, 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 : 190 ; 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, ]STH3. — In Kitasato's experiments the typhoid bacillus was 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 : 300 ; diphtheria bacillus, 1 : 250 ; glanders bacillus, 1 : 250 ; typhoid bacillus, 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 : 650. 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 oxide. Liborius had previously reported still more favorable results, but 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 patients. When added in the proportion of three per cent steriliza- tion was effected in six hours, and by six per cent in two hours. When milk of lime containing twenty per cent of calcium hydrate was 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 applied to walls has been determined by Jager. Silk threads soaked in cultures of various pathogenic bacteria were attached to boards and the lime-wash applied with a camel's-hair brush. Anthrax ba- cilli (without spores), the glanders bacillus, Staphylococcus pyogene? aureus, 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. 177 killed by three successive applications. In 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. 12 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 variety 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 chloride, . . . . . 1 : 14300 Silver nitrate, . . 1 : 12500 SUBSTANCES VERY STRONGLY ANTISEPTIC. Osmic acid, . . . . . . 1 : 6666 Chromic acid, . . . . . . . 1 : 5000 Chlorine, ...... 1:4000 Iodine, ....... 1:4000 Chloride of gold, . ..... 1:4000 Bichloride of platinum, . . „ • . . 1 : 3333 Hydrocyanic acid, . . . . . 1 : 2500 ACTION OP SALTS. 179 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, . . . . . 1:3 180 ACTION OF SALTS. ANTISEPTIC AND GERMICIDAL VALUE OF VARIOUS SALTS, ARRANGED ALPHABETICALLY. Alum. — Antiseptic in the proportion of 1 : 222 (Miquel). Aluminum Acetate. — According to De la Croix, this salt is an antiseptic in the proportion of i : 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- struction 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 five minutes by a solution containing 0.12 percent, anthrax bacilli in one minute by 0. 1 per cent, Staphylococcus pyogenes aureus in one minute by 0. 2 per cent, anthrax spores in thirty minutes by a ACTION OF SALTS. 181 five-per-cent 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 twenty-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 Bietsch). 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-cent 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 182 ACTION OF SALTS. cocci, but was fatal to Micrococcus tetragenus — two hours' exposure. Koch 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- cilli. 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. Grold 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 twenty-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 Glanders 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 Chloride. — Antiseptic in the proportion of 1 : 11 (Mi- quel). Manganese Protochloride. — 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 spores of Bacillus anthracis and of Bacillus subtilis in two hours. More recent experiments indicate that failure to grow in culture so- lutions cannot be accepted as evidence of the destruction of vitality in the case of spores 'exposed to the action of this agent, unless due precautions are taken to exclude the restraining influence of the small amount of mercuric chloride which remains attached to the spores. Koch had ascertained that the development of spores is restrained by ACTION OP SALTS. 183 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-gelatin, 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 oiie-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 : GO, 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-down 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 184 ACTION OF SALTS. 1 : 5,000 for pathogenic bacteria in the absence of spores; due regard being 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 oxycyanide by 1 : 16,000 (Behring). Boer obtained the following results with the oxycyanide — cul- tures in bouillon, twenty-four hours in incubating oven, time of exposure two hours : Restrained development. Destroyed vitality. Anthrax bacillus 1 : 80000 1 : 40000 Diphtheria bacillus 1 : 80000 1 • 40000 Glanders bacillus. . . 1 : 60000 1 : 30000 Typhoid bacillus . . . . , 1 : 60000 1 : 30000 Cholera spirillum .... 1 : 90000 1 • 60000 Mercuric Iodide. — The antiseptic value of this salt is placed by Miquel at 1 : 40,000, which is more than double that given by the same author to the bichloride. In the writer's experiments upon the antiseptic value of salts and oxides of mercury the following results were obtained : ACTION OF SALTS. 185 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 Hydroclilorate. — 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 ruicrococci 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 10.0 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 Chr ornate. — 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 186 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). Potassium Permanganate. — In the writer's 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 ( Loftier). 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 bacilli. 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. Anthrax bacillus 1 60000 1 • 20000 Diphtheria bacillus 1 • 60000 1 • 2500 Glanders bacillus .... . . 1 : 75000 1 : 4000 Typhoid bacillus 1 : 50000 1 : 4000 Cholera spirillum. 1 : 50000 1 : 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. 187 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-csnt 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 188 ACTION OF SALTS. were not destroyed by two hours' exposure in a ten-per-cent solution, but a solution of five per cent killed the 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. 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 Asiaticse by 1 : 62,500, Bacil- lus typhi abdominalis by 1 : 5,000. In blood serum stronger solutions were required (1 : 500,000 for Staphylococcus pyogenes aureus). Sta- phylococcus pyogenes aureus, Streptococcus pyogenes, and Bacillus 190 ACTION OF COAL-TAR PRODUCTS, 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. Anthrax bacillus 1 : 120000 1 • 40000 Diphtheria bacillus I • 40000 1 '8000 Glanders bacillus 1 : 5000 1 :300 Typhoid bacillus 1 : 5000 1 :300 Cholera spirillum . . 1 • 1 00000 1 • 5000 METHYL VIOLET (PYOKTANIN). Anthrax bacillus Restrains development. Destroys vitality. 1 : 70000 1 : 10000 1 : 2500 1:2500 1 : 30000 1 : 5000 1 : 2000 1 :150 1 :150 1 : 1000 Diphtheria bacillus Typhoid bacillus Cholera spirillum 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, C6H6. — 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 of, or tincture ?) has but little germicidal power. The typhoid ba- ESSENTIAL OILS, ETC. 191 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 192 ACTION OF COAL-TAR PRODUCTS, not destroy these spores in thirty clays. 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. ISTicati 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. Anthrax bacillus 1 • 750 1 • 300 Diphtheria bacillus 1 -500 1 • 300 Glanders bacillus 1 -500 1 '300 Typhoid bacillus 1 -400 1 • 200 Cholera spirillum 1 :600 1 -400 Leitz reports the following results : The dejections of patient suffering from typhoid fever, mixed in equal quantity with the disin- fecting solution, were sterilized by a five-per-cent solution of car bolic acid in three days. Pure cultures of the typhoid bacillus wei sterilized in fifteen minutes by a five-per-cent solution. In the experiments of Nocht upon anthrax spores it was founc that while at the room temperature these spores were not destroys by several days' exposure in a five-per-cent solution, they were dc stroyed in three hours by the same solution at a temperature of 37.5°. Carbolic acid prevents putrefactive changes in bouillon when prt sent 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 Liideritz on the antiseptic power of an infusion of coffee. The first- named author found that anthrax bacilli no longer developed after three hours' exposure in a ten-per-cent solution, but spores were not killed at the end of a week. Streptococci in a bouillon culture re- quired twenty-four hours' exposure, and the staphylococci of pus were not destroyed in this time. Liideritz found that a three-per-cent in- fusion restrained the growth in nutrient gelatin of the typhoid ba- cillus, and a five-per-cent infusion killed the bacillus in two days ; the cholera 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. 193 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 I : 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 pyocyanus 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 13 194 ACTION OP 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 power. A four-per-cent solution, containing equal parts of cresol and H2SO4, 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-per-cent solution destroyed the tubercle bacillus in cultures and in sputum. As a result of his experiments Bearing concludes that cresol has no advantage over carbolic acid as a ger- micide for the destruction of spores. Tested upon Staphylococcus aureus, 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. Disinfektol. — This is a coal-tar product similar to creolin which has been recommended in Germany for disinfecting purposes. It is an oily, dark-brown fluid having a specific gravity of 1.086. It forms an emulsion with water, which has a slightly alkaline reaction. It has 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 disinf ektol equals in value, for the disinfection of the liquid discharges of typhoid patients, 12.5 per cent of creolin, thirty-three per Cent of hydrochloric acid, five per cent of carbolic acid, 1 : 500 of mercuric chloride. Ether. — Anthrax spores may germinate after being immersed in sulphuric ether for eight days (Koch). The tubercle bacillus is de- stroyed by ten minutes' exposure to the action of ether (Yersin). Essential Oils. — Chamberlain has made an extended series of experiments to determine the antiseptic power of the vapor of vola- 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 OILS, ETC. 195 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 " 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. Kiedlin 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 Ferret 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. 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. 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 experi- ments a solution of soda was added to release the hydroxylamin. Marpmann found that 1 : 100 preserved milk without change for four 196 ACTION OF COAL-TAR PRODUCTS, to six weeks, and that alkaline fermentation of urine was prevented by 1:1,000. Indol. — When added in excess to water this agent failed to de- stroy anthrax spores in eighty days (Koch). 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. Naphthol. — In the proportion of 1: 10,000 naphthol prevents the development of the glanders bacillus, the anthrax bacillus, the ty- phoid bacillus, the micrococcus of fowl cholera, of Staphylococcus. aureus and albus, and of several other microorganisms tested by Maximovitch. 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 fermentation. In the experiments of Foote hydronaphthol was found to show some germicidal power in the proportion of 1 : 2,300, but the conclusion is reached that a saturated aqueous solution (1: 1,150) does not equal a one-per-cent solution of carbolic acid or of creolin. 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 i& 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- coccus aureus does not destroy this micrococcus in five hours (v. Christmas- Dirckinck- Holmf eld) . Skatol in excess in water has no germicidal power, as tested upon anthrax spores (Koch). Smoke. — The researches of Beu show that meats which have been preserved by smoking commonly contain living bacteria cap- able of growing in culture' media ; and Petri has shown that pork which has 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 inoculation experiments. It was not until about six months after smoking that the bacillus failed to give evidence of vitality. Thymol. — A five-per-cent solution in alcohol does not destroy ESSENTIAL OILS, ETC. 197 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- cilli may be 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- blooded animals. Thus the 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. 199 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 Behring 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 cf 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 45.6° 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 200 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. 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 milk 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 addition of alcohol. The precipitate is washed and again dissolved in salt solution. The result showed that this cell globulin possesses germicidal power similar to that of blood serum. Urine. — The experiments of Lehmann show that fresh urine has a decided germicidal power for the cholera spirillum and the anthrax bacillus, and no doubt for other bacteria as well. To what constitu- ent of the urine this is due has not been determined. XIII. PRACTICAL DIRECTIONS FOR DISINFECTION. THE Committee on Disinfectants of the American Public Health Association (appointed in 1884), after an extended investigation with reference to the germicidal value of various agents, in a final report submitted in 1887 submits the following " Conclusions": The experimental evidence recorded in this report seems to justify the following conclusions: The most useful agents for the destruction of spore-containing infectious material are — 1. Fire. Complete destruction by burning. 2. Steam underpressure. 105° C. (221° F.) for ten minutes. 3. Boiling in water for half an hour. 4. Chloride of lime.1 A four-per-cent solution. 5. Mercuric chloride. A solution of 1 : 500. For the destruction of infectious material which owes its infecting power to the presence of microorganisms not containing spores, the committee rec- ommends— 1. Fire. Complete destruction by burning. 2. Boiling in water for ten minutes. 3. Dry heat. 110' C. (230° F ) for two hours. 4. Chloride of lime. A two-per-cent solution. 5. Solution of chlorinated soda.* A ten-per-cent solution. 6. Mercuric chloride. A solution of 1 : 2,000. 7. Carbolic acid. A five per-cent solution. 8. Sulphate of copper. A five-per-cent solution. 9. Chloride of zinc. A ten-per-cent solution. 10. 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- rence 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. 3. Sulphate of copper in solution, five per cent. 1 Should contain at least twenty-five per cent of availab'e chlorine. a Should contain at least three per cent of available chlorine. 3 This will require the combustion of between three and four pounds of sulphur for every thousand cubic feet of air space. 202 PRACTICAL DIRECTIONS FOR DISINFECTION. (6) In privy vaults : 1. Mercuric chloride in solution, 1: 500. 1 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. (d) 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, wa«h 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.] 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. (&) 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 de- stroj'-ed 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- reasonable. But 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. 204 PRACTICAL DIRECTIONS FOR DISINFECTION. 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 supei-heating 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. PRACTICAL DIRECTIONS FOR DISINFECTION. 205 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 the 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- tion. Boll has recently (1890) reported favorable results from the fol- lowing method : 1. Cleanse the finger nails from visible dirt with knife or nail scissors. 2. Brush the hands for three minutes with hot water and potash soap. 3. 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 five-per-ceut 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. 2. 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. 3. The hands are immersed for one or two minutes in a warm saturated solution of permanganate of ootash and are rubbed over thoroughly with a sterilized swab. 306 PRACTICAL, DIRECTIONS FOR DISINFECTION. 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 : 1 '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 eick 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. " The following standard solution is recommended: " Dissolve chloride of lime of the best quality, l 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." 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 Gfood 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 i.« 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. PRACTICAL DIRECTIONS FOR DISINFECTION. 207 •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 in 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 denned. 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, because 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 six 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 208 PRACTICAL DIRECTIONS FOR DISINFECTION. 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 no objection to substituting it for sulphate of iron other than the question of cost. The first cost of chloride of lirne, 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, gave 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- half-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 fieces 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 excret* 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- PRACTICAL DIRECTIONS FOR DISINFECTION. 209 meiit, 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 per cent; 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 Jiiger 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- vius led him to place a high value upon recently burned quicklime as a dis- infectant. More recent experiments by Jager, Kitasato, Pfuhl, and others have shown that this agent has considerable germicidal 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 material, but is neutralized by phos- phates, carbonates, and other bases, and by carbonic acid. In the writer's experiments a saturated aqueous solution of calcium oxide 14 210 PRACTICAL DIRECTIONS FOR DISINFECTION. 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. Pf uhl 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. DISINFECTION IN DIPHTHERIA. At the meeting of the Tenth International Medial Congress Berlin (1890) Loffler made an important communication upon the PRACTICAL, DIRECTIONS FOR DISINFECTION. 211 measures to be taken to prevent the spread of diphtheria. His con- clusions 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 tbe 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 Loftier with reference to rubbing down 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 bacteria attached fo 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. PART THIRD. PATHOGENIC BACTERIA. I. MODES OF ACTION. II. CHANNELS OF INFECTION. III. SUSCEPTIBILITY AND IMMUNITY IV. PYOGENIC BACTERIA. V. BACTERIA IN CROUPOUS PNEU- MONIA. VI. PATHOGENIC MICROCOCCI NOT DESCRIBED IN SECTIONS IV. AND V. VII. THE BACILLUS OF ANTHRAX. VIII. THE BACILLUS OF TYPHOID FEVER. IX. BACTERIA IN DIPHTHERIA. X. BAC- TERIA IN INFLUENZA. XI. BACILLI IN CHRONIC INFECTIOUS DISEASES. XII. BACILLI WHICH PRODUCE SEPTICAE- MIA IN SUSCEPTIBLE ANIMALS. XIII. PATHOGENIC AEROBIC BACILLI NOT DESCRIBED IN PREVIOUS SECTIONS. XIV. PATHOGENIC ANAEROBIC BACILLI. XV. PATHOGENIC SPIRILLA. XVI. BACTERIA IN INFECTIOUS DISEASES NOT PROVED TO BE OF PARASITIC ORIGIN. XVII. CLASSIFICA- TION OF PATHO- GENIC BAC- TERIA. PART THIRD. PATHOGENIC BACTERIA. I. MODES OF ACTION. MANY of the saprophytic bacteria are pathogenic for man, or for one or more species of the lower animals, when by accident or ex- perimental inoculation they obtain access to the body ; these may be designated facultative parasites. Other species which, for a time at least, are able to lead a saprophytic mode of life have their nor- mal habitat in the bodies of infected animals, in which they produce specific infectious diseases. To this class belong the cholera spirillum, the anthrax bacillus, the bacillus of typhoid fever, and various other microorganisms which are the cause of specific infectious diseases in some of the lower animals. These we may speak of as parasites and facultative saprophytes. Still others are strict parasites and do not find the conditions for their development outside of the bodies of the animals which they infest, except under the special conditions in which bacteriologists have succeeded in cultivating some of them. The best known strict parasites are the tubercle bacillus, the bacillus of leprosy, the spirillum of relapsing fever, and the micrococcus of gonorrhosa. There can be but little doubt that even the strict parasites, at some time in the past, were also saprophytes, and that the adaptation to a parasitic mode of life was gradually effected under the laws of natural selection. In a previous chapter (Section III., Part Second) we have referred to the modifications in biological characters which may occur as a result of special conditions of environment. Thus we may obtain non-chromogenic varieties of species which usually produce pigment, or non-pathogenic varieties of bacteria which are usually pathogenic. There is also evidence that the tubercle bacillus, a strict 216 MODES OF ACTION. parasite, may be so modified, by cultivation for successive genera- tions in a culture medium containing glycerin, that it will finally grow in ordinary beef infusion, thus showing a tendency to adapt itself to a saprophytic mode of life. Some of the saprophytic bacteria are indirectly pathogenic bj reason of their power to multiply in articles of food, such as mill cheese, fish, sausage, etc., and there produce poisonous ptomaine which, when these articles are ingested, give rise to various morbk symptoms, such as vomiting, gastric and intestinal irritation, fever, etc. Or similar symptoms may result from the multiplication of bacteria producing toxic ptomaines in the alimentary canal. Nc doubt gastric and intestinal disorders are largely due- to this cause and may be induced by a variety of saprophytic bacteria when thes establish themselves in undue numbers in any portion of the ali- mentary tract. In Asiatic cholera the same thing occurs, but wit more fatal results from the introduction of the East Indian cholei germ discovered by Koch. This is pathogenic for man, because it is able to multiply rapidly in the human intestine, and there produces toxic substance which, being absorbed, gives rise to the morbid phenc mena of the disease. The spirillum itself does not enter the blood invade the tissues, except to a limited extent in the mucous coat of the intestine, and the true explanation of its pathogenic power is nc doubt that which has been given. Other microorganisms invade the tissues and multiply in cei tain favorable localities, but have not the power of developing in the blood, in which they are only found occasionally and in very small numbers or not at all. Thus the typhoid bacillus locates itself in the intestinal glands, in the spleen, and in the liver, forming colonies of limited extent, and evidently not finding the conditions extremely favorable for its growth, inasmuch as it does not take complete pos- session of these organs. The symptoms which result from its pre- sence are doubtless partly due to local irritation, disturbance of func- tion, and, in the case of the intestinal glands, necrotic changes induced by it. But in addition to this its pathogenic action depends upon the production of a poisonous ptomaine which has been isolated and studied by the German chemist Brieger (typhotoxine). Certain saprophytic bacteria, when injected beneath the skin of a susceptible animal, multiply at the point of inoculation and invade the surrounding tissues, giving rise in some instances to the forma- tion of a local abscess, in others to an infiltration of the tissues with bloody serum, and in others to extensive necrotic changes. These local changes are due not simply to the mechanical presence of the microorganisms which induce them, but to chemical products evolved during the growth of these pathogenic bacteria. Indeed, their patho- MODES OF ACTION. 217 genie power evidently depends, in some instances at least, upon these toxic products of their growth, by which the vital resisting power of the tissues is overcome. Among the bacteria which in this way produce extensive local inflammatory and necrotic changes are certain anaerobic species found in the soil and in putrefying material, such as the bacillus of malignant oedema and the writer's Bacillus cadaveris. The bacillus of symptomatic anthrax, an infectious disease of cattle, acts in the' same way. All of these produce toxic substances which have a very pronounced local action upon the tissues invaded by them. Other bacteria, while they develop chiefly in the vicinity of the point of entrance — by accident or by inoculation — produce a potent toxic sub- stance which gives rise to general symptoms of a serious character, such as tetanic convulsions (bacillus of tetanus) or intense fever and nervous phenomena (micrococcus of erysipelas). Again, the local irritation resulting from the presence of parasitic bacteria may pri- marily give rise to the formation of new growths having alow grade of vitality, which later may undergo necrotic changes, as in tubercu- losis, glanders, and leprosy. In this case constitutional symptoms are not present, or are of a mild character during the development of these new formations, which apparently result from the local ac- tion of substances eliminated during the growth of the parasite, rather than from its simple presence. This is an inference based upon the fact that non-living particles, or even living parasites, as in trichinosis, do not produce similar new growths composed of cells/ but become encysted in a fibrous capsule. In pneumonia we have a local process in which one or more lobes of the lung are invaded by a pathogenic micrococcus (Micrococcus pneumonise crouposse) which induces a fibrinous exudation that com- pletely fills the air cells. How far the symptoms of the disease are due to the local inflammation and disturbance of function, and to what extent they may be due to the absorption of a soluble toxic substance evolved as a result of the growth of the micrococcus, has not been determined. But the mild character of the general symp- toms when a limited area of lung tissue is involved leads to the in- ference that the pathogenic power of this particular pathogenic microorganism is chiefly exercised locally. The pus cocci and various other saprophytic bacteria, when intro- duced beneath the skin, give rise to the formation of abscesses, un- attended by any very considerable general disturbance ; and also to secondary purulent accumulations — metastatic abscesses. That this is not due simply to their mechanical presence is shown by the fact that powdered glass and other inert substances, when thoroughly sterilized, do not give rise to pus formation when intro- 218 MODES OF ACTION. duced beneath the skin or injected into the cavity of the abdomen. On the other hand, it has been demonstrated by the experiments of Grawitz, De Bary, and others that certain chemical substances which act as local irritants when brought in contact with the tissues may induce pus formation quite independently of microorganisms : nitrate of silver, oil of turpentine, and strong liquor ammonise have been shown to possess this power. And it has been demonstrated by the recent experiments of Buchner that sterilized cultures of a long list of different bacteria — seventeen species tested — give rise to sup- puration when introduced into the subcutaneous tissues. Buchner has further shown that this property of inducing pus for- mation resides in the dead bacterial cells and not in soluble products- present in the cultures. For the clear fluid obtained by passing these sterilized cultures through a porcelain filter gave a negative re- sult, while the bacteria retained by the filter, although no longer capable of development, having been killed by heat, invariably caused suppuration. Individuals suffering from malnutrition are more susceptible to invasion by specific disease germs or by the common pus cocci than are those in vigorous health. Thus the sufferers from starva- tion, from crowd poisoning, sewer-gas poisoning, etc., are not only liable to be early victims during the prevalence of an epidemic dis- ease, but are very subject to abscesses, boils, ulcers, etc. A slight abrasion in such an individual, inoculated by the ever-present pus cocci, may give rise to an obstinate ulcer or a phlegmonous inflam- mation. In the same way some of the ordinary saprophytes, which usually have no pathogenic power, may be pathogenic for an animal whose strength is reduced by disease or injury. Thus necrotic changes may occur in injured tissues, or in those which have a deficient blood supply — from occlusion of an artery, for example — due to the presence of putrefactive bacteria which are incapable of development in the circulation of a healthy animal or in healthy tissues. We may also have a, progressive gangrene, due to infection of wounds by bacteria which are able to invade healthy tissues. This is seen in the so- called hospital gangrene, which is undoubtedly due to microorgan- isms, 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, its 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 bromine in arresting the progress of the disease, all support this vir rabbits are usually without result, and the last-named animal also withstands injections of considerable quantities into the general cir- culation through a vein. When, however, the animal has previously been weakened by the injection of toxic substances the streptococcus may multiply in its body and cause its death (Flugge). Fehleisen has inoculated cultures, obtained in the first instance from the skin of patients with erysipelas, into patients in hospital suffering from lupus and carcinoma, and has obtained positive re- sults, a typical erysipelatous inflammation having developed 278 PYOGENIC BACTERIA. around the point of inoculation after a period of incubation of from fifteen to sixty hours. This was attended with chilly sensations and an elevation of temperature. Persons who had recently recovered from an attack of erysipelas proved to be immune. Sections made from the ear of an inoculated rabbit, or of skin taken from the affected area in erysipelas in man, show the streptococci in considerable numbers in the lymph channels, but not in the blood vessels. They are more numerous, according to Koch and to Fehl- eisen, upon the margins of the erysipelatous area, and may even be seen in the lymph channels a little beyond the red margin which marks the line of progress of the infection. The researches of Weichselbaum and others show that Strepto- coccus pyogenes is the infecting microorganism in a certain propor- tion of the cases of ulcerative endocarditis. The author named found it in four cases out of fifteen examined, and in two cases of endocarditis verrucosa out of thirteen. In a previously reported series of sixteen cases (fourteen of ulcerative endocarditis and two of ver- rucosa) the streptococcus was found in six. In diphtheritic false membranes this streptococcus is very com- monly present, and in certain cases attended with a diphtheritic exu- dation, in which the Bacillus diphtherias has not been found by com- petent bacteriologists, it seems probable that Streptococcus pyogenes is the pathogenic microorganism responsible for the local inflamma- tion and its results. Thus in a series of twenty-four cases studied by Prudden in 1889 the bacillus of Loffler was not found, "but a strep- tococcus apparently identical with Streptococcus pyogenes was found in twenty-two." Chantemesse and Widal have also reported cases in which a fibrinous exudate resembling that of diphtheria was as- sociated with a streptococcus. " These forms of so-called diphtheria are most commonly associated with scarlatina and measles, erysipe- las, and phlegmonous inflammation, or occur in individuals exposed to these diseases ; but whether exclusively under these conditions is not yet established" (Prudden). Loffler has described under the name of Streptococcus articu- lorurn a micrococcus obtained by him from the affected mucous membrane in cases of diphtheria, and which he believes to be acci- dentally present and without any etiological import in this disease. In its characters it closely resembles Streptococcus pyogenes and is perhaps a variety of this widely distributed species. Its characters are described by Fliigge as follows : " Cultivated in nutrient gelatin, it forms at the end of three days small, transparent, light-gray drops, upon the margin of which, under the micro- scope, the cocci in twisted chains may be observed. As many as one hun- PYOGENIC BACTERIA. 279 dred elements may be found in a single chain, and some of these are distin- guished by their size; occasionally whole chains are made up of these large cocci, and when closely observed some of these may present indications of division transversely to the axis of tbe chain. Subcutaneous inoculation of cultures into mice results in the death of a considerable number of these ani- mals— more than half ; and the streptococci are found in the spleen and other organs. Inoculation into the ear of rabbits causes an erysipelatous inflam- mation. When injected into the circulation of these animals through a vein joint affections are developed in from four to six days, and a purulent ac- cumulation occurs in which the streptococci are found. In two rabbits in- oculated in the same way with a culture of the streptococcus of erysipelas, Loffler has observed a similar result." Recent researches indicate that infection by Streptococcus pyo- genes through the endometrium is the usual cause of puerperal fever. Thus Clivio and Monti demonstrated its presence in five cases of puerperal peritonitis. Czerniewski found it in the lochia of a large number (thirty-five out of eighty-one) of women suffering from puerperal fever, but in the lochia of fifty-seven healthy puer- peral women he was only able to find it once. In ten fatal cases he found it in every instance, both in the lochial discharge during life and in the organs after death. Widal carefully studied a series of sixteen cases and arrived at the conclusion that this was the infect- ing microorganism in all. Bumm and other observers have given similar evidence. Eiselsberg and Emmerich have succeeded in de- monstrating the presence of the streptococcus in hospital wards con- taining cases of erysipelas. That puerperal fever may result from infection through the finger of the accoucheur, when he has previ- ously been in contact with cases of erysipelas, has long been taught, and, in view of the facts above recorded, is not difficult to under- stand. But in view of the fact that the streptococcus of pus has been found in vaginal mucus and in the buccal and nasal secretions of healthy persons, it may appear strange that cases of puerperal fever not traceable to infection from erysipelas or from preceding cases do not occur more frequently. This is probably largely due to an attenuation of the pathogenic power of the streptococcus when it leads a saprophytic existence. Widal asserts that, when cultivated in artificial media for a few weeks, the cultures no longer have their original virulence, and Bumm has made the same observation. On the other hand, in " streptococcus-peritonitis " occurring as a result of puerperal infection Bumm states that the thin, bright-yellow, odorless fluid contained in the cavity of the abdomen is extremely virulent ; a very slight trace, a fragment of a drop, injected into the abdominal cavity of a rabbit, is sufficient within twenty-four hours to cause a general septic inflammation with a bloody serous exuda- tion, quickly terminating in the death of the animal ; injected sub- cutaneously it gives rise to an enormous phlegmon which also 280 PYOGENIC BACTERIA. quickly proves fatal. But cultures of Streptococcus pyogenes, after it has been carried through successive generations in artificial media, injected beneath the skin of a rabbit, usually produce no result, or at most an abscess of moderate dimensions. It seems probable that the micrococcus isolated by Fliigge from necrotic foci in the spleen of a case of leucocythgemia, and described by him under the name of Streptococcus pyogenes malignus, was simply a very pathogenic variety of the streptococcus of pus. He was not able to differentiate it from Streptococcus pyogenes by its morphology or growth in culture media, but it proved far more pathogenic when tested upon animals. Mice inoculated subcutane- ously with a minute quantity of a pure culture died, without excep- tion, in three to five days. A large abscess was formed at the point of inoculation, and the blood of the animal contained numerous cocci in pairs and chains. Rabbits inoculated in the ear showed at first the same local appearances as result from inoculations with strepto- coccus of pus and of erysipelas, but after two or three days symp - toms of general infection were developed, and death occurred at the end of three or four days. At the autopsy the cocci were found in the blood, and frequently there were purulent collections in the joints containing the same microorganism. Krause has also de- scribed a streptococcus which only differs from Streptococcus pyo- genes of Rosenbach and Passet by the greater virulence manifested by its cultures. The fact that pathogenic bacteria may attain an intensified de- gree of virulence by cultivation in the bodies of susceptible animals was demonstrated by Davaine many years ago, and is fully estab- lished by the experiments of Pasteur and others. It is true of the anthrax bacillus, of the writer's Micrococcus Pasteuri, and of other well-known pathogenic microorganisms. The reverse of this — at- tenuation of virulence as a result of cultivation in artificial media- is also well established for several pathogenic species. Now it appears that the attenuated streptococcus is far less likely to give rise to erysipelas or to puerperal infection than is the same micro- organism as obtained from a case of one or the other of these infec- tious diseases. The same is probably true also of Staphylococcus aureus and other facultative parasites which are found as sapro- phytes upon the surface of the body and upon exposed mucous mem- branes in healthy persons. And it is not improbable that attenuated varieties of these micrococci which find their way into open wounds, or into the uterine cavity shortly after parturition, if they escape destruction by the sanguineous discharge, acquire increased patho- genic power from their multiplication in it, as a result of which they are able to invade the living tissues. But it appears probable that PYOGENIC BACTERIA. 281 infection through open wounds does not depend alone upon the potency of the pathogenic micrococci present in them, but also upon the absorption of chemical poisons produced by septic (putrefactive) bacteria, which weaken the vital resisting power of the tissues. Gottstein, as a result of experiments made by him, is of the opinion that the resorption of broken-down red blood corpuscles favors infec- tion by pathogenic bacteria present in wounds ; and he has shown that the injection into animals of certain toxic substances which de- stroy the red corpuscles in the circulation makes them susceptible to the pathogenic action of certain bacteria which are harmless for them under ordinary circumstances. Thus a guinea-pig, an animal which is immune against the bacillus of fowl cholera, succumbed to an inoculation made after first injecting subcutaneously 0.06 gramme of hydracetin dissolved in alcohol. At the autopsy hgemorrhagic exu- dations were found in the serous cavities, haemorrhagic infarctions in the lungs, and quantities of the bacillus injected were found in the blood and in fluid from the cavity of the abdomen. In man the ever-present pus cocci are more likely to invade the tissues, forming furuncles, carbuncles, and pustular skin eruptions, or erysipelatous and phlegmonous inflammations, when the standard of health is reduced from any cause, and especially when by absorp- tion or retention various toxic organic products are present in the body in excess. It is thus that we would explain the liability to these local infections, as complications or sequelae of various specific infec- tious diseases, in the victims of chronic alcoholism, in those exposed to septic emanations from sewers, etc. , and probably in many cases from the absorption of toxic products formed in the alimentary canal as a result of the ingestion of improper food, or of abnormal fermen- tative changes in the contents of the intestine, or from constipation. The Pus Cocci in Inflammations of Mucous Membranes. — To what extent the pus cocci are responsible for inducing and main- taining non-specific inflammations of mucous membranes has not been determined ; but having demonstrated the pyogenic properties of these cocci, their presence in the purulent discharges from inflamed mucous membranes can scarcely be considered as unimportant, not- withstanding the fact that they are also frequently found in secre- tions from healthy mucous surfaces. They are likewise found upon the skin of healthy persons, and yet we have unimpeachable experi- mental evidence that they may produce a local inflammation, at- tended with pus formation, when injected subcutaneously, or even when freely applied to the uninjured surface. In otitis media Levy and Schrader obtained Staphylococcus albus in pure cultures in three cases out of ten in which paracentesis was performed, and in two others it was present in association with 20 282 PYOGENIC BACTERIA. other microorganisms. In eighteen cases of otitis media in young children Netter found Staphylococcus aureus six times and Strepto- coccus pyogenes thirteen times. Scheibe, in eleven cases in which perforation had not yet taken place, found Staphylococcus albus in two and various other microorganisms in the remaining cases ; Sta- phylococcus aureus was not present in any. Habermann obtained aureus associated with other bacteria in a single case of purulent otitis media. In a series of eight cases occurring as a sequela of influenza Scheibe obtained Streptococcus pyogenes in two, " diplo- coccus pneumonias " in two, Staphylococcus aureus in one, Strepto- coccus pyogenes and Staphylococcus albus together in two, and Strep- tococcus pyogenes in association with an undescribed micrococcus in one. In all of these cases a slender bacillus was also present, as shown by microscopical examination, which did not grow in any of the culture media employed. Bordoni-Uffreduzzi and Gradenigo have tabulated the results obtained by various bacteriologists who have examined pus obtained through the previously intact tympanic membrane. In thirty -two cases of this character the microorganism most frequently found was diplococcus pneumonise (Micrococcus pneumonias crouposae of the present writer), which was present in a pure culture in thirteen and associated with Staphylococcus aureus in one, with Staphylococcus albus in one, and with Streptococcus pyogenes in one. In the other sixteen cases the pyogenic cocci were present in ah1 but two, in which bacilli were found — Bacillus tenuis in one, a non-liquefying bacillus in one. In twenty-seven cases in which the pus was withdrawn from one to thirty days after paracen- tesis or spontaneous rupture of the membrane, the pyogenic cocci were present in twenty and diplococcus pneumonias in seven. In acute nasal catarrh Paulsen found Staphylococcus aureus in seven cases out of twenty-four examined, and E. Frankel in two out of four ; but it must be remembered that Von Besser has shown that this micrococcus is frequently present in the secretions from the healthy nasal mucous membrane, and we have experimental evidence that the pus organisms, when introduced into the conjunctival sac of rabbits (Widmark), do not give rise to catarrhal inflammation. On the other hand, Widmark found that when inoculated into the comea of rabbits an intense conjunctivitis resulted, together with keratitis and perforation of the cornea in fifteen per cent of the cases. The same author in his bacteriological researches obtained the pyogenic staphylococci from the circumscribed abscesses of blepharadenitis, while in inflammation of the lacrymal sac Streptococcus pyogenes was usually present. Shougolowicz,in the bacteriological examination of twenty-six cases of trachoma, found Staphylococcus albus in twelve, Staphylococcus PYOGENIC BACTERIA. 283 aureus in nine, Staphylococcus citreus in three, and Staphylococcus cereus albus in three. These pus organisms were in a number of the cases associated with other well-known saprophytes, and in seven cases a short bacillus not previously described was found. That various bacilli are found in the conjunct! val sac of healthy eyes and in different forms of conjunctivitis has been shown by Fick, whose results do not correspond in this respect with those of Gif- ford, who found almost exclusively micrococci. Whatever may be the final conclusion as to the role of the pus cocci heretofore de- scribed in the etiology of acute or chronic conjunctivitis, there can be no doubt of the power of the " gonococcus " to induce a virulent in- flammation of the conjunctivas when introduced into healthy eyes. 6. MICROCOCCTJS GONORRHCEJ3 Synonym. — Gonococcus (Neisser). Discovered by Neisser (1879) in gonorrhceal pus and described by him under the name of " Gonococcus." Cultivated by Bumm (1885), and infective virulence proved by inocula- tion into man. Constantly present in viru- lent gonorrhoeal discharges, for the most part in the interior of the pus cells or at- tached to the surface of epithelial cells. Morphology. — Micrococci, usually join- ed in pairs or in groups of four, in which the elements are flattened — " biscuit- shaped. " The flattened surfaces face each other and are separated, in stained pre- parations, by an unstained interspace. SnT ,. , ,, . , , • /ii - - -. Ihe diameter or an associated pair ot cells pure culture, x about 1,000; 6,gono- varies from 0.8 to 1.6 /^ in the long dia- cocci in pus ceils and epithelial ceil .. _ from case of gonorrhoeal ophthal- meter — average about 1.25 ^— and trom mia. c formand mode Of division 0.6 to 0.8 jn in the line of the interspace of gonococci-schematic. (Bumm.) 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 FlG- 85- -«. gonococci from 284 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 from gonorrhoeal pus, spread upon a cover glass and " fixed," secunduin art em, 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'ft method of staining. But this character cannot be depended upon alone for establishing the diagnosis, for Bumin has shown that FIG. 86. — " Gonococcus " in gonorrhceal 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 hi 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. 285 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. — The "gonococcus" does not grow in flesh infusions, in nutrient gelatin or agar, but it may be cultivated upon blood serum, and, according to Bumm, grows more readily upon human blood serum than upon that of the lower animals. This he obtained for his experiments from the placenta of a recently de- livered woman by passing two ligatures around the cord before separating the child from its placental attachment, and dividing it between them. But even upon blood serum obtained in this way it is not a simple matter to obtain a pure culture. When other micro- cocci are present, even in small numbers, they take the precedence and overgrow the surface of the culture medium before the gono- coccus has made any visible growth. It is therefore necessary to start a culture with pus containing this micrococcus only and in considerable numbers. And the pus should not be spread out in a thin layer, but should be distributed upon the surface in little drops or masses, in which the development commences. A temperature of 30° to 34° C. is most favorable for the development of this micro- coccus, and Bumm recommends the transfer to fresh culture material in from eighteen to twenty-four hours. The cultures thrive best in a moist atmosphere, and it is well to place the tubes containing them in a large glass jar partly filled with distilled water and having a tightly fitting cover. The growth under the most favorable condi- tions is slow, and frequently no development occurs when pus con- taining numerous gonococci is placed upon blood serum in an incu- bating oven ; or after a slight multiplication 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 286 PYOGENIC BACTERIA. 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. 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 gonorrhoeal 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 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. FIG. 87.— Gonorrhceal conjunctivitis, second day of sickness; section through the mucous mem- brane of upper eyelid; invasion of the epithelial layer by gonococci. (Bumm.) In both cases a typical gonorrhoea developed as a result of the inocu- lation. 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. The mucous membranes in man which are subject to gonorrhoeal infection are those of the urethra, the conjunctiva, the cervix uteri, PYOGENIC BACTERIA. 287 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 blennorrhcea 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 CROU^OUS 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 : Acute pneumonia is now generally regarded by the leading clinical au- thorities and pathologists in this country and in Europe as a specific infec- tious disease. Green, in his article on " Inflammation of the Lungs" in Quain's "Dictionary of Medicine," says: "It is maintained by some ob- 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 D'Ansberg in 1880 (one hun- dred and sixty -one cases, with forty-six deaths, in a period of five months). Again, 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. 289 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 of ten 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 21 290 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 vis 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, Friedliinder 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 G-unther 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 'I should have said frequent instead of " constant presence." BACTERIA IN CROUPOUS PNEUMONIA. 291 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 fibrinous 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 daring 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 chai-acteristic 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: " 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 : ' ' Af anassiew 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) "whose researches we have just reviewed." This quotation indicates that Prof, See did not question the 292 BACTERIA IN CROUPOUS PNEUMONIA. identity of the oval or " lanceolate " coccus found by Talamon in pneumonic exudate, and which iu 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 dh'ectly 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 fibrinous 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 Friedlander, 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 demonstrated 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: "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, though 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 typical 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. . . . "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 these 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 : "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. 293 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 pathogenic varieties in the buccal secretions of different individuals. At this time I had not seen a culture of Friedlander's bacillus. Later, in the autumn of 1885, when I made its acquaintance in Dr. Koch's laboratory, I 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, Weichsel baum, Bordoni-Uffreduzzi, Netter, Grameleia, 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 bacilJus." The extended researches of Weichselbaum, published in 1886, give strong support to the view that this coccus is the usual infectious agent 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 Weichselbaifm's laboratory, reported the result of his researches in 1887. He found the "diplococcus pneumonias" 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 ray paper published in the American Journal of the Medical Sciences for July, 1886. 284 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 every 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 Pasteuri. 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 resume, 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 we have adopted, we believe ourselves au- thorized to conclude that fibrinous pneumonia is always dependent upon the microbe of Pasteur." Friinkel, 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 etiological 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, ought to be put to the account of the other." This opinion the present writer has entertained since his researches made in 1885. The experimental evidence offered by Gameleia 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: " The number of my rabbits iu which pneumonia was induced is about two hundred." BACTERIA IN CROUPOUS PNEUMONIA. 295 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 Fraukel 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 opdema), 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 death 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 intheexudateinto 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." 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. 296 BACTERIA IN CROUPOUS PNEUMONIA. 7. BACILLUS OF FRIEDLANDER. Synonyms. — Pneumococcus (Friedlander); Bacillus pneumonias (Fliigge). 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 ($JM recent cultures ; commonly united in pairs °^ ^° ^©(^/^ or chains of four, and under certain cir- ^0°o Q° ^(o) ffi cumstances surrounded by a transparent Ji j{ capsule. The gelatinous envelope — so- FIO. 88. -Baciiiusof Friedlander; called capsule — is not seen in preparations o, from a culture; 6, from blood of made f rom cultures in artificial media, but mouse, showing capsule. (Fliigge.) . . . 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 mounted in balsam. The bacillus is quickly stained in dried cover- glass preparations by immersion in aniline- water-gentiaii-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. 297 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 Flo- 89 — Friedlander'a 0 * 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 at 16°-18° c- (Baumgar- 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 rinto 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 298 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 pneumoiiise (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. 7-4, 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. 299 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- 300 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 of 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 pneumonias." 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. 301 Xetter 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 pneumonise." Micrococcus Pneumonice Crouposce in Ulcerative Endocar- ditis.— Weichselbaum, in a series of twenty-nine cases examined (1888), found " diplococcus pneumonias" 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. 92. FIG. 90.— Micrococcus pneumonise crouposae from blood of rabbit inoculated with normal human saliva (Dr. S.). X 1,000. FIG. 91. — Micrococcus pneumonise 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. &2.— Surface culture of Micrococcus pneumonise crouposse, on nutrient agar, showing the development of long cha:ns. 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. 302 BACTERIA IN CROUPOUS PNEUMONIA. (1889), in a case of purulent inflammation of the shoulder joint follow- ing pneumonia and pleurisy, obtained the " diplococcus pneumonise " 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 sometimes formed, especially in cultures upon the surface of nutrient agar, and 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 capsule. Owing to the elon- gated 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 indivi- dual cells more nearly approach a spherical form than in the blood of an inoculated animal. The " lanceolate " 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 pre- parations from the fibrinous exudate of croupous pneumonia or from the blood of an inoculated animal. It appears as an unstained mar- ginal band surrounding the elliptical cells, and varies greatly as to its extent in different preparations. This capsule probably consists of a substance resembling mucin, and, being soluble in water, its ex- tent depends partly upon the methods employed in preparing speci- mens for microscopical examination. It is occasionally seen in stained preparations from the surface of cul- tures on blood serum ; and in drop cultures examined under the microscope, by using a small diaphragm, it may be seen to surround the cocci as a scarcely visible halo. 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 bacillus, with which it has often been confounded on account of the morphological resemblance of the two microorganisms. 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 FIG. 93. — Micrococcus pneu- monise crouposae, showing cap- sule, attached to pus cells from exudate in pleural cavity of inoculated rabbit. (Salvioli.) BACTERIA IN CROUPOUS PNEUMONIA. 303 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 have a compact, finely granular central 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 FIG. 94 — Single colony of Micro- coccus pneumonise crouposse upon agar plate, twenty-four hours old. X 100. (Frankel and Pfeiffer.) 304 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 has recently (1891) reported 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 ren- dered 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-five-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. BACTEKIA IN CROUPOUS PNEUMONIA. 305 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 recent 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 injections in rabbits, for a period of nineteen days in one series of experiments and for fifty-five days in another. Exposed to direct sunlight the same material retained its virulence after twelve hours' exposure. Cultures have far less resistance, and the protec- tion afforded by the dried albuminous material in which the micro- cocci were embedded, in the experiments referred to, probably ac- counts for the virulence being retained so long a time. Recently (1892) Kruse and Pansini have published an elaborate paper giving an account of their researches relating to " diplo- coccus pneumonisB " and allied streptococci. We give below a sum- mary 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 22 306 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 milk. 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. "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 usutlly 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 cases which are of the longest duration. ' ' The blood commonly contain s an immense number of micrococci, usual! y joined in pairs and having a diameter of about 0. 5 /*. These are found in blood drawn from 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. 307 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 blood 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. 95. — Micrococcus pneumonia? crouposse 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 308 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 intrapulmonary 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 pneumoniae crou- posee. 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. Recently (1891) G. and F. Klemperer have published an impor- tant memoir relating to the pathogenic action of this micrococcus. They succeeded in conferring immunity upon susceptible animals by inoculating them with filtered cultures of the micrococcus, and in some instances this immunity had a duration of six months. A curious 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- munity 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. The un warmed 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. 309 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. 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 be transplanted to fresh material at short intervals — two days. Pathogenesis. — Mice are especially susceptible, and usually die within forty-eight hours after inoculation. Also pathogenic for guinea-pigs, rabbits, and dogs. PATHOGENIC MICROCOCGI NOT HERETOFORE DESCRIBED. 311 10. STAPHYLOCOCCUS SAL.IVARIUS 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. ft 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 A 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 " in diameter, usually associated in thick, cloud-like zooglcea masses. Biological Characters not given. Pathogenesis. — In rabbits an extensive abscess forms in the 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 tf_ — _ contents of the abscess injected into other FIG. 96.— Micrococcus of progressive rabbits produce a similar result. The micro- tissue necrosis in mice; section of the coccus does not invade the blood. o£>;ch5cart Be °el's; "' 8treptococci- 312 PATHOGENIC MICROCOCCI Fio. 97. — Micrococcus of pyaemia in rabbits, in capil- lary from the cortical portion of the kidney. X 700. (Koch.) 13. MICROCOCCUS OF PYJEMIA IN RABBITS. Obtained by Koch (1879) in rabbits inoculated subcutaneously with putrefying flesh infusion. Morphology. — Round cells, 0.25 « 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 autopsy 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 /z. 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 (Fliigge). 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. 313 of vesical catarrh, and in the vesicles of pemphigus ; also by Frankel in the vaginal secretion of children suffering from colpitis not of gonorrhoeal 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. BiQlogical Characters. — Grows at the room temperature upon the sui'face of nutrient gelatin; small, grayish-white colonies appear along the line of inoculation at the end of twenty-four hours, which later form a continent 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. — Inoculationsuoon mucous membranes susceptible to gon- orrhoeal infection are without result. But by injecting the diplococcus from pure cultures, in suspension in distilled water, beneath the skin in man, Bumrn 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 trachomatous 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 establi-h its etiological relation to the form of oph- thalmia with which it is associated (?). Morphology. — Very small, biscuit shaped micrococci, in pairs— diplococci — separated by a very narrow dividing line. (This description would apply to some of the more common pus cocci, e.g., Staphylococcus 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 incu- 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 conjunctivae 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 gonorrhoeal 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, 23 314 PATHOGENIC MICROCOCCI 18. MICROCOCCUS TETRAGENUS. First described by Gaffky (Fliigge). Obtained by Koch and Gaffky (1831) 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 yu, 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, FIG. 98. — Micrococcus tetragenus; section of lung of mouse, x 800. (Fliigge.) 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- shaped, finely granular, and with a mulberry-like surface. When they come to th6 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. 315 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 /f 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 316 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. PatJiogenesis. — 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 (188G) from the sputum of two cases of croupous pneumonia following measles. Morphology. — Oval micrococci, having a diameter of 0.6 to 1.0 p 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° 0., but ceases at a temperature of 48° to 50° C. Pathogenesis. — Pathogenic for dogs, rabbits, guinea-pigs, mice, and birds. In mammals the principal pathological appearance re- sulting from infection consists in the formation of " granulation tu- NOT DESCRIBED IN SECTIONS IV. AND V. 317 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 jit, 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 ' * Lungenseuche " (infectious nleuro-pneumonia of cattle). Morphology. — Micrococci, varying considerably in size — average dia- meter 0.9 M; 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. — oes 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-vellowish 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 lance,t, 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 microcpccus above described to the disease with which it was associated, in the researches of the authors mentioned, has not been established by subsequent investigations. 318 PATHOGENIC MICROCOCCI 23. STREPTOCOCCUS SEPTICUS (Flilgge). Found by Nicolaier and by G-uarneri in unclean soil during researches made in Flugge'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 in fected silkworms suffering from la flachene (maladie des morts-plats). Etiological relation established by Pasteur. Morphology. — Oval cells, not exceeding 1.5 /* 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. NO8EMA BOMBYCIS. Synonyms. — Micrococcus ovatus ; Panhistophyton ovatum. Found in the blood and all of the organs of silkworms infected with pebrine (Fleckenkrankheit). First observed by Cornalia. Etiological relation established by Pasteur. Morphology. — Shining, oval cells, three to four u 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 ju in length, surrounded by a capsule ; sometimes associated to form tetrads. Stains with the usual a,niline 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 forty -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. 319 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 bullse in a case of pemphigus. Morphology. — Micrococci of from 0.8 to 1.4 ju. 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 chib-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, about 0.9 n 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 surface of agar the growth resembles that of Streptococcus pyogenes, but is somewhat more abundant. Upon 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. Pathogenesis. — 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 ejpithelium, blood casts, albumin, and streptococci. 320 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, resembling1 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 37J C. , at the end of three or four days the superficial colonies consist of a small, brown, central mass sur- rounded by a granular, se;ni 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 layer 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 by 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 az-e being milked for the manufacture of cheese, at Roquefort 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 slowly developed ; the outline is irregular and the edges thicker than the central portion ; the central portion of this layer gradually acquires a yellow color, while the periphery remains of a dirty-white or grayish color. Blood serum is liquefied by this micrococcus. Pathogenesis. — A few drops of a pure culture injected subcutaneously or into 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 cubic centimetre iniected into the mammary gland of a goat produced no re- sult ; the horse, the ftalf , the pig, the cat, chickens, and guinea-pigs also proved to be immune. Subcutaneous injections in rabbits produce an extensive ab- scess at the point of inoculation. NOT DESCRIBED IN SECTIONS IV. AND V. 321 31. STREPTOCOCCUS OF MASTITIS IN COWS. Obtained by Nocard and Mollereau (1887) from the milk of cows suffering from a form of chronic mastitis (mammite contagieuse). Morphology. — Spherical or oval cocci, a little less than one // in diameter, usually united in long chains. Stains with the usual aniline colors and also by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying streptococcus. Grows in the usual culture media at the room temperature. Develops rapidly in milk or in bouillon at a temperature of 16° to 30° C. The milk of a cow suffering from the form of mastitis produced by this micrococcus, when drawn with proper precautions in sterilized test tubes, at the end of twenty-four hours is acid in reaction; the lower two- thirds of the tube is filled with an opaque, dirty- white, homogeneous deposit, and above this is an opalescent, serous fluid of a bluish or dirty-yellow or slightly reddish color, according to the age of the lesion. A drop of this milk examined under the microscope shows the presence of the streptococcus in great numbers. The addition of two to five per cent of glucose or of gly- FIQ. 99.— Streptococcus of mastitis in cows (Nocard). cerin to bouillon makes it a more favorable culture medium ; the reaction should be neutral or slightly alkaline, as this streptococcus does not grow readily in an acid medium, although it produces an acid reaction in media containing sugar, the acid formed being lactic. In gelatin stick cultures the growth upon the surface is scanty, in the form of a thin pellicle around the point of puncture ; along the line of inoculation minute, opaque, granular colonies are developed, which, being closely crowded, form a thick line with jagged mai'gins. In agar stick cultures the growth is similar but more abundant. Upon the surface of nutrient gelatin, agar, or blood serum a large number of mi- nute, spherical, semi-transparent colonies are developed along the impfstrich ; these have a bluish tint by reflected light ; they may become confluent, form- ing a thin layer with well-defined margins. Upon gelatin plates, at 16° to 18° C., colonies are first visible at the end of two or three days ; they are spherical and slightly granular, at first transparent and later of a pale-yellow color by transmitted light, which gradually becomes brown. At the end of five or six weeks the colonies are still quite small, well defined, and opaque. Pathogenesis. — Pure cultures injected into the mammary gland of cows and goats gave rise to a mastitis resembling in its development that from 24 322 PATHOGENIC MICKOCOCCI which the streptococcus was obtained in the first instance. Injections into the cavity of the abdomen or into a vein, of one cubic centimetre of a pure culture, gave a negative result in dogs, cats, rabbits, and guinea-pigs. 32. DIPLOCOCCUS OF PNEUMONIA IN HORSES. Obtained by Schiitz (1887) from the lungs of horses affected with pneu- monia. Morphology. — Oval cocci, usually in pairs, surrounded by a homogene- ous, transparent capsule. Does not stain by Gram's method. Biological Characters. — An aerobic, non-liquefying micrococcus. Grows at the room temperature. Upon gelatin plates forms small, spherical, white colonies. In gelatin stick cultures grows along the line of puncture as small, white, separate colonies, which grow larger without becoming confluent. Upon the surface of agar small transparent drops are developed along the impf- strich. Pathogenesis. — The injection of a pure culture into the lung of a horse produces pneumonia and causes its death in eight or nine days. Pathogenic for rabbits, guinea-pigs, and mice. 33. STREPTOCOCCUS CORYZ^ CONTAGIOSvE EQUORUM. Obtained by Shiitz (1888) from pus from the lymphatic glands involved in horses suffering from the disease known in Germany as Druse des Pferdes. Morphology. — Oval cocci, in pairs, in chains containing three or four elements, or in long chaplets. Stains with the usual aniline colors — very intensely with Weigert's or Ehrlich's solution. Biological Characters. — An aerobic and facultative anaerobic micrococ- cus. Grows slowly at the room temperature, more rapidly at 37° C. Upon gelatin plates at the end of three to five days minute colonies become visible ; these never exceed the size of a pin's head. In gelatin stick cultures growth upon the surface is scanty or absent; along the line of puncture minute colonies are developed in rows. Upon agar plates, at 37° C., at the end of twenty-four hours lentil-shaped colonies are developed the size of a pin's head; under a low power the superficial colonies are seen to have a well-de- fined, opaque nucleus surrounded by a grayish, transparent marginal zone, which represents a half -fluid, slimy growth which does not extend after the third day and later disappears entirely ; the deep colonies are at first well- defined, and later surrounded by wing-like outgrowths. Upon blood serum, at 37° C., yellowish, transparent drops are first developed; these become con- fluent and form a viscid and tolerably thick layer; this later becomes dry and iridescent. Pathogenesis. — Pathogenic for horses and for mice, producing in these animals an abscess at the point of inoculation, and metastatic abscesses in the neighboring lymphatic glands. Not pathogenic for rabbits, guinea-pigs, or pigeons. 34. H^MATOCOCCUS BOVIS (Babes). Obtained by Babes (1889) from the blood and various organs of cattle which had died of an epidemic malady (in Roumaiiia) characterized by ha;mo- 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 or united in groups ; the free cocci are surrounded by a pale- yellowish, shining aureole of 0.5 to 1 n in diameter. NOT DESCRIBED IN SECTIONS IV. AND V. 323 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. \Jponpotato, 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 hyperaemic, 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 PNEUMONIA. 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 from Micrococcus pneumoniae crouposae. 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 crouposae does not grow at the room temperature) . In gelatin stick cultiires very small colonies are developed along the line of puncture at the end Of twenty-four to twenty-eight hours. Upon the surface of agar a rather scanty, moist layer is developed along the impfstrich. Upon potato a thin, scarcely visible layer is developed. In bouillon tine development is abun- dant; the culture medium acquires a very acid reaction and gives oil a strong odor like that of perspiration. . Pathogenesis.— Pathogenic for mice, guinea-pigs, and rabbits, in wnicn animals it produces fatal septicaemia; the spleen is not enlarged, as is the case in animals inoculated with Micrococcus pneumoniae crouposae. 37. STREPTOCOCCUS SEPTICUS LIQUEFACIENS. Obtained by Babes (1889) from the blood and various organs of a child which died of septicaemia following scarlatina. 324 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° (1, small, white, tnin, 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 pneumonias crouposae 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° p., 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 hyperasmic ; 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. MICROCOCCUS NO. II. OF FISCHEL,. Obtained by Fischel (1891) from the blood of two cases of influenza. Morphology. — Micrococci of from 1 to 1.25 u 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- faction of the gelatin commences at the end of four days ; this progresses slowly. Upon agar plates, at 37° C., superficial colonies are developed re- sembling a drop of milk. Upon potato, at 37° C., at the end of eight days a 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. 325 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 Bonorne (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 not 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- nias crouposae. 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 bullae of pemphigus neonatorum, in nine children suffering from this disease during an epidemic which oc- curred at Goteborg. Morphology. — Micrococci from 0.5 to "L 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 bullae 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 bullse the same coccus was obtained in pure cultures. 42. STAPHYLOCOCCUS PYOSEPTICUS. Obtained by Hericourt and Blchet (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. 326 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 rapae), 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. 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 open 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 1804, 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 328 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 FIG. 100.— 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 /f 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 roda THE BACILLUS OF ANTHRAX. 329 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 25 FIG. 101.— Bacillus anthracis, from a culture, showing formation of spores. X 1,000. (Klein.) 330 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 FIG. 102.— Culture of Bacillus an- thracis in nutrient gelatin : a, end of four days ; b, end of eight days. (Baumgarten.) THE BACILLUS OF ANTHRAX. 331 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 J Fio. 103.— Colonies of Bacillus anthracite 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 332 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 albu- minous substance (Hankin) is formed during the growth of the an- thrax bacillus, and that cultures containing this toxalbumin, from which the bacilli have been removed by filtration through porcelain, produce immunity when injected into susceptible animals, similar to that resulting from inoculations with an attenuated virus. It is probable that the pathogenic power of the anthrax bacillus depends largely upon the presence of this toxalbumin, 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 quan- tity of acid than those which have been attenuated by any of the agencies above mentioned (Behring). THE BACILLUS OF ANTHRAX. 333 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 oedema, 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. Recently (1892) Petermann has made a series of experiments with filtered cultures of the anthrax bacillus which lead him to the con- clusion that "large quantities of a culture in serum from the ox, fil- tered through porcelain, injected into the veins of a susceptible animal, have a preventive 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 334 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 FIG. 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 septica3mia. 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. 335 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. Fio. 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 tune in the superficial layers of the soil. 336 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. 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 Friinkel 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 power ; 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 26 338 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 Gaff ky 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 twenty-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 OP TYPHOID FEVER. 339 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. 10G (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. Frankel and Simmonds have demonstrated that the bacilli multi- 340 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 Gaffky 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 FIG. 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. Frankel 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 dise se— 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 OP TYPHOID FEVER. 341 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 fsecal 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 Motschukoffsky 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 342 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- marks, 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), white 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 introduction 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 my opinion it is not at all necessary, when we experiment upon ani- mals, to obtain precisely the same symptoms as in man. In support of this opinion I may refer to the infectious diseases which we are able to induce experimentally in the lower animals. Anthrax runs a very different course in animals and 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, we cannot, in general, produce in animals ; and, nevertheless, we cannot assert that the animals experimented upon do not suffer from tuber- culosis, and that the conclusions which we draw from such experiments are not perfectly correct." In Frankel and Simmonds' experiments a considerable quantity of material was used, and the injections were, for the most part, made into the peritoneal cavity in mice, or into the circulation THE BACILLUS OF TYPHOID FEVER. 343 through a vein in rabbits. The influence of quantity of material used is especially shown in the case of the mice, and the question arises whether the pathogenic power of the bacillus for these ani- mals does not depend upon the simultaneous injection of the ptomaine developed in cultures as a result of the vital activity of the organ- ism. Thus we read that mouse No. 4 resisted an injection of a di- lute solution of culture No. 1, but succumbed to a more concentrated solution — one-fifth of a Pravaz syringe. Mouse No. 5 was not killed by the injection of one-third of a syringeful of a dilute solution, but subsequently died from the injection of one-third of a syringeful of a concentrated solution. Mouse No. 16, injected October 10th with half of a syringeful of a very diluted culture, did not die. The in- jection was repeated on the 17th of October with half a syringeful of a concentrated solution, with fatal result. In all, thirty-five mice were injected, with a fatal result in twen- ty-seven cases. In rabbits the injections were commonly made in the large vein of the ear, and the quantity of material injected was considerably greater — from one-third the contents of a hypodermatic syringe to two syringefuls. In some instances death occurred with- in a few hours, in others on the following day or after an interval of two or three days. It is noticeable that the results differ very great- ly as to the date of death and the relative quantity of material re- quired to produce a fatal result. This probably depends to some extent upon the size of the animal, and perhaps partly upon indi- vidual differences in resisting power. The experiments, considered together, show that the typhoid ba- cillus is not pathogenic for these animals in the same sense as is the anthrax bacillus or the bacillus of rabbit septicaemia. These organ- isms introduced beneath the skin or into the circulation in the small- est amount infallibly produce death, and at the expiration of a pe- riod of time which is tolerably uniform. In all, seventy-nine experiments upon rabbits were made, with the following result : Five injections into the intestine, five into the sub- cutaneous connective tissue, one into the lung, and two inhalation experiments, all without result; twenty injections into the peri- toneal cavity furnished two, and forty-six injections into the vein of the ear twenty positive results — i. e. , were fatal to the animal. In the fatal cases the bacilli were proved to be present in the spleen by culture experiments and by microscopical examination of properly stained sections. The colonies were identical in appearance with those found in the spleen of cases of typhoid in man. Col- onies were found in the spleens of the rabbits experimented upon exactly as in the human subject — sometimes in the trabeculae, some- times in the Malpighian bodies, sometimes free in the splenic pulp. 34:4 THE BACILLUS OF TYPHOID FEVER. Brieger has made some very interesting researches with reference to the chemical substances which are produced as a result of the physiological processes attending the growth of this bacillus. Having obtained a culture from the spleen of a typhoid patient, and assured himself by comparison with a pure culture given him by Koch that he was dealing with the right organism, Brieger planted the bacillus in a culture solution containing grape sugar and salts — Nahrsalzen — in which it thrived admirably. Such a solution at 30° C. became clouded at the end of twenty-four hours, and gave off an evident odor of ethyl alcohol, which increased from day to day. In addition to ethyl alcohol small quantities of the volatile fat acids were produced — among them acetic acid. Lactic acid was also formed from the grape sugar. The bacillus grew still better in al- buminous culture fluids. It did not in these give rise to the produc- tion of sulphuretted hydrogen or of any of the volatile products of putrefactive decomposition, such as indol and phenol. There was no gas formation in such cultures, even after standing for eight weeks, but a slight odor, resembling that of whey, was given off from the cultures. Repeatedly, but not in every case, Brieger suc- ceeded in obtaining from such cultures a very deliquescent basic product. This was obtained in only very small quantities, even when the cultures had remained in the incubating oven for a month. The physiological properties of this base have convinced Brieger that it is a new ptomaine. In guinea-pigs this ptomaine produced a slight flow of saliva and frequent respiration. Later the animals lost con- trol of their extremities and of the muscles of the trunk ; they fell upon their side, but when placed upon their legs were able to move forward a little ; they, however, soon fell again and remained help- less upon their side. The pupils gradually became widely dilated and failed to respond to light ; the flow of saliva became more pro- fuse ; no convulsions occurred. Little by little the pulsations of the heart and the breathing became more frequent. During the entire course of these symptoms the animals had frequent liquid discharges. Death occurred in from twenty-four to forty-eight hours. Post- mortem examination showed the heart to be contracted in systole, the lungs to be hypersemic, the intestine contracted and pale. The experimental evidence which we have presented, considered in connection with established facts relating to the propagation of typhoid fever, seems to the writer to be convincing as regards the etiological role of this bacillus. No other organism has been found, after the most careful search, in the deeper portions of the intestinal glands involved in this disease, or in the internal organs ; on the other hand, this bacillus has been demonstrated to be constantly present. It is undoubtedly present THE BACILLUS OF TYPHOID FEVER. 345 during the lifetime of the patients, and is found in greater abun- dance in those cases which terminate fatally at an early date. It is not a putrefactive organism, and is not developed in the tissues post mortem, although it has been shown by Frankel and Simmonds that it multiplies rapidly in the spleen after death, up to the time that putrefactive decomposition commences. This is quite in accord with what we should a priori have expected, in view of the facts relat- ing to the propagation of typhoid fever. These facts indicate that the disease in question is due to a microorganism which is capable of multiplication external to the human body in a variety of organic media, at comparatively low temperatures, and that it is widely dis- tributed. From the endemic prevalence of the disease over vast areas of the earth's surface we may infer that it is induced by a hardy microorganism. Eberth's bacillus complies with all of these conditions. There are numerous facts which indicate that the development of an attack of typhoid and the severity of the symptoms depend to some extent upon the quantity of the infectious material introduced into the alimentary canal. Milk or water which has been infected directly by the discharges of typhoid patients is especially danger- ous, and there is reason to believe that repeated or concentrated doses of such infectious material may be effective when a single draught of the contaminated fluid, or a greater degree of dilution, would be innocuous. Again, we have evidence that the typhoid germs may become effective as a result of certain favorable circumstances relating to the individual or to his environment. Those agencies which reduce the vital resisting power of the tissues, and especially exposure to the emanations from putrefying material, to sewer gas, to vitiated air in overcrowded and ill- ventilated apartments, etc., are recognized as favorable to the development of typhoid fever where the specific cause is present. All these facts seem to accord with the experi- mental evidence which indicates that the pathogenic power of the bacillus of Eberth depends upon the formation of a poisonous ptomaine rather than upon a special facility for multiplying in the tissues of a living animal. Indeed, it seems quite probable that its power to invade living animal tissues depends upon the toxic action of this ptomaine ; or, it may be, of other ptomaines produced under- certain circumstances in the body in excess or introduced from with- out. Such toxic agents may serve, when the specific germ is intro- duced into the intestine in comparatively small numbers, to give it the mastery over the vital resisting power of the tissues subject to in- vasion, and thus to induce an attack of the disease. ' 1 The above account of researches relating to the etiology of typhoid fever is from 27 346 THE BACILLUS OF TYPHOID FEVER. 46. BACILLUS TYPHI ABDOMINALIS. Synonyms. — Bacillus typhosus ; Typhus bacillus. Eberth (1880 and 1881) demonstrated the presence of this bacillus in the spleen and diseased glands of the intestine in typhoid cada- vers. Gaffky (1884) first obtained it in pure cultures from the same source and determined its principal biological characters. It is found, in the form of small, scattered colonies, in the spleen, the liver, the glands of the mesentery, the diseased intestinal glands, and in smaller numbers in the kidneys, in fatal cases of typhoid fever; it has also been obtained, by puncture, from the spleen during life, from the alvine discharges of the sick, and rarely from the urine. It is not found in the blood of the general circulation, unless, pos- sibly, in rare cases and in small numbers. FIG. 108. FIG. 109. FIG. 108.— Bacillus typhi abdomlnalis, from single gelatin colony. X 1,000. From a photo- micrograph. (FrankelandPfeiffer.) FIG. 109 —Bacillus typhi abdominalis, from single gelatin colony. X 1,000. From a photo- micrograph. (Sternberg.) Morphology. — Bacilli, usually one to three /* in length and about 0.5 to 0.8 yw broad, with rounded ends ; may also grow out into long threads, especially upon the surface of cooked potato. The dimen- sions of the rods differ considerably in different media. Spherical or oval refractive granules are often seen at the extremities of the rods, especially 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 the writer at the annual meeting of the Association of American Physicians, Washington, D. C., June 18th, 1886. THE BACILLUS OP TYPHOID FEVER. 347 terminal flagellum. These flagella are spiral in form, about 0. 1 >u 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 Loffler's method of staining (p. 32). Fie. 110.— Bacillus typhi abdominal! s, 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 Ziehl's 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. 348 THE BACILLUS OF TYPHOID FEVER. FIG. 111.— Singlecolony of Bacillus typhi abdominalis, ju nutrient gela- tin, (x?) From a photograph by Roux. •HUH 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 milk, 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- ance when cultivated in the 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. 112.— Bacillus typhi abdominalis ; stick culture in nutrient gelatin, eighth day at 16° -20° C. (Baum garten.) THE BACILLUS OF TYPHOID FEVER. 349 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 350 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- settled question. But, in view of the experimental evidence now THE BACILLUS OF TYPHOID FEVER. 351 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) arid 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 (C7H17NO2). 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. 352 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 113 —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. 353 they also show thai 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 the 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 27* 354 THE BACILLUS OP 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 per cent of carbolic acid to nutrient gelatin prevents the free development of the typhoid bacillus. Thoinot has 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, recently suggested by Hazen and White ^ has been tested with favorable results by Foote. This method de- pends upon the fact that most of the common water bacilli do not grow at a temperature of 40° C. , whereas this is a favorable tempe- rature for the development of the typhoid bacillus. A small quan- tity of the suspected 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, in a recent paper (Centralbl. /. Bakteriol., Bd. xii., page 367), claims that the typhoid bacillus may be differen- tiated from other similar bacilli (Bacillus coli communis, bacillus of hog cholera, etc. ) by the fact that it does not produce gas in culture PLATE V STERN BERG'S BACTERIOLOGY. Fig. ;;. PATHOGENIC UACTERIA, THE BACILLUS OF TYPHOID FEVER. 355 media containing sugar — grape sugar, cane sugar, or milk sugar. The medium recommended by Smith for making this test is a pep- tone-bouillon containing 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. PLATE V. PATHOGENIC BACTERIA. FlG. 1. — Bacillus authracis 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. FlG. 3. — Micrococcus gonorrhceae in gonorrhoeal pus. Stained with gen* tiaii 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 cholerse 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 Loffler's solution of methylene blue, x 1,000. Photomicrograph by Frankel and Pfeiffer. 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, arid 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 pyogeiies, 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 Loftier in 1884. In his first publication Loftier did not claim to have fully demon- strated the etiological relation of this bacillus, but this appears to be f ully 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 BACTERIA IN DIPHTHERIA. 357 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 Loftier 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 has recently (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, Von 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- braiious inflammation occurring in two children's asylums, in which for many months there had been no scarlatina and no measles, and in which there was no complicating suppurative inflammation and no erysipelas," Prudden (1890) obtained Loffler's bacillus in cultures from eleven, and he says : "We are now, it would seem, justified, as it did not appear to the writer 358 BACTERIA IN DIPHTHERIA. 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 % the bacillus called Bacillus diphtherias of Loffler." In this country Welch and Abbott (1891) have also demonstrated the presence of the Klebs-Loffler bacillus in a series of eight cases occurring in Baltimore, and have proved its specific pathogenic power by inoculations in animals. 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 from 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- BACTERIA IN DIPHTHERIA. 359 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 : "The only species of bacteria which we have found constantly in the cases of diphtheria has been the Loffler 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 Loffler bacillus, and a short, oval, often slightly pointed bacillus, growing in long chains running parallel to each other. There are often marked irregularities in shape and especially in size of this bacillus, even of individuals in the same chain. The colonies of this bacillus are grayish-white, moist, larger than those of the streptococcus, but smaller than those of the Loffler 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 these 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 oedema- 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 Loffler bacillus on glycerin-agar, 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 breath ing feebly. It was found dead on the morning of the third day. At the autopsy the wound was 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 dow'n 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, beginningat the larynx and extending down for six centimetres, wascovered with a tolerably firm, grayish-white, loosely attached pseudo-membrane, in all respects identical with the croupous membranes observed in the same situation in cases of human diphtheria." 360 BACTERIA IN DIPHTHERIA. 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 jw, 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 FIG. 114. — Bacillus diphtherias, of irregular spherical or oval segments. from a culture upon blood serum Multiplication occurs by fission only, From a photomicrograph, x 1,000. r nd Pfeiffer.) and the bacilli do not grow out into fila- 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 IN DIPHTHERIA. 361 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- ment is SO rapid in the incubating FIG. 115.— Colonies of Bacillus diphtherias ,, ,\_ i £ . in nutrient agar, end of twenty-four hours. oven that, at the end of twenty- x :0 (Frankei knd pfeiffer.) 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. The reaction of the bouillon becomes at first acid, but later it has an alkaline reaction (Welch). With reference to the growth on potato, authors have differed, probably because the growth is scarcely vis- ible ; upon this point we quote from Welch and Abbott : 28 362 BACTERIA IN DIPHTHERIA. " Our experience has 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. Loftier 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 hsemorrhagic suprarenal capsules, occasionally BACTERIA IN DIPHTHERIA. 363 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 osdematous 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 in 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 hours. 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 potent toxic substance, which being absorbed gives rise to toxaemia and 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 cultures which had been filtered through porous porcelain. Old 364 BACTERIA IN DIPHTHERIA. 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, and 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 toxine 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. Fraiikel 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. BACTERIA IN DIPHTHERIA. 365 The recent researches of Behring show that the blood of immune animals contains some substance which neutralizes the toxic product contained 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 fact, if fully con- firmed by further investigations, opens up a new field of experimen- tal research, and may lead to important results in the therapeutics of this and other infectious diseases. According to Roux and Yersin, " attenuated varieties " of the diphtheria bacillus maybe obtained by cultivating it at a temperature of 39.5° 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 from it chiefly in being non-pathogenic. The following account we take from the latest paper upon the subject by Roux and Yersin (troisieme memoire, 1890). Found by Roux and Yersin in mucus from the pharynx and ton- sils of children— from forty-five children in Paris hospitals, suffering from various affections, not diphtheritic, fifteen times ; from fifty- nine healthy children in a village school on the seaboard, twenty -six times. Of six children with a simple angina but two furnished cul- tures of this bacillus, while it was obtained in five out of seven cases of measles. 366 BACTERIA IN DIPHTHERIA. 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 diphtherias. 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- riae. 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. "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 been no local lesion. The most marked oedema resulted from cul- tures obtained from cases of measles. ' ' 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 in 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 has recently (1891) 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 f ollicular tonsillitis, eight from ordinary post- BACTERIA IN DIPHTHERIA. 367 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 diphtherias 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 I. , 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." 49. BACILLUS DIPHTHERIJE 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 fibrinous exudale. In pigeons the back part of the tongue, the fauces, and the corners of the mouth are especially affected ; in chickens the tongue, the gums, the nares, the larynx, and the conjunctival mucous membrane. The disease is especially fatal among chickens, the young fowls and those of choice varieties being 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. Grows 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 semi-transparent, grayish- white layer. Upon potato a thin layer is formed having a grayish tint. Pathogenesi*. — Pigeons inoculated with a pure culture in the mucous membrane of the mouth are affected exactly as are those which acquire the disease naturally. Subcutaneous inoculations in pigeons give rise to an in- flammation resulting in local necrotic changes. Pathogenic for rabbits and for mice. Subcutaneous injections in mice give rise toa fatal result in about five days. The bacillus is found in the blood and in the various organs, in the interior of the vessels, and sometimes in the interior of the leucocytes; they are especially numerous in the liver. The lungs are dotted with red spots, the spleen is greatly enlarged, and the liver has a marbled appearance from the presence of numerous irregular white masses scattered through the pale-red parenchyma of the organ. These white masses are seen, in sec- tions, to consist of necrotic liver tissue, in. the centre of which the bacilli 368 BACTERIA IN DIPHTHERIA. are found in great numbers, in the interior of the vessels. This appearance is so characteristic that Loftier considers inoculations in mice to be the most reliable method of establishing the identity of the bacillus. Not pathogenic for chickens, guinea-pigs, rats, or dogs. There seems to be some doubt whether the form of diphtheria which pre- vails among pigeons, and which Loftier has shown to be due to the bacillus above described, is identical with the diphtheria of chickens. Diphtheria in man has been supposed by some authors to be identical with that which prevails among fowls, and possibly this may be the case under certain cir- cumstances. But the evidence seems to be convincing that there is an infectious diphtheria of fowls which is peculiar to them, and which, under ordinary circumstances, is not communicated to man. 50. BACILLUS DIPHTHERIA VITULORUM. Described by Loffier (1884) and obtained by him from the pseudo-mem- branous exudation in the mouths of calves suffering f rom an infectious form of diphtheria. The disease is characterized by the appearance of yellow patches upon the mucous membrane of the cheeks, the gums, the tongue, and sometimes of the larynx and nares of infected animals. There is a yel- lowish discharge from the nose, an. abundant flow of saliva, occasional at- tacks of coughing, and diarrhoea. Death may occur at the end of four or five days, but usually the animal survives for several weeks. Diphtheritic patches similar to those in the mouth are also found in the large intestine, and scattered abscesses in the lungs. Loffier, in a series of seven cases examined, obtained from the deeper por- tions of the pseudo-membranous deposit a long bacillus which appears to be the cause of the disease. Morphology. — Bacilli, five to six times as long as broad, usually united in long filaments. The diameter of the rods is about half that of the bacillus of malignant oedema. Biological Characters.— Attempts to cultivate this bacillus in nutrient gelatin, blood serum from sheep, and various other media were unsuccessful. But when fragments of tissue containing the bacillus were placed in blood serum from the calf a whitish border, consisting of the long bacilli, was de- veloped. These could not, however, be made to grow when transferred to fresh blood serum. Pathogenesis. — Mice inoculated subcutaneously with the fresh diph- theritic exudation died in from seven to thirty days. The autopsy disclosed an extensive infiltration of the entire walls of the abdomen, which often pene- trated the peritoneal cavity and enveloped the liver, the kidneys, and the intestine in a yellowish exudate. ^ The bacillus was found in this exudate, and by inoculating a little of it into another animal of the same species a similar result was obtained. Not pathogenic for rabbits or guinea-pigs. 51. BACILLUS OF INTESTINAL DIPHTHERIA IN RABBITS. Described by Bibbert (1887) and obtained by him from the organs of rab- bits which succumbed to an affection characterized by a diphtheritic inflam- mation of the mucous membrane of the intestine. The autopsy revealed also swelling of the mesenteric glands and minute necrotic foci in the liver and spleen. Morphology. — Bacilli with slightly rounded ends, from three to four/* long and 1 to 1.4 u in diameter; often united in pairs or in filaments con- taining several elements. Stains with the aniline colors, but not so readily in sections as some other microorganisms. Ribbert recommends staining with aniline-water- fuchsin solution, washing in water, then placing the sections in methylene blue solution, and decolorizing in alcohol. Does not stain by Gram's method. BACTERIA IN DIPHTHERIA. 369 Biological Characters. — An aerobic, non-liquefying (non-motile ?) ba- cillus. Upon gelatin plates semi-transparent, grayish colonies are formed, which later have a brownish color; the surface of these is finely granular and of a pearly lustre. In stick cultures in nutrient gelatin the growth along the line of puncture is very scanty. On potato a flat, whitish layer is formed, which extends slowty over the surface. Grows best at a temperature of 30° to 35° C. Pathogenesis. — Pure cultures injected into the peritoneal cavity or sub- cutaneously in rabbits caused the death of these animals in from three to fourteen days, according to the quantity injected. At the autopsy necrotic foci are found in the liver and spleen, and the mesenteric glands are en- larged, but the intestine presents a healthy appearance. But when cultures are introduced into the alimentary canal the characteristic diphtheritic in- flammation of the mucous membrane of the intestine is induced. This re- sult was obtained both by direct injection into the lumen of the intestine and by injecting cultures into the mouth. X. BACTERIA IN INFLUENZA. A NUMBER of bacteriologists have made careful researches during the recent extended epidemic of influenza, and quite recently (1892) a bacillus has been discovered, both by Pfeiffer and by Canon, of Berlin, which there is good reason to believe is the specific cause of this disease. Before describing this we shall refer briefly to previous researches. 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 pneumoniae 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 which 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 pneumoniae," 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 pneumoniae crouposae ; 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 pneumoniae " was constantly found in great numbers. Fiscnel (1891) obtained in cultures from the blood of two cases two dif- BACTERIA IN INFLUENZA. 371 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, page 324). Kirchner (1891) found constantly in the sputum of recent cases a diplo- coccus enclosed in a jelly-like capsule, which differed in its biological and pathological characters from Micrococcus pneumonias crouposae (see Micro- coccus of Kirchner, No. 38, page 324). 52. BACILLUS OP 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 ZiehPs 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 by Gram's method. 372 BACTERIA IN INFLUENZA. 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 b'acilli 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). Pathogenesis. — 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. PLATE VI STERN BERG'S BACTERIOLOGY. ?y &*&* Jut Vlg. 1. Fiji. 3. w/ - *• *.? 1 v • . ' - x ..-v i 9 ^ 9?' \' * * • m ff+.fj • m.^ * - • ' * PATHOGENIC BACTERIA. BACTERIA IN INFLUENZA. 373 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." PLATE VI. PATHOGENIC BACTERIA. FIG. 1.— Bacillus tuberculosis in giant cell, x 1,000. Photomicrograph made at the Army Medical Museum, Washington, by Gray. FIG. 2. — Bacillus tuberculosis from a culture 011 glycerin-agar. x 1,000. Photomicrograph by Frankel and Pfeiffer. FIG. 3.— Bacillus tetani from an agar culture, x 1.000. Photomicro- graph by Frankel and Pfeiffer. FIG. 4. — Micrococcus pneumonias crouposae in sputum of a patient with pneumonia, x 1,000. Stained by Gram's method. Photomicrograph by Frankel and Pfeiffer. FIG. 5. — Bacillus septicsemise haemorrhagicce ("bacillus of fowl cholera") in blood from the heart of an inoculated pigeon, x 1,000. Photomicro- graph by Frankel and Pfeiffer. 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. XI. 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- factory, 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 Society 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 characters were given. This achievement, the result of patient and intelligent scientific investigation, will always rank as one of the most important in the history of medicine. The previous demonstration by Villemin (1865) — confirmed by Cohnheim (1877) and others — that tuberculosis might be induced in healthy animals by inoculations of tuberculous mate- rial, had paved the way for this great discovery, and advanced BACILLI IN CHRONIC INFECTIOUS DISEASES. 375 pathologists were quite prepared to accept it. The more conservative have since been obliged to yield to the experimental evidence, which has received confirmation in all parts of the world. To-day it is generally recognized that tuberculosis is a specific 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- 376 BACILLI IN CHRONIC INFECTIOUS DISEASES. Fia. 116. — Bacillus tuberculosis. X 1,000. From a photomicrograph. 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 {A. 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- tary, but may be united in pairs, or 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. 377 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 should 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. m.— Bacillus tubercuio- • ••1 ij-i -i -ii f sis in sputum, X 1,000. (Baum- as possible ; although a delay of some days garten!) 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 (Ziehl's 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 29 378 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 Ziehl's 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-fuchsin solution, or for twelve hours in the Ehrlich-Weigert tubercle stain — eleven cubic centimetres of saturated alcoholic solution of methyl violet, ten cubic centimetres of absolute alcohol, one hundred cubic centimetres of ani- line water. They should then be decolorized by placing them for BACILLI IN CHRONIC INFECTIOUS DISEASES. 379 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. (Baumgarten.) 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 defined 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 380 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 often 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 regards anthrax spores and many others, the 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. 381 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 Nbcard 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. For 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 382 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- ber 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° C.), 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 FIG. no.— Tubercle impressions by carefully applying a dry cover glass bacilli from surf ace of J J V / & . . culture upon blood se- to the surface. Upon staining the preparation in mm. x 5oo. (Koch.) 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 been 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. 383 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 384 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 Roux. 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, dry-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, FIG. 120.— Culture of tu- bercle bacillus upon glyce- rin-agar. Photograph by Roux. BACILLI IN CHRONIC INFECTIOUS DISEASES. 385 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 1)y 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. 30 386 BACILLI IN CHRONIC INFECTIOUS DISEASES. Sawizky has recently (1891) made a series of experiments to de- termine the length of time during which dried tuberculous sputum retains its virulence. He arrives at the conclusion that virulence is not suddenly 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-m.entioned 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 diminished production of a toxic product to which the bacillus owes its pathogenic power appears to be very certain, in view of the fact that the late cultures in a series have a more vigorous and abundant development than the more pathogenic cultures obtained directly from the animal body. The discovery by Koch of a toxine in cultures of this bacillus, which is soluble in glycerin, and which in very minute doses pro- duces febrile reaction and other decided symptoms when injected sub- cutaneously into tuberculous animals, must rank as one of the first BACILLI IN CHRONIC INFECTIOUS DISEASES. 387 importance in scientific medicine, whatever the final verdict may be as to its therapeutic value in tubercular diseases in man. The toxic substance contained in Koch's glycerin extract from cultures of the tubercle bacillus, now generally known under the name of tuberculin, is soluble in water, insoluble in alcohol, and passes readily through dialyzing membranes. It is not destroyed by the boiling temperature. According to the chemical examination of Jolles, the " lymph " contains fifty per cent of water and does not contain alkaloids or cyanogen compounds. Ib contains albuminates, which are thrown down as a voluminous white precipitate by tannic acid, and are redissolved by hot water containing sodium chloride and very diluted potash solution. The elementary analysis gave N" 5.90 per cent, C 35.19 per cent, and H 7.02 per cent. The re- sults obtained are believed to show that the active substance present in the lymph is a toxalbumin. In experiments made with Koch's lymph in Pasteur's laboratory by Bardach, a very decided elevation of temperature was produced in tuberculous guinea-pigs by the sub- cutaneous injection of 0.1 gramme, and a fatal result by the injec- tion of 0.2 to 0.5 gramme. In man a decided febrile reaction is pro- duced in tuberculous patients by very much smaller doses — 0.001 cubic centimetre. Hammerschlag, in his chemical researches, found that the tubercle bacillus yields a larger proportion of substances soluble in alcohol and ether than any other bacilli tested (twenty -seven per cent). The alcoholic extract contains fat, lecithin, and a toxic substance which produces convulsions in rabbits and guinea-pigs. The portion in- soluble in alcohol and ether contains cellulose and an albuminoid substance. No ptomaines were found, but a toxalbumin was isolated, which caused an elevation of temperature in rabbits of 1° to 2° C., lasting for a day or two. Hunter reports the following results of his chemical examination of tuberculin. It contains — 1. Albumoses, chiefly protoalbumose and deuteroalbumose, along with heteroalbumose, and occasionally a trace of dysalbumose. 2. Alkaloidal substances, two of which can be obtained in the form of the platinum compounds of their hydrochlorate salts. 3. Extractives, small in quantity and of unrecognized nature. 4. Mucin. 5. Inorganic salts. 6. Glycerin and coloring matter. The following conclusions are reached with reference to its toxic properties- : 1. Tuberculin owes its activity, not to one principle, but to at least three, and probably more, different substances. 2. Its action in producing local inflammation, fever, and general consti- tutional disturbance is not a simple but an extremely complex one. 388 BACILLI IN CHRONIC INFECTIOUS DISEASES. 3. Its active ingredients are of the nature of albumoses, alkaloidal sub- stances, and extractives. The action of these is in certain instances antag- onistic. 4. Its remedial and inflammatory actions are connected with the presence of certain of its albumoses, while its fever producing properties are chiefly associated with substances of non-albuminous nature. 5. The albumoses are not lost by dialysis ; the latter are. By the adoption of suitable methods it is thus possible to remove the substances which cause the fever, while retaining those which are beneficial in their action. 6. The fever produced by tuberculin is thus absolutely unessential to its remedial action. In a recent communication (October, 1891) Koch has given a full account of his method of preparing crude tuberculin, and also the process by which he obtains from this a tuberculin which appears to be pure, or nearly so. To obtain considerable quantities of the crude product the tubercle bacillus is cultivated in an infusion of calves' flesh or of beef extract to which one per cent of peptone and four to five per cent of glycerin have been added. This culture liquid must be made slightly alkaline, and it is placed in flasks with a flat bottom, which should not be more than half -filled — thirty to fifty cubic centi- metres. The inoculation is made upon the surface with small masses from a culture upon blood serum or glycerin-agar. By accident Koch discovered that these masses floating upon the surface give rise to an abundant development and to the formation of a tolerably thick and dry white layer, which finally covers the entire surface. At the end of six to eight weeks development ceases and the layer after a time sinks to the bottom, breaking up meanwhile into fragments. These cultures, after their purity has been tested by a microscopical examination, are poured into a suitable vessel and evaporated to one- tenth the original volume over a water bath. The liquid is then fil- tered through porcelain. The crude tuberculin obtained by this pro- cess contains from forty to fifty per cent of glycerin, and consequently is not a suitable medium for the development of saprophytic bacteria, if they should by accident be introduced into it. It keeps well and preserves its activity indefinitely. From this crude tuberculin Koch has obtained a white precipitate, with sixty-per-cent alcohol, which has the active properties of the crude tuberculin as originally prepared. This is fatal to tuberculous guinea-pigs in doses of two to ten milligrammes. It is soluble in water and in glycerin, and has the chemical reactions of an albu- minous body. In preparing it one and a half volumes of absolute alcohol are added to one volume of the crude tuberculin, and, after stirring it to secure uniform admixture, this is put aside for twenty-four hours. At the end of this time a flocculent deposit will be seen at the bottom of the vessel. The fluid above this is carefully poured off, and an equal quantity of sixty-per-cent alcohol poured into the vessel BACILLI IX CHRONIC INFECTIOUS DISEASES. 389 for the purpose of washing the precipitate. This is again allowed to settle and the procedure is repeated three or four times, after which the precipitate is washed with absolute alcohol. It is then placed upon a filter and dried in a vacuum exsiccator. An analysis of this purified tuberculin by Proskauer gave 18.46 per cent of ash, consisting almost entirely of potassium and magne- sium phosphate. The elementary analysis gave 48.13 per cent of carbon, 7.06 per cent of hydrogen, 14.46 per cent of nitrogen, and 1.17 per cent of sulphur. Recently (1892) Tizzoni and Oattani have presented some ex- perimental evidence which indicates that injections of Koch's tuber- culin into guinea-pigs may produce in these animals a certain degree of immunity against tuberculosis ; and that this immunity depends upon the presence of an anti-tuberculin formed in the body of the partially immune animal. Numerous experiments made by veterinary surgeons upon tuber- culous cows show that the injection of Koch's tuberculin in these animals, in doses of thirty to forty centigrammes, produces a rise of temperature of from 1° to 3° C. The febrile reaction usually occurs in from twelve to fifteen hours after the injection. Its duration and intensity do not depend upon the extent of the tuberculous lesions, but is even more marked when these are slight than in advanced cases. In non-tuberculous animals no reaction occurs, and the ex- periments made justify the suspicion that tuberculosis exists if an elevation in temperature of a degree or more occurs as a result of the subcutaneous injection of the dose mentioned. When the number of tubercle bacilli in sputum is comparatively small they may easily escape observation. Methods have therefore been suggested for finding them under -these circumstances. Ribbert (1886) proposed the addition to the sputum of a two-per-cent solution of caustic potash, and boiling the mixture. The tenacious mucus is dissolved, and when the mixture is placed in a conical glass vessel the bacilli are deposited at the bottom and may easily be found in the sediment after removing the supernatant fluid. The same object is accomplished by Stroschein (1889) by the addition to sputum of three times its volume of a saturated solution of borax and boracic acid in water. A method of estimating the number of bacilli in sputum has re- cently been proposed by Nuttall, which appears to give sufficiently accurate results and to be useful in judging of the progress of a case or of the results of treatment. For the details of this method we must refer to the author's paper (Johns Hopkins Hospital Bulle- tin, vol. xi., No. 13, 1891). It consists essentially in first making the sputum fluid by the addition of a solution of caustic potash ; in 390 BACILLI IN CHRONIC INFECTIOUS DISEASES. then shaking it thoroughly in a bottle containing sterilized gravel or pounded glass ; in carefully measuring the total quantity of fluid, and in dropping upon glass slides uniform drops by means of a grad- uated pipette ; in spreading these uniformly by means of a platinum needle and a turn table ; in covering the dried film with a film of blood serum, and coagulating this by heat ; and, finally, in staining and counting the bacilli in a series of slides from the same specimen, and from the average number found in a single drop estimating the total number in the sputum for twenty-four hours. Pathogenesis. — Man, cattle, and monkeys are most subject to contract the disease naturally, and it may be communicated by in- oculation to many of the lower animals — guinea-pigs, field mice, rab- Fio. 121.— Limited epithelioid celled tubercle of the iris, x 950. (Baumgarten.) bits, and cats are among the most susceptible animals ; and in larger doses dogs, rats, white mice, and fowls may also be infected. When tuberculous sputum is introduced beneath the skin of a guinea-pig the nearest lymphatic glands are found to be swollen at the end of two or three weeks, at the same time there is a thickening of the tissues about the point of inoculation ; later a dry crust forms over the local tuberculous tumefaction, and beneath this is a flattened ulcer covered with cheesy material. The animals become emaciated and show difficulty in breathing, and 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. 391 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 392 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 Bellinger, 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 impossible to confound 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 be 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. 393 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 observed in pathogenic bacteria, when these are culti- vated in pure cultures outside of the body for a 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, wassuffering 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. Recently Cadiot, Gilbert, and Roger have made a series of ex- periments 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 31 394 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 cf 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. ' ' 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° arid 45° C., and the thermal death-point is 70° C. "4. At 45° to 50D 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." 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 anaesthetic form of the disease, in the thickened portions of nerves involved in the leprous process. Some observers have also reported finding them in the blood, but this appears to be quite exceptional. In the leprous cells they are commonly found in great numbers, and they may also be seen in the lymph spaces outside of these cells. They are not found in the epidermal layer of the skin, but, according to Babes, they may penetrate the hair follicles. Morphology, — The bacillus of leprosy resembles the tubercle ba- cillus in form, but is of more uniform length and not so frequently BACILLI IN CHRONIC INFECTIOUS DISEASES. 395 FIG. 122.— Section of a recent lepra nodule of the skin. X 950. (Baumgarten.) bent or curved. The rods have pointed ends ; and in stained pre- parations unstained spaces, similar to those observed in the tubercle bacillus and generally assumed to be spores, are to be seen, although not quite so distinctly as in the latter. The bacilli are said by Fliigge to be from four to six yu in length and less than one /* in width — probably considerably less, for the same author states that the tubercle bacillus has about the diameter of the bacillus of mouse septi- caemia, and this is given as 0.2 J*. This bacillus stains readily with the aniline colors and also by Grain's method. Although it differs from the tubercle bacillus in the ease with which it takes up the ordinary aniline colors, it re sembles it in retaining its color when subset] uently treated with strong solutions of the mineral acids. Double-stained prepara- tions are therefore easily made by first staining sections or cover- glass preparations in Ziehl's carbol-fuchsin solution or in an aqueous solution of methyl violet, decolorizing in acid, washing in alcohol, and counter-staining with methylene blue — or, if methyl violet was used in the first instance, with vesuvin. Biological Characters. — The earlier attempts to cultivate this bacillus were without success, but recently Bordoni-Uffreduzzi has obtained from the marrow of the bones of a leper a bacillus which he believes to be the leprosy bacillus, and which he was able to culti- vate upon blood serum to which a certain amount of peptone and of glycerin had been added. At first this bacillus only grew with diffi- culty and in the incubating oven ; but after it had been cultivated artificially through a number of generations it is said to have grown upon ordinary nutrient gelatin at the room temperature. The bacillus obtained in this way is said to have retained its color when treated with acids, after having been stained with aniline-f uchsin, correspond- ing in this respect with the bacillus of leprosy and the tubercle ba- cillus. But it differed considerably in its morphology from the Ba- cillus leprse as seen in the tissues of lepers, being considerably thicker, and it was not so promptly stained by the aniline colors as is the bacillus found in the tissues. Moreover, attempts to cultivate the same bacillus from leprous tubercles of the skin were unsuccessful, as were also inoculation experiments into the anterior chamber of the eye in rabbits. It is therefore a matter of doubt as to whether the bacillus obtained by Bordoni-Uffreduzzi is identical with that present 396 BACILLI IN CHRONIC INFECTIOUS DISEASES. in such numbers in the cells of the leprous tubercles, to which the name Bacillus leprse has been given. Quite recently the announce- ment has been made that the " India Leprosy Commission " has suc- ceeded in cultivating the leprosy bacillus in blister serum. Not hav- ing seen a detailed account of the experiments of this Commission, the writer is unable to estimate the value of their work and the reli- ability of the alleged success in cultivating this bacillus. Some of the earlier observers described the bacillus of leprosy as motile, but this assertion seems to have been based upon some error of observation, and it is now generally agreed that, like the tubercle bacillus, it is without proper movements. The question of spore for- mation has not been definitely settled. As before remarked, un- stained portions, occurring at regular intervals, are seen in the rods in stained preparations ; but no satisfactory evidence has been presented to show that these are truly reproductive spores. Pathogenesis, — The inference that the bacillus above described bears an etiological relation to the disease with which it is associated is based upon the demonstration of its constant presence in leprous tissues — which has now been repeatedly made in various and distant parts of the world — and of its absence from the same tissues involved in different morbid processes. As it has not been obtained in pure cultures, the final proof of such etiological relation is still wanting. We have, however, experimental evidence to show that leprous tis- sues containing this bacillus are infectious and may reproduce the disease. The experiment has been made upon man by Arning, who inoculated a condemned criminal subcutaneously with fresh leprous tubercles. The experiment was made in the Sandwich Islands, and the man was under observation until his death occurred from leprosy at the end of about five years. The first manifestations of the disease became visible in the vicinity of the point of inoculation several months after the experimental introduction of the infectious material. Positive results have also been reported in the lower animals by Damsch, by Vossius, and by Melcher and Ortmann. The last-named investigators inoculated rabbits in the anterior chamber of the eye with portions of leprous tubercles excised for the purpose from a leper. The animals died from general infection at the end of several months, and the characteristic tubercles containing the bacillus were distributed through the various organs. 56. BACILLUS MALLEI. Synonyms. — The bacillus of glanders; Der Rotzbacillus, Ger. ; Bacille de la morve, Fr. Discovered by Loftier and Schiitz (1882), and proved to be the cause of glanders by the successful inoculation of pure cultures. BACILLI IN CHRONIC INFECTIOUS DISEASES. 39? Y Found especially in the recent nodules in animals infected with glanders ; also in the same after ulceration, and in the discharge from the nostrils, pus from the specific ulcers, etc. ; sometimes in the blood of infected animals (Weichselbaum). Morphology. — Bacilli with rounded ends, straight or slightly curved, rather shorter and decidedly thicker than the tubercle bacil- lus ; usually solitary, but occasionally united in pairs, or in filaments containing several elements (in potato cultures). In stained preparations unstained or feebly stained spaces are seen in the rods, alternating with the deeply stained protoplasm of the cell. As in the tubercle bacil- lus, which presents a similar appearance, these spaces have been supposed by some bacteriolo- gists to represent spores ; but Loftier believes them to represent rather a degeneration of the protoplasm. Baumgarten and Rosenthal claim to have demonstrated the presence of spores by the use of Neisser's method of staining, but they do not consider it established that the unstained spaces in the rods referred to are of this nature. The glanders bacillus may be stained with aqueous solutions of the aniline colors, but the staining is more intense when the solution Fia. 123.— Bacillus mal- lei, x 1,000. From a pho- tomicrograph. CFrankel and Pfeiffer.) FIG. 134.— Section of a glanders nodule, x 700. (Flugge.) is made feebly alkaline. Add to three cubic centimetres of a 1 : 10,000 solution of caustic potash, in a watch glass, one cubic centimetre of a saturated alcoholic solution of an aniline color (methylene blue, gentian violet, or fuchsin) ; or the aniline-water-fuchsin, or methyl violet solution of Ehrlich may be used, with the addition just be- fore use of an equal quantity of 1 : 10,000 solution of caustic potash. Loffler recommends that cover-glass preparations be placed in Ehr- lich's solution and heated for five minutes; then decolorized in a one- 398 BACILLI IN CHRONIC INFECTIOUS DISEASES. per-cent solution of acetic acid to which sufficient tropseolin has been added to give it the yellow color of Rhine wine ; then quickly washed in distilled water. This bacillus presents the peculiarity of losing very quickly in decolorizing solutions the color imparted to it by the aniline staining solutions. For this reason the staining of the bacillus in sections is attended with some difficulty. Loffler recom- mends his alkaline methylene-blue solution for staining sections ; and for decolorizing, a mixture containing ten cubic centimetres of distilled water, two drops of strong sulphuric acid, and one drop of a five- per-cent solution of oxalic acid. Thin sections should be left in this acid solution about five seconds. The method more recently recom- mended by Kuhne also gives good results in skilful hands (see p. 34). Biological Characters. — An aerobic, non-motile, parasitic bacillus, which may be cultivated in various artificial media at a temperature of 37° C. The lowest temperature at which develop- ment occurs (22° C. — Loffler) is a little above that at which nutrient gelatin is liquefied ; the highest limit is 43° C. According to Frankel, the glanders bacillus is a facultative anaerobic. Baumgarten and Rosenthal claim to have demonstrated the presence of spores by Neisser's method of staining. Loffler was led to doubt the forma- tion of spores from the results of his experiments upon the thermal death-point of this bacillus, and its comparatively slight resistance to desiccation and destructive chemical agents. He found that ex- posure for ten minutes to a temperature of 55° C., or for five minutes to a three- to five-per-cent solution of carbolic acid, or for two min- utes to a 1 : 5,000 solution of mercuric chloride, was effectual in de- stroying its vitality. As a rule, the bacilli do not grow after having been preserved in a desiccated condition for a few weeks ; and in a moist condition the cultures cannot be preserved longer than three or four months — usually not so long as this (Loffler). The bacillus does not grow in infusions of hay, straw, or horse manure, and it is doubtful whether it finds conditions in nature favorable for its sap- rophytic existence. It grows, in the incubating oven, in neutral bouillon, in nutrient gelatin, or in nutrient agar, and still better in glycerin-agar. Upon the last-mentioned medium it grows, even at the room temperature (Kranzfeld), but better still in the incubating oven, as a pale- white, transparent streak along the line of inocula- tion, which at the end of six or seven days may have a width of seven to eight millimetres. According to Raskina, nutrient agar made with milk forms an extremely favorable medium, upon which a thick, pale- white layer develops in two or three days, which on the third or fourth day acquires an amber-yellow color, and the deeper layers acquire a brownish-red tint. The growth upon solidified blood serum, in the course of three or BACILLI IN CHKONIC INFECTIOUS DISEASES. 399 four days at 37° C., consists of yellowish, transparent drops, which later coalesce into a viscid layer, which has a milky appearance from the presence of numerous small crystals (Baumgarten). The growth upon cooked potato is especially characteristic. In the incubating oven, at the end of two or three days, a rather thin, yellowish, trans- parent layer develops, which resembles a thin layer of honey. Later this ceases to be transparent, and the amber color changes, at the end of six to eight days, to a reddish-brown color ; and outside of the reddish-brown layer, with more or less irregular outlines, the potato for a short distance acquires a greenish-yellow tint. Pathogenesis. — Glanders occurs principally among horses and asses, but may be contracted by man from contact with infected animals ; it has also been communicated, in one instance with a fatal result, by subcutaneous inoculation, resulting accidentally from the use of an imperfectly sterilized hypodermic syringe which had pre- viously been used for injecting cultures of the bacillus into guinea- pigs. The field mouse and the guinea-pig are especially susceptible to infection by experimental inoculations ; the cat and the goat may be infected in the same way. Lions and tigers in menageries are said to have contracted glanders from being fed upon the flesh of in- fected animals (Baumgarten). Rabbits have but slight susceptibility, and the same is true of sheep and dogs ; swine, cattle, white mice, and common house mice are immune. The etiological relation of the bacillus is fully established by the experiments of Loffler and Schutz, confirmed by other bacteriologists, which show that pure cultures injected into horses, asses, and other susceptible animals, produce genuine glanders. The disease is char- acterized in the equine genus by the formation of ulcers upon the nasal mucous membrane, which have irregular, thickened margins and secrete a thin, virulent mucus ; the submaxillary lymphatic glands become enlarged and form a tumor which is often lobulated ; other lymphatic glands become inflamed, and some of them suppurate and open externally, leaving deep, open ulcers ; the lungs are also involved and the breathing becomes hurried and irregular. In farcy, which is a more chronic form of the same disease, circumscribed swellings, varying in size from a pea to a hazelnut, appear on differ- ent parts of the body, especially where the skin is thinnest ; these suppurate and leave angry -looking ulcers with ragged edges, from which there is an abundant purulent discharge. The specific bacillus can easily be obtained in pure cultures from the interior of suppurat- ing nodules and glands which have not yet opened to the surface, and the same material will give successful results when inoculated into susceptible animals. But the discharge from the nostrils or from an open ulcer contains comparatively few bacilli ; and as these are 400 BACILLI IN CHRONIC INFECTIOUS DISEASES. associated with various other bacteria which grow more readily in our culture media, it is not easy to obtain pure cultures, by the plate method, from such material. In the guinea-pig subcutaneous inoculation is followed in four or five days by tumefaction at the point of inoculation, and after a time a prominent tumor with caseous contents is developed ; ulceration of the skin follows, and a chronic, purulent ulcer with irregular, indu- rated margins results ; after a time this may cicatrize. Meanwhile the lymphatic glands become involved, and the symptoms of general Fio. 125.— Section through a glanders nodule in liver of field mouse. Tissue X 250. Bacilli X 500. (Baumgarten.) infection are developed at the end of four or five weeks ; the glands suppurate, and in males the testicles are also involved ; finally a dif- fuse inflammation of the joints occurs, and death results from ex- haustion. In the guinea-pig the specific ulcers upon the nasal mu- cous membrane, which characterize the disease in the horse, are rarely developed to any great extent. In field mice general infection occurs at once as a result of the subcutaneous injection of a small quantity of a pure culture, and the animal dies at the end of three or four days. Upon post-mortem BACILLI IN CHRONIC INFECTIOUS DISEASES. 401 examination the principal changes are found in the liver and in the greatly enlarged spleen. Scattered through these organs are minute gray points which are scarcely visible to the naked eye. In the guinea-pig, which succumbs at a later date, these nodules are larger and closely resemble miliary tubercles, both macroscopically and under the microscope, in stained sections of the tissues. Similar nodules are also found in the kidneys and in the lungs ; they have a decided tendency to undergo purulent degeneration. The bacilli are found principally in these nodules, of recent formation, and are com- monly associated in groups, as if they had been enclosed in the inte- rior of a cell the membranous envelope of which had undergone degeneration and disappeared. As before remarked, it is not an easy matter to demonstrate the bacillus in sections of the tissues containing these nodules, owing to the facility with which they lose their color in alcohol and other de- colorizing agents. For this reason it will be best to dehydrate sec- tions by the use of aniline oil (Weigert's method) or to resort to Kiihne's method of staining. It is also difficult to demonstrate the presence of the bacillus in nodules which have undergone purulent degeneration, in the secre- tions from the nostrils of horses suffering from glanders, or in the pus from the specific ulcers and suppurating glands ; for they are present in comparatively small numbers. But the virulent nature of these discharges is shown by inoculations into guinea-pigs or mice, and it is easier to obtain a pure culture from such virulent material by first inoculating a susceptible animal than directly by the plate method; for the small number of bacilli present, and their associa- tion with other bacteria which develop more rapidly in our culture media, make this a very uncertain procedure. For establishing the diagnosis of glanders, therefore, Loffler recommends the inoculation of guinea-pigs with pus from a suppurating gland or ulcer, or the nasal discharge from a suspected animal, rather than a direct attempt to demonstrate the presence of the bacillus by staining and culture methods. The method proposed by Strauss gives more prompt results. This consists in the intraperitoneal injection of cultures or of the suspected products into the cavity of the abdomen of male guinea- pigs. If the glanders bacillus is present the diagnosis may be made within three or four days from the infectious process established in the testicles. At the end of this time the scrotum is red and shining, the epidermis desquamates, and suppuration occurs, the pus some- times perforating the integument. This pus is found to contain the glanders bacillus. The animal usually dies in the course of twelve to fifteen days. When the animals are killed three or four days 402 BACILLI IN CHRONIC INFECTIOUS DISEASES. after the inoculation, the two layers of the tunica vaginalis testis are found to be covered with a purulent exudate containing the glanders bacillus and to be more or less adherent. Even as early as the second day the tunica vaginalis is seen to be covered with granulations. An attenuation of virulence occurs in cultures whicn have been kept for some time, and inoculations with such cultures may give a negative result ; or, when considerable quantities are injected, may produce a fatal result at a later date than is usual when small amounts of a recent culture are injected into susceptible animals. Kalning, Preusse, and Pearson have obtained from cultures of the glanders bacillus a glycerin extract similar to the crude tubercu- lin of Koch — mallein. This, when injected into animals suffering from glanders, gives rise to a considerable elevation of temperature, and it has been proposed to use it as a means of diagnosis in cases of suspected infection in animals in which the usual symptoms have not yet manifested themselves. The value of the test has already been demonstrated by the experiments of Heyne, Schilling, and others. 57. BACILLUS OF LUSTGARTEN. Synonym. — Syphilis bacillus. Found by Lustgarten. (1884) in syphilitic lesions and in secretions of syphilitic ulcers, and believed by him to be the specific infectious agent in this disease. No satisfactory experimental evidence that this is the case has yet been obtained. Morphology. — Straight or curved bacilli, which bear considerable resem- FIG. 126. FIG. 127. FIG. 126.— Migrating cell containing syphilis bacilli. (Lustgarten. ) FIG. 127 —Pus from hard chancre containing syphilis bacilli (Lustgarten.) blance to tubercle bacilli, but differ from them in the staining reactions. They are usually more or less curved, or bent at a sharp angle, or S- shaped ; the ends often present slight knob-like swellings ; the length is from three and one-half ft to four and one-half /<, and the diameter is from 0 25 to 0.3 //. With a high power the contour is seen to be not quite regular, but wavy in outline, and bright, shining spaces in the deeply stained roda may be ob- served ; these, from two to four in a single rod, are believed by Lustgarten BACILLI IN CHRONIC INFECTIOUS DISEASES. 403 to be spores. The bacilli are not found free in the tissues, but are enclosed in cells of a round-oval or polygonal form, which are said to be about double the size of a white blood corpuscle. The bacilli are not numerous, and very commonly only one or two are found in a single cell, but groups of six or eight may sometimes be seen, especially upon the margins of a syphilitic lesion, and in the tissues in the immediate vicinity of the infiltration, which show but little change or are apparently healthy (Lustgarten). The presence of these bacilli in syphilitic lesions was demonstrated by Lustgarten by the following staining method : The thin sections are placed in the Ehrlich-Weigert gentian-violet solution (one hundred parts aniline water, eleven parts saturated alcoholic solution of gentian violet) for from. twelve to twenty four hours at the room temperature, and two hours in the incubating oven at 40° C. The sections are then thoroughly washed in alco- hol and placed for ten seconds in a 1.5-per-cent solution of potassium per- manganate; in this solution a precipitate of peroxide of manganese is formed, which adheres to the section; this is dissolved and washed off in a dilute aqueous solution of pure sulphuric acid ; the sections are then washed in water, and, if not completely decolorized, are returned for a few seconds to the permanganate solution and again washed off in the acid; it may be necessary to repeat this operation three or four times. Finally the sections are dehydrated and mounted in balsam in the usual manner. Cover-glass preparations are made in the same way, except that, after being- taken from the staining solution, they are washed off in water instead of in alcohol. Another method of staining, recommended by De Giacoma, consists in placing the sections for twenty -four hours in aniline-water-fuchsin solution (cover-glass preparations may be stained in the same solution, hot, in a few minutes), then washing them in water, and decolorizing in a solution of per- chloride of iron — first in a dilute and then in a saturated solution. The method of staining employed by Lustgarten serves to differentiate his bacillus from many other microorganisms, but not from the tubercle ba- cillus and the bacillus of leprosy, which, as he pointed out, may be stained in the same way. And it has since been shown by Alvarez and Tavel, and by Matte rstock, that in smegma from the prepuce or the vulva, bacilli are found which have the same staining- reaction and are similar in their mor- phology to the bacillus of Lustgarten. This by no means proves that the smegma bacilli found under the prepuce of healthy persons are identical with the bacilli found by Lustgarten and others in sections of tissues involved in syphilomata. In the absence of pure cultures and inoculation experiments it is impossible to establish identity, however similar may be the characters referred to. Several well-known pathogenic bacilli resemble quite as closely in these particulars other bacilli which have, nevertheless, been differentiated from them by culture and inoculation experiments. We may mention especially in this connection the bacillus of diphtheria, as obtained from the pseudo-membranous exudation in a genuine case of this disease, and the pseudo diphtheria bacilli found by Eoux and Yersinin the fauces of healthy children. On the other hand, since it has been shown that similar bacilli are common in preputial srnegma, we cannot attach great importance to the finding of Lustgarten's bacillus in primary syphilitic sores ; and it has not been found in sufficient numbers, or with sufficient constancy, by those who have searched for it subsequently to the publication of Lustgarten's inves- tigations, to give strong support to the view that it is the specific infectious agent in syphilis. Baumgarten, who has searched in vain for Lustgarten's bacillus in uncomplicated visceral syphilomata, suggests that the bacilli found occasionally in such lesions were perhaps tubercle bacilli and repre- sented a mixed infection. As the bacillus under consideration has not been obtained in cultures, we have no information as to its biological characters and pathogenesis. THE SYPHILIS BACILLUS OF EVE AND LINGARD. Eve and Lingard (1886) report that they have obtained in cultures from 4:04 BACILLI IN CHRONIC INFECTIOUS DISEASES. the blood and diseased tissues of syphilitics who have not undergone mer- curial treatment, bacilli which in their form and dimensions resemble the tubercle bacilli, but which stain readily by the common aniline colors and by Gram's method, and are not stained by Lustgarten's method. They grow readily upon solidified blood serum, forming a thin, pale-yellow or brown- ish-yellow layer. Inoculations of pure cultures into apes were without result. The negative results which have attended the culture experiments and microscopical examinations of the blood and diseased*tissues, made by many competent bacteriologists in other parts of Europe, make it appear probable that the bacilli described by the English investigators named belong to some saprophytic species, and that they are not usually present in syphilo- mata or the blood of syphilitic patients. MICROCOCCI OF DISSE AND TAGUCHI. . Disse and Taguchi (1886) claim to have obtained from the blood of syphi- litics micrococci which they were able to cultivate in artificial media at 20 to 40° C., and which formed on the surface of such media a grayish- white layer consisting of diplococci which are motile and of larger motion less cocci. The diplococci are said to originate from division of the larger cocci. Inocu- lations into rabbits, dogs, and sheep gave rise to chronic interstitial inflam- matory processes in the lungs and liver, to granulomata in various organs, and to fatty degenerative changes in the walls of the arteries, which, in the opinion of the authors named, correspond with the pathological changes produced by syphilitic infection in man. We remark, with reference to the supposed etiological relation of this coccus, that bacteriologists in Europe have not confirmed the authors named as to the presence of this micrococcus in the blood of syphilitics, and that the micrococcus of progressive granuloma formation described by Manfredi produces similar pathological changes in inoculated animals ; also that there is no evidence that the animals experi- mented upon are subject to syphilitic irifection. 58. BACILLUS OF RHINOSCLEROMA (?). First observed by Von Frisch (1882) in the newly formed tubercles of rhinoscleroma. Cultivated by Paltauf and Von Eiselberg (1886). Rhinoscleroma is a chronic affection of the skin, and especially of the mucous membrane of the nares, which is characterized by the formation of tubercular thickenings of the skin and tumefaction of the nasal mucous membrane, followed sometimes by ulceration. It prevails in Italy, Austria, and to a slight extent in some parts of Germany. Pathologists generally regard it as an infectious process, although this has not been proved. The bacilli, first described by Von Frisch, appear to be constantly present in the newly formed tubercles. They are commonly found in certain large hyaline cells peculiar to the disease, and may also be observed in the lym- phatic vessels or scattered about in the involved tissues. Morphology. — Short bacilli with rounded ends, usually united in pairs, and surrounded by a gelatinous capsule resembling that of Friedlander's bacillus. According to Eisenberg, the bacilli are two to three times as long as broad, and may grow out into filaments. These bacilli stain readily with the aniline colors and by Gram's method. The capsule may be demonstrated by the methods usually employed in stain- ing Friedlander's bacillus, or by the following method which is especially recommended by Alvarez: The excised portions of tissue involved in the dis- ease are placed for twenty-four hours in a one-per-cent solution of osmic acid and then in absolute alcohol. When properly hardened thin sections are made; these are stained in a hot solution of aniline-water-methyl-violet for a few minutes, and then decolorized, by Gram's method, in iodine so- lution. Biological Characters,— A.n aerobic, non-motile, non-liquefying bacillus (facultative anaerobic ?). BACILLI IN CHRONIC INFECTIOUS DISEASES. 405 In gelatin stick cultures the growth, resembles that of Friedlander's ba- cillus— i.e., a nail-like growth, consisting of densely crowded, opaque colonies along the line of puncture, and a heaped-up, white, glistening mass upon the surface, hemispherical in form and viscous in consistence. Upon gelatin plates yellowish-white, spherical colonies are developed within two or three days, which under the microscope are seen to be granular. Upon potato a cream-like growth occurs along the line of inoculation, which is white or yellowish-white in color, and in which gas bubbles may be developed. De- velopment is most rapid at a temperature of 35° to 38°, but also occurs at the room temperature. Pathogenesis. — The etiological relation of this bacillus to the disease with which it is associated has not been established. It is pathogenic for mice and for guinea-pigs, less so for rabbits ; in this regard, as in its morphology and growth in various culture media, it bears a close resemblance to Fried- lander's bacillus, which is also found not infrequently in the nasal secretions of healthy persons and in those suffering from chronic nasal catarrh or ozaena. The principal points of difference, as pointed out by Baumgarten, are as follows : The bacillus of rhinoscleroma is usually more decidedly rod-shaped FIG. 128.— Bacillus of rhinoscleroma in lymphatic vessels of the superficial part of tumor. X 1,200. (Cornil and Babes ) than Friedlander's bacillus, although both may be of so short an oval as to resemble micrococci. The first-mentioned bacillus constantly presents the appearance of being surrounded by a transparent capsule, even in the cul- tures in artificial media, while Friedlander's bacillus in such media does not usually present this appearance, unless as a result of special treatment. Finally, the bacillus of rhinoscleroma may retain its color, in part at least, when treated by Gram's method, while Friedlander's bacillus is completely decolorized when placed in the iodine solution employed in this method. Notwithstanding these points of difference, Baumgarten is not entirely satisfied that this bacillus is a distinct species, and several bacteriologists have maintained that it is identical with the bacillus of Friedlander. 59. BACILLUS OF KOUBASOFF. Obtained by Koubasoff (1889) from new growths in the stomach of a person who died of cancer of the stomach. Morphology. — Bacilli with round ends, or with one end pointed, two or three times as long as the tubercle bacillus and three or four times as thick. Stains readily with the aniline colors. Biological Characters. — A.n aerobic and facultative anaerobic, non- liquefying, motile bacillus. Forms spores in the centre of the rods. Grows in the usual culture media at the room temperature, more rapidly at 36° C. 406 BACILLI IN CHRONIC INFECTIOUS DISEASES. In stick cultures in, glycerin-gelatin, the growth resembles an in verted stetho- scope; at the surface a circular, bluish membrane is formed, which is de- pressed in the form of a funnel, while along the line of puncture a slender, yellowish, jagged column is developed. Upon agar, at 36° C., a bluish- white layer is quickly developed. Upon potato the growth resembles that of the typhoid bacillus at first; later a granular membrane is formed; under a low power the granules appear to be formed of intertwined masses of fila- ments. The growth upon blood serum is similar to that upon agar. Pathogenesis. — Subcutaneous injections in guinea-pigs cause their death in one to two weeks, in rabbits in one to two months, in cats and dogs in two months or more. Death occurs in a shorter time in animals which, have been fed upon cultures than as a result of subcutaneous injections. The animals become very much emaciated and have paralysis of the sphincter muscles. At the autopsy flat or nodular elevations, which are often ulce- rated, are seen here and there upon the mucous membrane of the stomach and intestine ; the mesentery, especially of the small intestine, is hyperaemic ; the mesenteric glands are swollen, as are also the inguinal glands. In the liver and sometimes in the ovary, uterus, and spleen larger or smaller nod- ules are seen. 60. BACILLUS OP NOCARD. Obtained by Nocard (1888) from pus collected from the superficial ab- scesses in cattle suffering from a chronic infectious disease which prevails especially upon the island of Guadaloupe — known as " farcin du boeuf"; Ger. "Wurmkrarikheit." Morphology. — A long and slender bacillus, about as thick as the bacillus of rouget (Bacillus murisepticus) ; usually seen in tangled masses which consist of an opaque central portion surrounded by long filaments, which apparently give off lateral ramifications. (This description of the morphol- ogy gives rise to the suspicion that the microorganism described by Nocard is a microscopic fungus rather than a bacillus.) According to Nocard, the branching is more apparent than real, and is in fact a false dichotomization, such as is seen in the genus Cladothrix. Stains best by Weigert's method ; is decolorized by Gram's method. Does not stain readily with most aniline colors. Biological Characters. — An aerobic, non-motile bacillus, which does not grow in nutrient gelatin at the room temperature. Grows in the usual culture media at a temperature of 30 3 to 40° C. Forms small oval spores. Is destroyed in ten minutes by a temperature of 70° C. Upon the surface of agar it forms irregular, opaque, yellowish-white colonies, which are thickest at the margin, have a dull, dusty- looking, mammillated surface, and after a time become confluent, forming a thick, wrinkled, membranous layer. Upon potato development is rapid in the form of prominent, dry, pale- yellow plaques. In bouillon whitish flocculi are formed, most of which fall to the bottom, while some float upon the surface, where they form dry, dusty-looking, rounded pellicles of adirty-gray color withagreenish reflection. ^Pathogenesis. — The guinea-pig is the most susceptible animal. When injected into th^ peritoneal cavity of one of these animals it produces, in from nine to twenty days, lesions which closely resemble those of miliary tuberculosis. At the autopsy the peritoneu m is found to be covered with nodules, in the centre of which the bacillus is found in tangled masses ; the liver, spleen, kidneys, and intestine are also studded with pseudo-tubercles, but these are only found in the peritoneal coat and not in the parenchyma of the various organs, or in the organs of the thoracic cavity. Intravenous injections give rise to lesions similar to those of general miliary tuberculo- sis, the organs generally containing a considerable number of nodules, in the centre of which_tufts of bacilli are found. In cattle and sheep similar lesions result from intravenous injections, but without causing the death of the animal. The dog, the cat, the horse, the ass, and the rabbit are immune. Subcutaneous inoculations in guinea pigs produce an extensive local abscess, followed by a chronic induration of the neighboring lymphatic glands. XII. BACILLI WHICH PRODUCE SEPTICAEMIA IN SUSCEPTIBLE ANIMALS. WHEN, as a result of accidental (natural) or experimental inocula tion, a microorganism is introduced into the body of a susceptible animal which is able to multiply in its blood, producing a general in- fection, we speak of this general blood infection as a septicfemia. When pathogenic microorganisms which are unable to multiply in the blood establish themselves in some particular locality in the ani- mal body which is favorable for their growth, and by the formation of toxic products, which are absorbed, give rise to general symptoms of poisoning, we designate the affection toxcemia. As examples of this mode of pathogenic action we may mention diphtheria and tetanus. As a rule, the various forms of septicaemia are quickly fatal, and, as the microorganisms to which they are due multiply in the blood of the infected animal, this fluid possesses infectious pro- perties, and, when inoculated in the smallest quantity into another susceptible animal, reproduces the same morbid phenomena. A typi- cal example of this class of diseases is found in anthrax, to which disease a special section has already been devoted (VII.). But in this and other forms of septicaemia subcutaneous inoculations do not, as a rule, result in the immediate invasion of the blood by the para- sitic microorganism. Often a local inflammatory process of consider- able extent is first induced ; and in some cases general infection only occurs a short time before the death of the animal, depending, per- haps, upon a previous toxaemia from the absorption of toxic products developed at the seat of local infection. The pathogenic action, then, in acute forms of septicaemia appears to result, not alone from the presence and multiplication of the pathogenic microorganism in the blood, but also from the toxic action of products evolved during its growth. Some of the pathogenic bacilli of this class now known to bac- teriologists have been discovered by studying the infectious diseases induced by them in lower animals among which these diseases pre- vail naturally — i.e., independently of human interference. Many 4:08 BACILLI WHICH PRODUCE SEPTICAEMIA more are known to us from experiments made in pathological labora- tories, in testing by inoculations into animals bacteria obtained from various sources, with reference to their pathogenic power. We in- clude in this group only those bacilli which induce fatal septicaemia in susceptible animals when injected into the circulation or sub- cutaneously in a comparatively small quantity — e.g., less than half a cubic centimetre of a bouillon culture. 61. BACILLUS SEPTICAEMIA HJEMORRHAGIC^E. Synonyms. — Bacillus of fowl cholera • Microbe du cholera des poules (Pasteur) ; Bacillus cholerae gallinarum (Flugge); Bacillus der Hiihnercholera ; Bacillus of rabbit septicaemia ; Bacillus cuniculi- cida (Flugge) ; Bacillus der Kaninchenseptikamie (Koch) ; Bacillus der Rinderseuche (Kitt) ; Bacillus der Schweineseuche (Loffler and Schiitz) ; Bacillus der Wildseuche (Hueppe) ; Bacillus der Biiffel- seuche (Oreste-Armanni) ; (Bacterium of Davaine's septicaemia ?) It is now generally admitted by bacteriologists that Koch's ba- cillus of rabbit septicaemia (1881) is identical with the bacillus ("micrococcus") of fowl cholera previously described by Pasteur (1880). The similar bacilli found in the blood of animals dead from the infectious diseases known in Germany as Wildseuche (Hueppe), Rinderseuche (Kitt), Schweineseuche (Schiitz), and Buffelseuche (Oreste-Armanni) appear also to be identical with the bacillus of rabbit septicaemia and fowl cholera. This view is sustained by Hueppe and by Baumgarten, and by the recent comparative re- searches of Caneva (1891) and of Bunzl-Federn (1891). This is evidently a widely distributed pathogenic bacillus ; it was obtained by Koch from rabbits inoculated with pu- ^ ^ ,-.. • * ,0 tref ying flesh infusion, by Gaffky from impure river *O'»*O** W water, and by Pasteur from the blood of fowls suffer- '*>rS***\ *r~»* ing from the infectious disease known in France as '•» v40 ' " O * cholera des poules. It is not infrequently found in t(\J t/* •» • ' putrefying blood, and its presence in the salivary FIQ 129 Bacillus secretions of man has occasionally been demonstrated septicaemia? hsemor- (Baumgarten) . rhagic* in the blood With reference to the American swine plague of a rabbit. X 950. ° (Baumgarten.) 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 IN SUSCEPTIBLE ANIMALS. 409 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 /* in diameter and about 1.4 ^ 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- 33 410 BACILLI WHICH PRODUCE SEPTICAEMIA cubating oven a rather thin, transparent, grayish-white or yellowish, waxy layer is developed hi the course of a few days. According to Bunzl-Federn, the bacillus of fowl cholera and that yjjtf Jjy|i||j 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- caBmia 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 different 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 FIG. 130. — Bacillus septicaemias haemor- rhagicse; stick culture in nutrient gelatin, end of four days at 16°- 18° C. (Baumgarten > FIG. 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 CO. (Smith.) IX SUSCEPTIBLE ANIMALS. 411 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 virulence 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 fatal 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 membrane 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 hsemorrhagic 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- 413 BACILLI WHICH PRODUCE SEPTICAEMIA 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-(J-, when they are inoculated with a small quantity of an attenuated cul- ture. In less susceptible animals — guinea-pigs, sheep, dogs, horses •.,-£.,"" .:./••;, ] ''" **£• »*.* I" FIG. 133.— Bacillus of Schweineseuche, 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. IN SUSCEPTIBLE ANIMALS. 413 According to Baumgarten, bacilli from Wildseuche or from Rinder- 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 septicjemia. 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 Coruil 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 diarrhoaa, 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 hsemorrhagicae) ; short rods with rounded ends, from 1 to 1.5 n 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- perature. In its growth 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, grayish 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 OF 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 (1S84) ; it was first obtained in pure cultures and its principal char- acters determined by Salmon and Smith (1885), and has since been 414 BACILLI WHICH PRODUCE SEPTICAEMIA 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 //in length and 0. 6 to 0. 7 n 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. — Anaerobic (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. IN SUSCEPTIBLE ANIMALS. 415 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 septicsemise hsemorrhagicse. 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 " frequently 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- 416 BACILLI WHICH PRODUCE SEPTICAEMIA 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 haemorrhagic 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 IN SUSCEPTIBLE ANIMALS. 417 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. 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 septicaemias 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° C., 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 and Schimmelbusch) ; Bacillus der Amerikanischen Binderseuche (Caneva) ; Bacillus of spontaneous rabbit septicaemia (Eberth). The recent 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 septicaemia 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 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 34 418 BACILLI WHICH PRODUCE SEPTICAEMIA 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 hsemorrhagica 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 : 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 Loffler-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. \Jponpotato it formed a thick, opaque, yellowish layer, while the growth of the hog-cholera bacillus was much thinner and that of the Loffler-Schiitz bacillus scarcely to be seen. In bouillon the Loffler-Schiitz bacillus, 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." IX SUSCEPTIBLE ANIMALS. 419 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 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 (Nos. 61 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 bad been manured. Morphology. — 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 septicae- miae hsemorrhagicae, but not so constantly and not so sharply denned. Biological Characters. — An aerobic, (non-liquefying f), 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 the growth consists of a thin layer which is not at all characteristic. 420 BACILLI WHICH PRODUCE SEPTICJ3MIA 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 heart 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, Schutz) ; Bacille du rouget du pore (Pasteur) ; Ba- cillus of mouse septicaemia; Bacillus murisepticus (Fliigge) ; 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 Schutz (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 resemble each other, apparently do not consider them identical and describe them separately. Baumgarten, on the other hand, de- scribes them under one heading and considers it highly probable that they are identical, although he also admits slight differences in the morpho- logical characters and growth in culture media. These differences are, however, no greater than FIG. 135.-Bacillus of , . .,. . ' , .°.. . mouse septicaemia in we have in artificially produced varieties or other leucocytes from blood well-known microorganisms, and we think it best of mouse. X 700. (Koch.) e 11 T» j •!_• .LI j 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 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 Schutz from the blood and various organs of swine which had succumbed to the infectious malady known in Germany as rothlauf and in France as rouget. Morphology. — Extremely minute bacilli, about 1 p 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 SUSCEPTIBLE ANIMALS. 421 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 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 ordinary aniline staining agents and also by Gram's method. Bioloqical Characters. — A FlQ- Ise.-Bacillus of rouget, from a pure ,. , f 7 . 7 . culture. X 1,000. From a photomicrograph. facultative anaerobic, non-hque- (Roux.) fying bacillus. According to Schottelius, the rothlauf bacilli are sometimes mo- ^utf^^M tile, but Fliigge states that other observers have oo not seen them in active motion. Frankel says they have the power of voluntary motion. Eisen- berg says that the bacillus of mouse septicaemia is motionless, and Frankel says " they seem to be in- capable of voluntary motion/' Baumgarten re- marks : "Whether the bacilli exhibit voluntary movements has not been determined/' Although this bacillus is not strictly anaerobic, it grows better in the absence of oxygen than in its pre- sence. Development occurs in various culture me- dia at the room temperature, but is more rapid in the culture oven. In gelatin stick cultures no development occurs upon the surface, but the growth along the line of puncture is very charac- teristic; this consists of a delicate, cloud-like, ra- diating 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; Fliigge compares it to the brush of bristles used for cleansing test tubes. In old cultures in nutrient gelatin a slight soften- ing of the gelatin occurs along the line of growth, and as a result of FIG. 137.— Bacillus of mouse septicaemia ; culture in nutrient gela- tin, end of four days at 18° C. (Baumgarten.) 422 BACILLI WHICH PRODUCE SEPTICAEMIA 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 developed in the course of two or three days in the deeper layers of the gelatin, but not upon the surface ; these are ne- bulous, grayish-blue, radiating masses, which are so delicate as to be scarcely visible 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 Fliigge 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 mia; single colony outset, and later a scanty, grayish- white deposit upon x 80Utr(Fmg|otm' tnebottom 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. PatTiogenesis. — 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 IN SUSCEPTIBLE ANIMALS. 423 usually inflamed and spotted with haemorrhagic extravasations ; the serous membranes also may be inflamed, and the cavities of the pleurae, pericardium, and peritoneum usually contain more or less fluid. The bacilli are found in the blood vessels throughout the 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 FIG. 139.— Section of diaphragm 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. 4:24 BACILLI WHICH PRODUCE SEPTICAEMIA The bacilli are found in the blood vessels generally, and are very 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 and by Schiitz ; but the last-named bacteriologists, and Schot- telius also, found the characteristic rothlauf bacillus in cultures from his laboratory which had been 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 occurrence of death 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 use 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 the 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 PARVUS. 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 micrococcus. 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 oedema is developed at the IN SUSCEPTIBLE ANIMALS. 425 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 identical with Bacterium coli commune of Escherich. Obtained by Brieger (1884) from human faeces. 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- berg). 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 CAVICIDA HAVANIENSIS. This bacillus was obtained by the writer from the contents of the intestine of a yel low-fever cadaver, in Havana, 1889, through inoculated guinea-pigs. Morphology. — A bacillus with rounded ends, from tiro to three ft long and about 0.7 ju 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 : 3 very scanty and thin, not extending far from tb 3 point of puncture ; along the line of puncture are developed small, translucent, pearl- like, sph irical colonies, which later become opaque and sometimes granular. In gelatin roll tubes, at the end of twenty- four hours at 22° C., FIG. 140.— Bacillus cavicida the deep colonies are very small spheres, of a pale Havaniensis; from a potato straw col )r ; later they become opaque, light brown culture, x 1,000. From a pho- spheres, or may have a dark central mass sur- tomicrograph. (Sternberg.) rounded by a transparent zone. The superficial colonies it the end of five days are small, translucent masses of a pale straw color tow ards the centre, with thin and irregular margins, sometimes with 35 426 BACILLI WHICH PRODUCE SEPTICAEMIA 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, arid 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, often bent or twisted — "sausage-shaped." Immediately after division the bacilli are about one-half longer than they are broad, but before dividing sC%z^*2»£ilJ?i5;r <*•'• »-H o.'-v:--; •>'••• r) ->vC:;vV;^ /r - •'•;{«Ci S^p^S-P^ %^v,.;:^,;;;:^-v^.-: "'?l^iift ?*•. %V<' 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 35° C." (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 IN SUSCEPTIBLE ANIMALS. 427 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. — Mice inoculated with a small quantity of a pure culture die from acute septicaemia in about forty-eight 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 /* long and 0.58 u 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, anu 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- fying, 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 capsule ; 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 15° C. 428 BACILLI WHICH PRODUCE SEPTICAEMIA FIG. 142,— Proteus hominis capsulatus, from liver of mouse. X 1,000. (Bordoni-Uffre duzzi.) This bacillus grows as well in an acid medium as in one which is slightly alkaline. In gelatin plates, at the end of eighteen to twenty-four hours, 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 of 15° to 17° C., the colonies may be as large as a pin's head and still remain spherical 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 ; m the vicinity of the point of inoculation is a subcutaneous oedema 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 IN SUSCEPTIBLE ANIMALS. 429 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 hasmorrhagic 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 characterized 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 of 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>ulong; 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 430 BACILLI WHICH PRODUCE SEPTICAEMIA room temperature — better in the incubating1 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 "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 ft 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. G-rows 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 along1 the line of puncture, and at the end of forty-eight hours a slight liquefaction, in form of a funnel, occurs near the IN SUSCEPTIBLE ANIMALS. 431 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° 0. , 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 cultures, 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 pleuras ; 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 n 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 and cover the sur- face ; an abundant white deposit is seen in the condensation water. Upoti 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 hyperaamic. 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 ia artificial media. 80. BACILLUS CAPSULATUS. 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 grow 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 fuchsin or gentian violet solu- 432 BACILLI WHICH PRODUCE SEPTICAEMIA FXG 143.— Bacillus capsulatus, from peritoneal exudate of an inoculated guinea-pig. X 1,000. From a photomicrograph. (Ffeiffer.) 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. Biological 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 porcQ- lain-white color. In gelatin stick cultures growth occurs to the bot- tom of the line of puncture, and on 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 HYDROPHILUS 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 ju in length ; often short oval; may grow out into filaments of 12 to 20 // in length. Biological Characters. — An aerobic, liquefying, motile bacillus. Grows in the usual culture media at the room temperature. In gelatin stick cul- IN SUSCEPTIBLE ANIMALS. 433 tures, at 18° to 203 C., liquefaction has already commenced along the line of puncture at the end of twelve hours, and at the end of thirty-six to forty- eight hours half of the gelatin is liquefied in funnel shape ; on the third or fourth day the gelatin is completely lique- fied, and a thick, white, flocculent deposit is seen at bottom of the tube. In glycerin- agar, at 37° C., a slight, bluish, diffuse fluorescence is seen upon the surface at the end of twelve hours, and soon after a luxu- riant growth, which soon covers the entire surface, is developed ; at the end of twenty- four to thirty-six hours large gas bubbles begin to form in the agar; gradually the fluorescence disappears, the surface growth becomes thicker and has a dirty -gray color which changes later to brownish. Blood serum is a favorable medium and is rapidly liquefied by this bacillus. Upon potato the growth is most characteristic. At the end of twelve hours a thin straw-yellow layer FlG 144._Baciiius hydrophiius fus- is developed along the impfstrich; this cus, in blood of triton. (Sanareiiu gradually becomes yellow, and at the end of four to five days has a brown color, resembling that of the glanders bacil- lus upon potato. Pathogenesis. — Pathogenic for frogs, toads, lizards, and oth "cold- ^^ blooded" animals; also for guinea-pigs, rabbits, dogs, cats, mice, chickens, and pigeons. When a few drops of a bouillon culture are injected into the muscles of the thigh, swelling and redness at the point of inoculation are quickly developed, and death usually occurs in eight to ten hours. The bacilli are found in great numbers in the blood and in all of the organs. Guinea pigs die from general infection within twelve hours after receiving a subcutaneous injection of a small amount of a pure cul- ture ; the spleen is enlarged and the liver and spleen hy- persemic ; an extensive inflammatory oedema in the vicin- ity of the inoculation wound is frequently observed; the bacilli are very numerous in the blood and in all the or- gans. Rabbits die in five to six hours from an intravenous injection. Adult dogs are immune, but new-born dogs (three to four days old) die infallibly, after receiving a subcutaneous injection of a small quantity of a pure cul- ture, in twelve to thirty -six hours. Young cats also suc- cumb to similar inoculations. Chickens and pigeons die within five to seven hours after receiving an intravenous injection, but resist subcutaneous injections. 82. BACILLUS TENUIS SPUTIGENUS. Obtained by Pansini (1890) from sputum. Morphology. Short bacilli, usually in pairs and sur- FIG. i45.-Baciiius rounded by a capsule, hydrophiius fuscus; Stains by Gram's method. culture in nutrient Biological Characters. — An aerobic, non-liquefying, gelatin, end of six- non- motile bacillus. Grows in nutrient gelatin at the teen hours. (Sana- room temperature. Develops abundantly on potato. reiii.) Coagulates milk and produces an acid reaction in this medium. Pathogenesis. —Pathogenic for rabbits and white rats; not for guinea- pigs or for Avhite mice (in small doses). 36 434 BACILLI WHICH PRODUCE SEPTICAEMIA 83. BACILLUS OF LASER. Obtained by Laser (1892) from mice which succumbed to an epidemic dis- ease in Frankel's laboratory at Konigsberg. In its characters this bacillus closely resembles the bacillus of swine plague (No. 65), and is perhaps identical with it. Morphology. — A small bacillus, with rounded ends, about twice as long as broad. Has flagella both at the extremities and sides. Stains by the usual aniline colors and also by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, actively motile bacillus. Spore formation not observed. Grows either in the incubating oven or at the room temperature. Thermal death - point 65° to 70° C. — ten minutes' exposure. Upon gelatin plates, at the end oftwo days, the deep colonies are spherical, finely granular, and brownish in color; the superficial are transparent, fiuely granular, and leaf -like. In gelatin stick cultures growth occurs along the entire line of puncture as well as upon the surface. At the end of three days a considerable evolution of gas is usually observed. In agar an abundant development is seen at the end of twenty- four hours in the incubating oven; upon the surface a gray- ish-white, shining layer with dentate margins is formed along the track of the needle. In bouillon, at 37D C., development is abundant and rapid; a thin film is formed on the surface at the end of the second day. Upon potato a brownish layer is formed at the end of twenty-four hours. In milk an acid reaction is produced. Pathogenesis. — Pathogenic for field mice, guinea-pigs, rabbits, and pigeons. The bacillus is found in the blood and various organs of infected mice. The spleen is found to be greatly enlarged. 84. BACILLUS TYPHI MURIUM (Loffler). Obtained by Loffler (1889) from mice which died in his laboratory from an epidemic disease due to this bacillus. Morphology. — Short bacilli, resembling the bacillus of diphtheria in pigeons, and varying considerably in dimensions— like the bacillus of typhoid fever; grows out into flexible filaments. Stains with the aniline colors — best with Loffler's solution of methylene blue. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, motile bacillus. Spore formation not determined. Has flagella around the periphery of the cells, like those of the typhoid bacillus, and ex- hibits similar active movements. In gelatin stick cultures, at the room temperature, growth occurs upon the surface, at the end of forty-eight hours, in the form of a flat, grayish- white, round, semi-transparent mass the size of a pin's head ; later the surface colony increases in extent and has more or less irregular margins. In gelatin plate cultures the deep colonies are at first round, slightly granular, transparent, and grayish; later they are of a yellowish-brown color arid decidedly granular. The superficial colonies are very granular and marked by delicate lines — similar to colonies of the typhoid bacillus. Upon agar a grayish- white layer is developed which is not at all characteristic. Upon potato a rather thin, whitish layer is formed, and around this the potato acquires a dirty bluish-gray color. In milk an abundant development occurs, and a decidedly acid reaction is produced withoiit causing any perceptible change in the appearance of the fluid. Pathogenesis. — Pathogenic for white mice, which die in from one to two weeks after infection ; also to field mice, which succumb to subcutaneous in- jections of a pure culture, and also, in from eight to twelve days, when fed upon potato cultures or bread moistened with a small quantity of a bouillon culture. Loffler believes that this bacillus may be used for the destruction of field mice in grain fields, inasmuch as they invariably die after ingesting food which has been contaminated with it, and also from eating the bodies IN SUSCEPTIBLE ANIMALS. 435 of other mice which have died as a result of infection. House mice are also susceptible. Rabbits, guineapigs, pigeons, and chickens were found by Loffler not to be susceptible to infection by feeding. 85. BACILLUS OP CAZAL AND VAILLARD. Obtained by Cazal and Vaillard (1891) from cheesy nodules upon the peritoneum and in the pancreas of an individual who died in the hospital at Val de Grace. Morphology. — Bacilli with rounded ends, but little longer than they are broad ; solitary, in pairs, or in chains of ten to fifteen or more elements. Stains with the usual aniline colors, but not by Gram's method ; the extremities of the rods are more deeply stained than the central portion — " polar staining." Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile bacillus. Does not form spores. Grows in the usual culture media at the room temperature — more rapidly in the incubating oyen at 37° C. In gelatin stick cultures, at the end of twenty-four hours, a series of puncti- form, white colonies is developed along the line of puncture ; upon the sur- face development is more abundant, and at the end of forty-eight hours liquefaction commences ; this progresses slowly from above downward, and a white, flocculent deposit accumulates at the bottom of the liquefied gelatin. Upon the surface of agar, at the end of twenty-four hours at 37° ., a moist, transparent, opalescent layer is developed, which rapidly ex- tends over the entire surface ; later this layer becomes somewhat thicker, whitish, and cream- like in consistence, without losing its transparency. Upon potato a thick, prominent, moist, and slightly viscid layer is devel- oped, which at first has a pale-yellow and later a yellowish-brown color. In bouillon development is abundant, producing a milky opacity of the liquid; a thick, flocculent deposit accumulates at the bottom of the tube ; the reaction of the culture liquid becomes very alkaline. All of the cultures give off a peculiar odor, slightly ammoniacal and resembling that of putrid urine. The cultures retain their vitality for several months — in a closed tube for more than a year. The thermal death-point is 60° C. with fifteen minutes' exposure. Pathogenesis. — Pathogenic for rabbits and mice, but not for guinea-pigs. In mice death occurs from general infection, at the end of forty -eight to sixty hours, from the subcutaneous injection of one eighth cubic centimetre of a recent bouillon culture. In rabbits injection of one cubic centimetre into the circulation causes the death of the animal in thirty-six to fifty hours. The symptoms induced are a foetid diarrhoea and paralysis of the extremities. When smaller doses are injected (0.5 cubic centimetre) a chronic malady is developed, characterized at the outset by diarrhoea and emaciation, then by the development of tumors which resemble those found in the man from whom the cultures were first obtained. These tumors are for the most part located in the subcutaneous connective tissue ; after a time they attain the size of a chestnut and ulcerate, allowing the escape of a semi-fluid, purulent material. The animals usually recover. Similar tumors are developed as a result of subcutaneous injections of one to three cubic centimetres of a recent bouillon culture. 86. BACILLUS OF BABES AND OPRESCU. Obtained by Babes and Oprescu (1891) from a case of septicaemia haemor- rhagica presenting some resemblance to exanthematic typhus. Morphology. — In agar cultures the bacilli are from 0. 4 to 0. 5 ju. thick, and are frequently united in pairs ; associated with these rod -shaped bacteria are forms which are of a short oval. In gelatin cultures oval forms are more numerous ; they have a diameter of 0.3 to 0.4 ju, and often appear to be surrounded by a capsule. In fresh cultures the bacilli are often in form of 43G BACILLI WHICH PRODUCE SEPTICAEMIA 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, very 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 — ;i 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 n long, usually in pairs. Stains with the usual aniline colors, but not by Gram's method. Biological Characters. — An aerobic an d facultative anaerobic, non-lique- fying, non-motile bacillus. 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 RNBERG'S BACTERIOLOGY. Plate VII. V* Y> l $VS^ W&K .2. Fig. I. Fig 3. BACILLUS OF GLANDERS (LOEFFLER) IN SUSCEPTIBLE ANIMALS. 437 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 OP 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 ; Pfeiff er's bacillus is pathogenic for these animals. PLATE VII. BACILLUS OF GLANDERS. FlG. 1. — Bacillus mallei from the liver of a field mouse, cover-glass pre- paration. (Loftier.) 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. (Loffler.) 37 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 subcutaneously, 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 of the colon bacillus, are reported to have produced fatal septicaemia in guinea-pigs when injected subcutaneously 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 PATHOGENC AEROBIC BACILLI NOT BEFORE DESCRIBED. 439 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 fort}T-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). 440 PATHOGENIC AEROBIC BACILLI 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 faeces of healthy children ; since shown to be constantly 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 /* broad ; but under certain cir- . cumstances the length does not exceed the breadth — » . about 0.5 j.i — and it might be mistaken for a micrococ- i'> cus ; again the prevailing form in a culture is a short ^ oval ; filaments of five jj. or more in length are often FIG. 146. — Ba- observed in cultures, associated with short rods or oval C°x i°ooo~ cells. The bacilli are frequently united in pairs. The (Escherich.) presence of spores has not been demonstrated. In un- 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 NOT DESCRIBED IN PREVIOUS SECTIONS. 441 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 vary 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 to wards 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 Nvith 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 442 PATHOGENIC AEROBIC BACILLI 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 FIQ. 147. FIG. 148. FIG. 147. — Bacillus coli communis in nutrient gelatin containing twenty per cent of gelatin, end of two weeks, 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 six 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. 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 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 NOT DESCRIBED IX PREVIOUS SECTIONS. 443 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 purictiform ecchymoses (Weisser). According to Escherich, the spleen is often somewhat enlarged. The small intestine is hyper- aemic, 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. 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." These are described as follows : BACILLUS d OF BOOKER. ' ' Found in two cases of cholera inf antum and the predominating1 form in one serious case of catarrhal enteritis. " Morphology. — Resembles Bacterium coli commune. ' ' Growth 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 differ 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 borders. 444 PATHOGENIC AEROBIC BACILLI " Action on Milk. — Coagulated into a gelatinous coagulum in twenty- four hours at 38° C., 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 pink on top. The pink color extends in a few days about one-half down the clot. " 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 surf ace 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. NOT DESCRIBED IX PREVIOUS SECTIONS. 445 "Agar: The colonies are large, round, and bluish- white. Slightly magni- fied, a light-yellow color. ' ' 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. " Potato: Bright-yellow, glistening, moist surface with well-defined bor- ders, and but slightly raised above the surrounding 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 l>een 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 spoi-e 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 more active 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 litnms 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. ''Relation to Gelatin. — Does not liquefy gelatin." 44(> PATHOGENIC AEROBIC BACILLI BACILLUS h 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 exten'sively. 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 H 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 commune. " 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. NOT DESCRIBED IN PREVIOUS SECTIONS. 447 " 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 causs 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. Synonym — Bacillus lactis aerogenes (Escherich). 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. Morphology. — Short rods with rounded ends, from 1 to ^ 2 n in length and from 0.1 to 0.5 V- broad; short oval and e£t %/ spherical forms are also frequently observed, and, under 99 certain circumstances, longer rods — 3 fi — may be developed : $"* / g usually united in pairs, and occasionally in chains contain- ing several elements. In some of the larger cells Escherich lias observed unstained spaces, but was not able to obtain FlG< any evidence that these represent spores. This bacillus stains readily with the ordinary aniline Si*-*?1 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, shining masses which, examined under the microscope, are found to be homogeneous 448 PATHOGENIC AEROBIC BACILLI 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 afflicted 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 fasces. 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 infaiitum. 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 conimunis and Bacillus lactis aerogenes of Escherich. He says: " 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 NOT DESCRIBED IN PREVIOUS SECTIONS. 449 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. " 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 FIG. 150. FIG. 151. Fi». 150.— Bacillus acidiformans, from a potato culture. X 1,000. From a photomicrograph* (Sternberg.) FIG. 151.— Culture of Bacillus acidiformans in nutrient gelatin, end ofjfour days at 22° C. From a photograph. (Sternberg.) liver preserved in the same way from two comparative autopsies — i.e., not cases of yellow fever. Morphology. — A short bacillus with rounded corners, sometimes short oval in form ; from li to 3 y" in length and about 1.2 # in breadth ; may grow out into filaments of 5 to 10 jj. , 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- liquefying, non-motile bacillus. Does not form spores. 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. It 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 450 PATHOGENIC AEROBIC BACILLI 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 HAVAXIENSIS. 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 x, Havana, 1889. Morphology. — Thisbacillusresembles the colon bacillus in form, but is some- what larger, from 2 to 4 n 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 m .and facultative anaerobic, non-lique- ' 'fying bacillus. Under certain circum- stances may exhibit active movements, but is usually motionless. A very curious thing with reference 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 FIG. 152.— Bacillus cuniculicida Havani- ensis, from a single colony in nutrient gela- tin, x 1,000. From a photomicrograph. (Sternberg.) NOT DESCRIBED IX PREVIOUS SECTIONS. 451 •chai-acters. 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 flagellum. 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. 153. FIG. 154. Fig. 153.— Bacillus cuniculicida Havaniensis; colonies in gelatin roll tube, third day at 20° C. X 6. From a photograph. (Sternberg.) FIG. 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- parent margins and 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° 0. (68° F.), but more rapidly and luxuriantly at a higher temperature — 30° to 35° C. It grows well in agar cultures, and especially in glycerin-agar, in which it produces some gas and an acid reaction. The growth on the surface of glycerin-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. 453 PATHOGENIC AEROBIC BACILLI 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 owing1 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 — t\vo 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 the 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 NOT DESCRIBED IX PREVIOUS SECTIONS. 453 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 ft in length and about 0.5 H 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 centi'al 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 cultui-e 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 in blood serum. Pathogenesis. — 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 injected 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 38 454 PATHOGENIC AEROBIC BACILLI 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 resting 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 cubic 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 the 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 PYOCYANUS. Synonyms. — Bacillus of green pus ; Microbe du pus bleu; Bacil- len des griinblauen 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- 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- NOT DESCRIBED IN PREVIOUS SECTIONS. 455 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 — PATHOGENIC AEROBIC BACILLI 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 pyocyanus. 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 the Bacillus pyocyanus, made soon after infection with the anthrax bacillus. 96. BACILLUS OP FIOCCA. Found by Fiocca in the saliva of cats and dogs. Closely resembles the influenza bacillus of Pfeiffer and of Canon. Morphology. — Resembles the bacillus of rabbit septicaemia, but is only half as large — from 0.2 to 0.33 /* 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 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 NOT DESCRIBED IN PREVIOUS SECTIONS. 457 these colonies are a little larger and less transparent; they remain distinct, especially along1 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 tne 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 Fia. 156.— Proteus vulgaris; " swarming islands " from a gelatin culture, x 285. (Kauser.y 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 " Bacterium termo," which was applied to any minute motile bacilli found in putrefying infusions. Morphology. — Bacilli with rounded ends, about 0.6 /u broad, and 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 /u 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- 458 PATHOGENIC AEROBIC BACILLI 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. — An aerobic 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 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 NOT DESCRIBED IN PREVIOUS SECTIONS. 459 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 Vulgaris 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 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." Another bacillus found by Booker in a considerable number of his cases he has designated by the letter A (No. 103). •±60 PATHOGENIC AEROBIC BACILLI 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 and 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 have 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 clefts ; 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 aboutone 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 n 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 and 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 NOT DESCRIBED IN PREVIOUS SECTIONS. 401 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 varkms 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 FIG. 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. 157.— Spiral zoogloea 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. 39 463 PATHOGENIC AEROBIC BACILLI 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 toxemia. 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. Gi'adually 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 animals 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 /j. broad and varying greatly in length; slightly 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 contents 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 ju. thick ; often swollen in the middle, like a lemon or a flask ; forms short, flexible filaments which also present similar swellings. f Stains with the usual aniline colors and also by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, 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 NOT DESCRIBED IN PREVIOUS SECTIONS. 463 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 cigar 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 cedema is found at the point of inoculation. Injections into the rectum of rabbits gave rise to hsemorrhagic enteritis, peritonitis, and death at the end of four days. 103. BACILLUS A OF BOOKER. Obtained by Booker (1889) from the al vine discharges of children suffer- ing from cholera infantum. Morphology. — Bacilli with round ends, varying greatly in length, usually three to four n long and 0.7 ju 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 three 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 gi'adually 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-liquefying, 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 the 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 464 PATHOGENIC AEROBIC BACILLI 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. — Anaerobic, 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. 10G. 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 ju long and 0.75 to 1 H 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. NOT DESCRIBED IN PREVIOUS SECTIONS. 405 107. 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 jj. 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 formedin from forty-eight to sixty hours, which 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 oedema (Klein). Obtained from garden earth by inoculation in guinea-pigs. Morphology. — Bacilli from 0.8 to 2.4 /win length and 0.7 u thick; grow out into long filaments. Stains with the usual aniline colors, but not by Gram's method. Biological Characters. — 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 cedema is produced by subcutaneous inoculations ; the spleen is but slightly enlarged. In rabbits but slight oedema 466 PATHOGENIC AEROBIC BACILLI 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 OF 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- liquefy ing 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 OZJENM. 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 NOT DESCRIBED IN PREVIOUS SECTIONS. 467 somewhat irregular outlines; later liquefaction commences and crater-like depressions in the gelatin ai'e 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 2 n 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 days; 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 OP 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 /* 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- 408 PATHOGENIC AEROBIC BACILLI 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. Iri 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 pleura! 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 Loftier (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 through 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 Schweinerothlauf (rouget). NOT DESCRIBED IN PREVIOUS SECTIONS 469 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. \Jponpotato 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 toxaemia 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 closes 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 haemorrhagic 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 of 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 /* long; solitary, in pairs, or in short chains. 40 470 PATHOGENIC AEROBIC BACILLI 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 li^ht-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 whooping1 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 an in- fectious form of pneumonia. Morphology. — Slender, short bacilli, which rather resemble micrococci when cultivated in gelatin. Stains with the usual aniline colors. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, non-motile bacillus. Spore formation not observed ; is killed by ex- posure 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 tissue 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. 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 f uchsin and Lofner's solution of inethyleiie 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 defined 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 colonies, more or less crowded above, and often isolated below, where by NOT DESCRIBED IN PREVIOUS SECTIONS. 471 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 ai'e 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 GINGIVJE PYOGENES. Synonym. — Bacterium gingivse ^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 paii-s. 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 and suppuration. 124. BACILLUS PULP.E PYOGENES. Obtained by Miller from .gangrenous tooth pulp. 472 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 KERATOMALACOS. 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 Loffler'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 along 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 and in the various organs. 127. BACILLUS SEPTICUS ULCERIS GANGRJENOSI. Obtained by Babes (1889) from the blood and various organs of a boy who 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. NOT DESCRIBED IN PREVIOUS SECTIONS. 473 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 ju long and one « 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. 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. ~Upon 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 ft broad; also grow out into filaments of six ft or longer. Stains with the usual aniline colors andby Gram's method. Biological Characters.— An aerobic, 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- 474 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 conjunctiva! sac of man. Morphology. — Large bacilli with round ends, from two to eight n long and about one n 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 87° 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 micleus 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 contovir. 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 highly 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 IN PREVIOUS SECTIONS. 475 thin, moist, and transparent in appearance ; later they have a grayish color, a coarsely granular surface, and are made up of Hap-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 impfstrich ; 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 VESICLE. Obtained by Clado (1887) from the urine of a person suffering from cys- titis. Morphology. — Bacilli with round ends, 1.6 to 2 /J. long and 0.5 /^ 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 poAver 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 impfstrich. 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 470 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 /j. 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 " nail-shaped " growth similar to that of Fried- lander's bacillus (Bacillus pneumonias) 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 u. long and 0.8 to 1 /* 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 bacil- 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 n long and 1. 5 /z thick, NOT DESCRIBED IN PREVIOUS SECTIONS. 477 often united in chains of six to eight elements. The cells are surrounded by a transparent capsule resembling that of Friedlaiider'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 conjunctival 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 eye 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 " Zwischendeckenfullung " ; also by Gessner from the contents of the small intestine in man. Morphology. — Bacilli with round ends, 1.25 to 1.5 ju. long and 0.75 to 1 ju. 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 inflammatory osdenia, resulting in the death of the animals. 140. BACILLUS ALVEI. Synonym. — Bacillus of foul brood (of bees). Obtained by Cheshire and Cheyne (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 // in length (aver- age about 3.6 fi) 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- 478 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 cultures, 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 oven at 37°, and does not develop at temperatures below 16 3 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 injectea beneath the skin of mice or rabbits produce no appa- rent result. 141. BACILLUS OF ACNE CONTAGIOSA OF HORSES. Obtained by Dieckerhoff and Grawitz (1885) from pus and dried scales from the pustules of ' ' acne contagiosa " of horses. Morphology. — Short rods, straight or slightly bent, 0.2 jj. in diameter. Stains best with an aqueous solution of fuchsin, and also by Gram's method ; does not stain well with Loffler's alkaline solution of methylene blue. Biological Characters. — An aerobic, non-liquefying bacillus. In gelatin stick cultures a very scantv growth occurs along the line of puncture ; upon the surface a white mass forms about the point of puncture. Upon blbod 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 toxaemia. Subcutaneous injections in guinea-pigs caused toxaemia and death at theend of twenty-four hours. At the autopsy a haemorrhagic 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, from which the animal usually recovered. Subcutaneous injections in rabbits sometimes caused a fatal toxtemia. House mice, field mice, and white mice were not affected by the application of cultures, by NOT DESCRIBED IN PREVIOUS SECTIONS. 479 rubbing, to the uninjured skin, but succumbed to subcutaneous injections in twenty-four hours or between the flfth and tenth days. Those which died at a late date presented the pathological appearances which characterize pyaemia. 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 fj, 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 colon 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 ; thev 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 xisual 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 480 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 Brieger, and from Emmerich's bacillus which it greatly resembles, by the fact that it does not grow upon potato. 145. BACILLUS OP 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 )J- long and 0.2 to 0.4 ju 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. TJpon. 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 subcutaneously. 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 HSEMORRHAGICA 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 M 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. 481 Pathogenesis. — Inoculations in the conjunctivas of rabbits produce ecchy- moses of the conjunctiva. At the autopsy numerous haemorrhagic 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.EMORRHAGICA 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 fever, purpura, and albuminous urine. Morphology. — Oval bacilli, usually in pairs, 08 to 1.5/f long and O.S/* 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 in 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 patho- genic 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 u long; in anaerobic cultures from eight to twenty u. Stains with the usual aniline colors. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, slightly motile bacillus. Spore formation n.ot 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. XIV. PATHOGENIC ANAEROBIC BACILLI. STRICTLY anaerobic bacilli are not able to multiply in the blood of living animals ; but some of them may multiply in the subcuta- neous connective tissue or in the muscles, when introduced by in- oculation, and are pathogenic because of the local inflammatory or necrotic processes to which they give rise, or because they produce soluble toxic substances which are absorbed and cause death by their special action upon the nervous system or by general toxaemia. 149. BACILLUS TETANI. Synonyms. — The bacillus of tetanus ; Tetanusbacillus, Ger. Nicolaier (1884) produced tetanus in mice and rabbits by intro- ducing garden earth beneath their skin, and showed that the disease might be transmitted to other animals by inoculations with pus or cultures in blood serum containing the tetanus bacillus, which, how- ever, he did not succeed in obtaining in pure cultures. Carle and Rattone (1884) showed that tetanus is an infectious disease, which may be transmitted by inoculation from man to lower animals — a fact which has since been verified by the experiments of Rosenbach and others. Obtained in pure cultures by Kitasato (1889). The writer produced tetanus in a rabbit in 1880 by injecting be- neath its skin a little mud from the street gutters in New Orleans. The tetanus bacillus appears to be a widely distributed microorgan- ism in the superficial layers of the soil in temperate and especially in tropical regions. In Nicolaier 's experiments it was not found in soil from forests or in the deeper layers of garden earth. 'Morphology. — Slender, straight bacilli, with rounded ends, which may grow out into long filaments. Spores are developed at one extremity of the bacilli, which are spherical in form and consid- erably greater in diameter than the rods themselves, giving the spore-bearing bacilli the shape of a pin. Stains with the usual aniline colors and also by Gram's method. The method of Ziehl may be employed for double-staining bacilli and spores. PATHOGENIC ANAEROBIC BACILLI. 483 Biological Characters. — An anaerobic, liquefying, motile bacillus. Forms spores. Grows at the room temperature, in the absence of oxygen, in the usual culture media. Grows best at a temperature of 36° to 38° C.; in nutrient gelatin, at 20° to 25° C., development is first seen at the end of three or four days ; does not grow at a temperature below 14° C. Spores are formed in cultures kept in the incubating oven at 36° C. , at the end of thirty hours ; in gelatin cultures at 20° to 25° C., at the end of a week (Kitasato). The bacilli exhibit voluntary movements which are not very active ; those containing spores are not motile. It may be cultivated in an atmosphere of hydrogen, but does not grow in the presence of oxy- gen— strictly anaerobic — or in an atmosphere of carbon dioxide. The addition of one and one-half to two per cent of grape sugar to nutrient agar or gelatin causes the development to be more rapid FIG. 159. FIG. 160. FIG. 159.— Tetanus bacillus, from a gelatin culture, x 1,000. From a photomicrograph by Pfeiffer. FIG. 160. — Tetanusbacillus, from an agar culture ; spore-bearing rods, x 1,000. From a photo- micrograph by Pfeiffer. and abundant. The culture medium should have a feebly alkaline reaction. Colonies in gelatin plates, in an atmosphere of hydrogen, re- semble somewhat colonies of Bacillus subtilis, the opaque central portion being surrounded by a circle of diverging rays ; liquefaction is, however, much slower, and the resemblance is lost after a short time. Older colonies resemble the colonies of certain microscopic fungi, being made up of diverging rays. In long gelatin stick cul- tures development occurs along the line of puncture, at a consid- erable distance below the surface, in the form of a radiate out- growth ; the gelatin is slowly liquefied, and a small amount of gas is at the same time formed. In peptonized bouillon having a slightly alkaline reaction, under hydrogen gas, the development is abundant 484 PATHOGENIC ANAEROBIC BACILLI. and the cultures give off a characteristic odor — " brenzlichen Ge- ruch " (Kitasato). According to Kitasato, blood serum is not a very favorable me- dium for the growth of the tetanus bacillus, and — contrary to the statement of Kitt, Tizzoni, and others — it does not cause liquefaction of this medium. The spores of the tetanus bacillus re- tain their vitality for months in a desic- cated condition, and are not destroyed in two and one-half months when present in putrefying material (Turco). They withstand a temperature of 80° C. main- tained for an hour, but are killed by five minutes' exposure to steam at 100° C. They are not destroyed in ten hours by a five-per-cent solution of carbolic acid, but did not grow after fifteen hours' ex- posure in the same solution. A five- per-cent solution of carbolic acid, to which 0. 5 per cent of hydrochloric acid has been added, destroys them in two hours ; in sublimate solution containing 1 : 1,000 of mercuric chloride they are destroyed at the end of three hours, or in thirty minutes when 0.5 per cent of hydrochloric acid is added to the solu- tion. Kitasato succeeded in obtaining pure cultures from the pus formed in the vicinity of inoculation wounds, by destroying the associated bacilli after the tetanus bacilli had formed spores. This was effected by heating cultures from this source for about an hour at a temperature of 80° C. The spores of the tetanus bacillus survived this exposure, and colonies were obtained from them in flat flasks especially devised for anaerobic cultures ; from these colonies pure cultures in nutrient agar or gelatin — long stick cultures — or in peptonized bouillpn were easily obtained. Brieger (188G) first succeeded in obtaining from impure cultures of the tetanus bacillus a crystallizable toxic substance, called by him tetanin, which was found to kill small animals in very minute doses and with the characteristic symptoms of tetanus. More recently Kitasato and Weyl have obtained the same substance, by following Briegers method, from a pure culture of this bacillus. From a FIG. 161.— Culture of Bacillus tetanl in nutrient gelatin. (Kitasato.) PATHOGENIC ANAEROBIC BACILLI. 485 bouillon made from one and one-fourth kilogrammes of lean beef, with the addition of twenty-five grammes of peptone, they obtained 1.7118 grammes of hydrochlorate of tetanin. This proved fatal to white mice in six hours in the dose of 0.05 gramme, and a dose of 0.105 gramme caused characteristic tetanic convulsions and death within an hour. The bacteriologists last named also obtained from their cultures the tetanotoxin of Brieger. Two mice were inoculated sub- cutaneously with 0.003 gramme of this substance ; one died at the end of five hours without the development of tetanic symptoms ; the other survived. In addition to these substances, indol, phenol, and butyric acid were demonstrated to be present in cultures of the tetanus bacillus. According to Kitasato, the tetanus bacillus does not become at- tenuated in its pathogenic potency by cultivation in artificial media, as is the case with many other pathogenic bacteria. The more recent researches of Brieger and Frankel, and of Kitasato, show that the toxic ptomaine discovered by Brieger in 1886 is not the substance to which cultures of the tetanus bacillus owe their great and pecu- liar pathogenic power. The distinguished German chemist and his associate have succeeded in isolating from tetanus cultures a toxal- bumin which is far more deadly than tetanin. Pathogenesis. — The experiments of Kitasato (1889) show that pure cultures of the tetanus bacillus injected into mice, rabbits, or guinea-pigs produce typical tetanic symptoms and death. As the presence of this bacillus at the seat of injury, in cases of tetanus in man, has now been demonstrated by numerous observers, there is no longer any question that tetanus must be included among the traumatic infectious diseases, and that the bacillus of Mcolaier and of Kitasato is the specific infectious agent. Kitasato's recently pub- lished experiments (1890) show that cultures of the tetanus bacillus which have been sterilized by filtration through porcelain produce the same symptoms, and death, in the animals mentioned, as result from inoculation with cultures containing the bacillus. It is evi- dent, therefore, that death results from the action of a toxic sub- stance produced by the bacillus. This is further shown by the fact that the bacillus itself cannot be obtained in cultures from the blood or organs of an animal which has succumbed to an experimental in- oculation with an unfiltered culture ; but the blood of an animal killed by such an inoculation contains the tetanus poison, and when injected into a mouse causes its death with tetanic symptoms. When a platinum needle is dipped into a pure culture of the tetanus bacillus and a mouse is inoculated with it subcutaneously, the animal invariably falls sick within twenty-four hours and dies of typical tetanus in two or three days. Rats, guinea-pigs, and rabbits 41 486 PATHOGENIC ANAEROBIC BACILLI. are killed in the same way by somewhat larger quantities — 0.3 to 0.5 cubic centimetre (Kitasato). Pigeons are very slightly susceptible. The tetanic symptoms are first developed in the vicinity of the point of inoculation ; if the animal is inoculated in the posterior portion of the body the hind legs first show tetanic contraction, if in the fore part of the body the muscles of the neck are first affected. At the autopsy there is a certain amount of hyperaemia at the point of in- oculation, but no pus is formed ; in inoculations with garden earth, or accidental inoculations in man, pus is commonly found in the vicinity of the inoculation wound. The various organs are normal in appearance. Kitasato says that he has not been able to demon- strate the presence of the bacillus or of spores in the spinal marrow, the nerves, muscles, spleen, liver, lungs, kidneys, or blood from the heart ; nor has he been able to obtain cultures from the various organs. In mice which were inoculated at the root of the tail Kitasato was able to demonstrate the presence of the bacilli at the point of inoculation by the microscopical examination of an excised piece of the tissues for eight to ten hours after the inocula- tion ; later than this they were not found. In pus from the inocu- lation wounds of men and animals accidentally infected the bacilli are present, but the formation of spores does not always oc- cur. According to Kitasato, the sooner death has occurred after accidental inoculation the less likely are spores to be found in the rods, but from pus in which no spores are seen cultures of the bacillus may be obtained in which spores will develop in the usual manner. Guinea-pigs are even more susceptible to the tetanus poison than mice, and rabbits less so. The amount of filtrate from a slightly alkaline bouillon culture required to kill a mouse is extremely minute — 0.00001 cubic centimetre (Kitasato). The tetanic symptoms are de- veloped within three days ; if the animal is not affected within four days it escapes entirely. The tetanus poison is destroyed by a tem- perature of 65° C. maintained for five minutes, or 60° for twenty minutes, or 55° for an hour and a half ; in the incubating oven at 37° C. it gradually loses its toxic potency ; in diffuse daylight, also, its toxic power is gradually lost ; in a cool, dark place it retains its original potency indefinitely ; in direct sunlight it is completely de- stroyed in from fifteen to eighteen hours ; it is not injured by being largely diluted with distilled water ; it is destroyed in an hour by hydrochloric acid in the proportion of 0. 55 per cent ; terchloride of iodine destroys it in the proportion of 0.5 per cent, cresol in 1 per cent — one hour's exposure. In general it is destroyed by acids and by alkalies. Blood serum from cattle, horses, sheep, rabbits, rats, or guinea-pigs does not modify its toxic properties. PATHOGENIC ANAEROBIC BACILLI. 487 Recent researches by Tizzoni and Cattani show that tetanus spores preserved upon silk threads become attenuated after a time when preserved in a dark place in free contact with the air. "Very virulent cultures liquefy gelatin, give off a very disagreeable odor, and have a decidedly alkaline reaction. Less virulent cultures quickly acquire an acid reaction. Cultures of which the virulence is very much attenuated grow more rapidly and abundantly than virulent cultures and produce more gas — in hydrogen at 37° C. ; they do not liquefy gelatin and have no odor. In attenuated cultures de- generation forms are often seen, and the spores are frequently elon- gated or almost rod-shaped. Cultures preserved in various gases for thirteen to fourteen months invariably become attenuated. Immunity. — Kitasato was not able to produce immunity in mice by inoculations with minute doses of the poison, or with a filtrate which had been exposed to various degrees of temperature by which its activity was diminished or destroyed. But immunity lasting for about two months was produced in rabbits by inoculating them "with the filtrate from a culture of the tetanus bacillus and subse- quently, in the same locality, with three cubic centimetres of a one- per-cent solution of terchloride of iodine ; this last solution was in- jected subcutaneously in the same dose at intervals of twenty-four hours for five days. Of fifteen rabbits treated in this way six proved to be immune against large doses of a virulent culture of the tetanus bacillus. The same treatment was not successful in producing im- munity in mice or guinea-pigs, but the important discovery was made that a small quantity of blood (0.2 cubic centimetre) from an immune rabbit, when injected into the abdominal cavity of a mouse, gave it immunity from the effects of inoculations with the tetanus bacillus. Moreover, mice which were first inoculated with a virulent culture of the bacillus, and, after tetanic symptoms had appeared, re- ceived in the cavity of the abdomen an injection of blood serum from an immune mouse, were preserved from death. The power of the blood of an immune animal to neutralize the tetanus poison was fur~ ther shown by mixing the filtrate from a virulent culture with blood serum from an immune animal and allowing it to stand for twenty- four hours ; a dose three hundred times greater than would have sufficed to kill a mouse proved to be without effect after such admix- ture with blood serum — as before stated, the blood serum of animals which are not immune has no effect upon the poison. The duration of immunity induced in this way was from forty to fifty days. Blood serum from an immune rabbit, preserved in a cool, dark room, retains its power of neutralizing the tetanus poison for about a week, after which time it gradually loses it. Having found that chickens have a natural immunity against tetanus, Kitasato made experiments 488 PATHOGENIC ANAEROBIC BACILLI. to ascertain whether their blood serum would also neutralize the tetanus poison; the result was negative. That the tetanus poison is present in the blood of individuals who die from tetanus has been proved by Kitasato by injecting a small quantity (0.2 to 0.3 cubic centimetre) of blood from the heart of a fresh cadaver into mice; the animals develop typical tetanic symp- toms and die in from twenty hours to three days. Tizzoni and Cattani have recently (1891) reported results similar to those obtained by Kitasato. By repeated inoculations with grad- ually increasing doses of the tetanus poison they succeeded in mak- ing a dog and two pigeons immune, and found that blood serum from this immune dog, in very small amount, completely destroyed the toxic power of a filtrate from cultures of the tetanus bacillus — one to two drops of serum neutralized 0. 5 cubic centimetre of filtrate after fifteen to twenty minutes' contact. They also ascertained that small amounts of blood serum from this immune dog injected into other dogs or white mice produced immunity in these animals ; but they were not able to produce immunity in guinea-pigs or rabbits by the same method. In a later communication (May, 1891) Tizzoni and Cattani give an account of their experiments made with a view to determining the nature of the substance in the blood serum of an immune animal which has the power of destroying the toxalbumin of tetanus — " tet- anus antitoxin/* They found, in the first place, that this antitoxin in blood serum is destroyed in half an hour by a temperature of 68° C. ; further, that it does not pass through a dialyzing membrane ; that it is destroyed by acids and alkalies. As a result of their re- searches they conclude that it is an albuminous substance having the nature of an enzyme. Vaillard has succeeded in producing immunity in rabbits by re- peated injections into the circulation of filtered cultures — in all twenty cubic centimetres — which had been exposed for one hour to a temperature of 60° C. At a temperature of 65° C. both the toxic and the immunizing action is destroyed. 150. BACILLUS CEDEMATIS MALIGNI. Synonyms. — Bacillus of malignant oedema; Vibrion septique (Pasteur). Discovered by Pasteur (1877); carefully studied by Koch (1881). This bacillus is widely distributed, being found in the superficial layers of the soil, in dust, in putrefying substances, in the blood of animals which have been suffocated (by invasion from the intestine), in foul water, etc. It may usually be obtained by introducing beneath the skin of a PATHOGENIC ANAEROBIC BACILLI. 489 rabbit or a guinea-pig a small quantity of garden earth. The animal dies within a day or two, and this bacillus is found in the bloody serum effused in the subcutaneous connective tissue for a consider- able distance about the point of inoculation. Morphology. — Bacilli from 3 to 3.5 jn long and 1 to 1.1 j* broad; — y \ FIG. 162.— Bacillus oedsmatis maligni, from subcutaneous connective tissue of inoculated guinea-pig. X 950.- (Baumgarten.) <"• oo frequently united in pairs, or chains of three elements ; may grow out into long filaments 15 to 40 /* long — these are straight, or bent at an angle, or more or less curved. They resemble the bacillus of anthrax, but are not quite as broad, have rounded ends, and in stained preparations the long filaments are not segmented as is the case with the anthrax bacillus. By Loffler's method of staining they are seen to have flagella arranged around the periphery of the cells. Large, oval spores may be de- veloped in the bacilli (not in the long fila- ments), which are of greater diameter than the rods, and produce a terminal or central swelling of the same, according to the loca- tion of the spore. Stains readily by the aniline colors usu- ally employed, but is decolorized when treated by Gram's method. In stained preparations the long filaments may present a somewhat granular appearance from unequal action of the staining agent. Biological Characters. — A strictly anaerobic, liquefying, mo- tile bacillus. Forms spores. Grows in the usual culture media FIG. 163.— Bacillus cedema- tis maligni, from an agar cul- ture, showing spores. X 1,000 From a photomicrograph. (Frankel and Pfeiffer.) 490 PATHOGENIC ANAEROBIC BACILLI. when oxygen is excluded — in an atmosphere of hydrogen. Grows at the room temperature — better in the incubating oven at 37° C. The spores are formed most abundantly in cultures kept in the in- cubating oven, but may also be formed at a temperature of 20° C. In the bodies of animals which succumb to an experimental inocula- tion no spores are found immediately after death, but the bacilli multiply rap- idly in the cadaver, and form spores when the temperature is favorable. The malignant- oedema bacillus may be cultivated in ordinary nutrient gela- tin, but its development is more abun- dant when one to two per cent of grape sugar has been added to the culture medium. In deep stick cultures in this medium development occurs at first only near the bottom of the line of puncture ; the gelatin is liquefied and has a grayish- white, clouded appearance ; an abundant development of gas occurs, and as this accumulates the growth and liquefaction of the gelatin extend upward. A very characteristic appearance is obtained when the bacilli are mixed in a test tube with gelatin which has been liquefied by heat, and which is then allowed to solidify. Spherical colonies are developed, in the course of two or three days, in the lower portion of the gelatin ; these are filled with liquefied gelatin of a grayish- white color, and when ex- amined with a low power are seen to be permeated with a network of filaments, while the periphery presents a radiate appearance. In. nutrient agar growth also occurs at the bottom of a deep punc- ture ; it has an irregular, jagged outline and a granular appearance; the considerable development at the deepest portion and gradual thinning out above give the growth a club shape ; in the incubating oven there is an abundant development of gas, which often splits up the agar medium and forces the upper portion against the cotton stopper. An abundant development of gas also occurs in cultures in blood serum, and the medium is rapidly liquefied ; at a tempera- ture of 37° it is changed in a few days to a yellowish fluid, at the bottom of which some irregular, corroded fragments of the solidified serum may be seen. In agar plates, placed in a close receptacle from which oxygen is excluded, cloudy, dull-white colonies are formed which have irregular outlines and under the microscope are seen to be made up of branching and interlaced filaments radi- Fio. 164.— Bacillus oedematis ma- ligni, cultures in nutrient gelatin; a, long stick culture; b, colonies at bot- tom of gelatin tube. (Flugge.) PATHOGENIC ANAEROBIC BACILLI. 491 ating from the centre. Cultures of the malignant-oedema bacillus give off a peculiar, disagreeable odor, which cannot, however, be designated as " putrefactive." Pathogenesis. — Pathogenic for mice, guinea-pigs, rabbits, and, according to Kitt, for horses, dogs, goats, sheep, calves, pigs, chick- ens, and pigeons. According to Arloing and to Chauveau, cattle are immune. The disease is rarely developed except as a result of ex- perimental inoculations, but horses occasionally have malignant oedema from accidental inoculation, and cases have been reported in man — "gangrene gazeuse." A small quantity of a pure cul- ture injected beneath the skin of a susceptible animal gives rise to an extensive inflammatory oedema of the subcutaneous connective tissue and of the superficial muscles, which extends from the point of inoculation, especially towards the more dependent portions of the body. The bloody serum effused is without odor and contains little if any gas. But when malignant oedema results from the in- troduction of a little garden earth beneath the skin of p, guinea-pig or other susceptible animal, the effused serum is frothy and has a pu- trefactive odor, no doubt from the presence of associated bacteria. Injections into the circulation do not give rise to malignant oedema, unless at the same time some bacilli are thrown into the connective tissue. While small animals usually die from an experimental in- oculation with a moderately small quantity of a pure culture, larger ones (dogs, sheep) frequently recover. At the autopsy, if made at once, the bacilli are found in great numbers in the effused serum, but not in blood from the heart or in preparations made from the parenchyma of the various organs ; later they may be found in all parts of the body as a result of post-mortem multiplication. This applies to rabbits and to guinea-pigs, but not to mice ; in these little animals the bacilli may find their way into the blood during the last hours of life, and their presence may be demonstrated in smear prepa- rations of blood from the heart or from the parenchyma of the spleen or liver. In mice the spleen is considerably enlarged, dark in color, and softened ; in rabbits and guinea-pigs less so. With this excep- tion the internal organs present no very notable pathological changes. Animals which recover from malignant oedema are said to be subsequently immune (Arloing and Chauveau). Roux and Cham- berlain have shown that immunity may be induced in guinea-pigs by injecting filtered cultures of the malignant-oedema bacillus (about one hundred cubic centimetres of a bouillon culture in three doses) into the abdominal cavity ; or, better still, by the injection of fil- tered serum from animals which have recently succumbed to an ex- perimental inoculation (one cubic centimetre repeated daily for even or eight days). 493 PATHOGENIC ANAEROBIC BACILLI. 151. BACILLUS CADAVERIS. Obtained by the writer (1839) from pieces of liver and kidney, from yel- low-fever cadavers, which had been preserved for forty-eight hours in an antiseptic wrapping, at the summer temperature of Havana; also in two <. V t P"» ^^. ^ %' Fio. 165. — Bacillus cadaveris; smear preparation from liver of yellow-fever cadaver, kept twenty-four hours in an antiseptic wrapping, x 1,000. From a photomicrograph. (Sternberg.) cases from pieces of yellow-fever liver immediately after the autopsy ; also from liver preserved in an antiseptic wrapping from comparative autopsies made in Baltimore. Morphology. — Large bacilli with square or slightly rounded corners, from 1.5 to 4 # in length and about 1.2 u broad; frequently associated in pairs ; may grow out into straight or slightly curved filaments of from 5 to 15 /* in length. Biological Characters. — An 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. Fia. 166.— Bacillus cadaveris, from an anae- robic culture in glycerin-agar. X 1,000. From a photomicrograph. (Sternberg.) PATHOGENIC ANAEROBIC BACILLI. 493 Pathogenesis. — Liver 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. 152. 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). FlQ. 167. FIG. 168. FIG. 167.— Bacillus of symptomatic anthrax, from an agar culture. X 1,000. From a photomi- crograph. (Frfinkel and Pfeifler.) FIG. 168.— 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. f "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 42 494 PATHOGENIC ANAEROBIC BACILLI. 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 Ziehl's method, and then the bacilli with a solution of methylene blue. Biological Characters. — An anaerobic, liq^- uefying^motilebacillus. 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. 169. — Bacillus of symptomatic an- thrax; long stick cul- ture in nutrient gela- tin, ten days at 18°- 80° 0. (Kitasato.) PATHOGENIC ANAEROBIC BACILLI. 495 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 496 PATHOGENIC ANAEROBIC BACILLI. according to the method of Toussaint and Chauveau ; a temperature of 4:2° 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. STERNBERG'S BACTERIOLOGY. \ Vv^i5^\ u \J Spirillum Obermeieri in blood of two monkeys, inoculated after removal of spleen.. ( S oudakewi \rch). XV. PATHOGENIC SPIRILLA. 153. SPIRILLUM OBERMEIERI. Synonyms. — Spirochsete Obermeieri ; Spirillum of relapsing fe- ver ; Die Recur rensspirochate. 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 /* 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 Loffler'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. 498 PATHOGENIC SPIRILLA. In experiments made by Heydenreich the spirillum was found to preserve its vitality (motility) for fourteen days at a temperature of FIG. 170.— Spirillum Obermeieri in blood of man. x 1,000. From a photomicrograph. (Frankel and Pfeiffer. ) 1G° to 22° C., for twenty hours at 37°, and at 43.5° for two or three hours only. Pathogenesis. — Causes in man the disease known as relapsing fever. Munch and Moczutkowsky have produced typical relapsing FIG. 171.— 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 PATHOGE^TIC SPIRILLA. 499 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. Onl attack did not preserve the animals experimented upon from a similar attack when they were again inoculated after an interval of a few days. Recently Soudakewitch (1891) has made successful in- oculation experiments in monkeys, and has shown that in monkeys from which the spleen has previously been removed the spirilla con- tinue 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 tlie access of the febrile paroxysm and the animal recovers. 154. 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 w.hich 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, remaining1 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. 500 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. 155. SPIRILLUM CHOLERA ASIATICS. Synonyms. — Spirillum (" bacillus ") of cholera; Comma bacillus of Koch ; Kommabacillus der Cholera Asiaticse ; 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 FIG. 172. FIG. 173. FIG. 172.— Spirillum choleras Asiaticae. X 1,000. From a photomicrograph. (Koch.) FIG. 178.— Spirillum cholera? Asiaticse, involutic-i 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 /* in length and about 0.3 to 0.4 /^ in 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. 501 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 " 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. 174. FIG. 175. FIG. 174.— Spirillum cholerae Asiatic®; colonies upon gelatin plate, end of thirty hours, x ICO. Photograph by Frankel and Pfeiffer. FIG. 175.— Spirillum cholerse Asiaticae, 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 43 502 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. 176.— Colonies of the cholera spirillum; o, end of twenty hours; 6, end of thirty hours; c, end of forty- eight hours; d, after liquefaction of the gelatin. (Flugge.) FIG. 177.— Spirillum cholerse Asiaticee; 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. 503 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 W FIG. 178.— Cultures in nutrient gelatin, at the room temperature (16° to 18° C.), at the com- mencement of thefourth day; a. Spirillum choleras 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 504 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 minutes' 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. 505 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 Neffelman and by Kitasato, they only survive for a few days when mixed with normal fseces. 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 Metschnikoff's 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 may be 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 506 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 has recently (1892) published his extended researches re- lating 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 inclose relation with the bacterial cells and is perhaps an integral part of the same. The spirilla may be killed by chloroform, thymol, or by desiccation without apparent injury to the toxic potency of this substance. It is destroyed, however, by absolute alcohol, by concentrated solutions of neutral salts, and by the boiling tempera- ture, and secondary toxic products are formed which have a similar physiological 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 spi- rillum is not found in the blood or in the various organs of individu- als who have succumbed to an attack of cholera, but it is constantly found in the alvine discharges during life and in the contents 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 fol- licles and Peyer's patches were reddened around their margins, and PATHOGENIC SPIRILLA. 507 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 ^dCi k*»WW(<$0 %tft "a FIG. 179.— 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. (Flugge.) 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 508 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 Rietsch, 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 Rietsch 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. 509 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 Kietsch 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 recent researches made in Calcutta (1891), arrives at the conclusion that Koch's " comma bacillus" can- not 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. 156. SPIRILLUM OF FINKLER AND PRIOR. Synonym. — Vibrio proteus. Obtained by Finkler and Prior (1884) from the faeces of patients with cholera nostras, 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. 44 510 PATHOGENIC SPIRILLA. Biological Characters. — Anaerobic 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, which under the microscope are seen to be finely granular and yellowish or 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 whitish film is seen. Upon agar a moist, slimy layer, covering the entire surface, is quickly developed. The growth in blood serum is rapid and FIG. 180. Fia. 181. FIG. 182. Fio. 180.— Spirillum of Finkler and Prior, from a gelatin culture. X 1,000. From a photomicro- graph. (Frankel and Pfeiffer.) Fia. 181.— Spirillum of Finkler and Prior; colonies upon gelatin plate; a, end of sixteen hours; b, end of twenty-four hours; c; end of thirty-six hours. X 80. (Flugge ) Fia. 182.— 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 ^rows at the room temperature and produces a slimy, grayish-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 Finkler- 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 than 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 such injec- tions (Koch). At the autopsy the intestine is pale, and its watery contents, PATHOGENIC SPIRILLA. 511 which contain the spirilla in great numbers, have a penetrating, putrefactive odor. 157. 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 fuchsin. 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. 183. — Spirillum tyroge- — more rapidly than the cholera spirillum and num. x ~oo. (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 denned 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 i84.-Spiriiiumtyrogenum; 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; b, end of twen- surface ; complete liquefaction usually occurs in ty-four hours; c, ead of thirty- about two weeks. Upon the surface of agar a six hours. X80. (Flugge.) thin5 veliowish iayer 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. 158. 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 may be seen at one extremity of the spiral filaments or curved rods in properly stained preparations. 512 PATHOGENIC SPIRILLA. Stains with the usual aniline colors, but not hy Gram's method. Biological Characters. — An aerobic (facultative anaerobic ?), liquefy- ing, motile spirillum. According1 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° O. 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 resembling1 colonies of the cholera spirillum of the same age. Under the microscope the larger liquefied areas are seen, to contain yellowish- 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 produced in cultures of the cholera spirillum, and even more pronounced. In 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 redissolved. The milk acquires a strongly acid reaction and the spirilla quickly perish. Pathogenesis. — 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 ; they 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 Gameloa, chickens may be infected by giving them food contaminated FIG 185.— Spiril- lum Metschnikovi ; culture in nutrient gelatin, end of forty- eight hours Froma photograph. (Fran- kel and Pfeiffer.) PATHOGENIC SPIRILLA. 513 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 4 ' 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 Lthem 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. XVI. BACTERIA IN INFECTIOUS DISEASES NOT PROVED TO BE DUE TO SPECIFIC MICROORGANISMS. 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 a number of diseases, in which these researches have thus far failed to settle in a definite manner the etiology of the diseases named. For convenience of reference we shall arrange these diseases in alphabetical order. 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 ju, usu- ally united in pairs and associated in zoogloea masses. They were located for the most part in the lymph spaces of the central portion of the chorium. They stained with the usual aniline colors and also by G-ram'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 hairs withdrawn from the diseased patches, and was easily cultivated in various media. Vaillard 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 one ju ; 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. 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. Pekefharing 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-bei'i 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 BACTERIA IN CERTAIN INFECTIOUS DISEASES. 515 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 Rio 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. BRONCHITIS. Lumnitzer (1888) obtained from the sputum of a patient with putrid oronchitis 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. Picchini (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-liquef ying ; the third form was always associated with these two. CARCINOMA. Various microorganisms 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 pneumo- nia croupossB (" diplococcus pneumonia") is the microorganism most frequently found, and next to this the Diplococcus intercellula- ris 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 micrococ- cus closely resembling Micrococcus pneumonise crouposse, but which he believes not to be identical with it (see Micrococcus of Bonome, No. 40). For further details see the descriptive accounts of the micro- organisms above referred to. 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 516 BACTERIA IN INFECTIOUS DISEASES NOT PROVED from these ulcers. Various common microorganisms were obtained in cul- tures 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 chancroidal virus he found constantly a bacillus which did not grow in artificial cultures. This was about 1.48 fi longandO.5/* thick, with round ends. It was readily stained with a solution of f uchsin, but not by Gram's method. 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 microor- ganism 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 multiplication 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 character- ize the disease. Jeffries, after reviewing the various theories which have been advanced in explanation of the etiology of cholera infan- tum, 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 his memoir he says : " Passing a step further, the symptoms, patho- logy, 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 pu- trid 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 nostras. This has not been shown to be due to the presence in the alimentary canal of a particular micro- organism ; but it can scarcely be doubted that the morbid pheno- mena 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 faeces of patients with cholera nostras a spirillum which they supposed to be the spe- cific cause of this disease, but subsequent researches have not con- firmed their conclusion. Thus, in seven cases studied by bacterio- TO BE DUE TO SPECIFIC MICROORGANISMS. 517 logical methods in Koch's laboratory during the years 1885, 1886y and 1887, the spirillum of Finkler and Prior was not found in a single instance (Frank). CONJUNCTIVITIS. The various forms of conjunctivitis have been ascribed to the specific action of bacteria. That this is true as regards gonorrhoeal ophthalmia is now generally admitted, and there is some reason to believe that the bacillus discovered by Koch and studied by Kartulis (see Bacillus of Kartulis) is the cause of one form of " Egyptian catarrhal 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 aggra- vated and become chronic as a result of the presence of various microorganisms, and especially of the pyogenic micrococci. With reference to the so-called bacillus of xerosis, the researches of Schreiber, made in Neisser's laboratory, show that it is not pecu- liar to xerosis, but that it is often found quite as abundantly in other eye affections and also in the healthy con-'unctival sac. 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 tbey play an important part in maintaining such inflammations ; and in ozsena 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 tbe morbid process which gives rise to the chronic discharge. (See Bacillus fcetidus ozaenas of Hajek.) CYSTITIS. Various species of bacteria have been found in the urine in cases of cystitis, and it appears probable that some of these are directly or indirectly concerned in keeping up the vesical inflammation in chronic cases of this disease. Whether any one of the species found is capable of producing cystitis when introduced into the healthy bladder is uncertain. On the other hand, we have rather numerous observations which show that there may be bacteriuria without cys- titis. Thus Schottelius and Reinhold report a case of heart disease in which certain bacilli were found in the urine in considerable numbers, and in a pure culture, during the entire time that the 45 518 BACTERIA IN INFECTIOUS DISEASES NOT PROVED patient was under observation, and in which there was no cystitis or other symptoms that could be ascribed to the presence of this bacillus. In the extended researches of Rovsing — thirty cases of cystitis — the following results were obtained : In one case diagnosed as cys- titis 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 bacillus, Staphylococcus pyogenes aureus, Staphylococcus pyogenes albus, Staphylococcus pyogenes citreus, Streptococcus pyogenes urese (n. sp.), Diplococcus pyogenes urese (n. sp.), Coccobacillus pyogenes ureas (n. sp.), Micrococcus pyogenes urese flavus (n. sp.), Diplococcus urese trifoliatus (n. sp.), Streptococcus urese rugosus (n. sp.), Diplo- coccus urese (n. sp.), Coccobacteria urese (n. sp.). Pure cultures of all of these species introduced into the bladder of rabbits failed to induce cystitis, even when injected in considerable 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 in- troduced 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 (1890) isolated from alkaline urine obtained from patients with cystitis two species of staphylococci — Staphylococcus urese candidus and Staphylococcus urese liquefaciens ; from albu- minous, acid urine he obtained Streptococcus pyogenes. Krogius obtained from the urine of individuals suffering from cystitis a bacillus which he calls Urobacillus liquefaciens septicus. Schnitzler (1890) 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 TO BE DUE TO SPECIFIC MICROORGANISMS. 519 instance 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 rabbits almost always gave rise to a severe purulent cystitis — large rabbits 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 ammomacal fermentation had occurred, no results followed his injections into the bladder. DEXGUE. McLaughlin (1886) has claimed to find micrococci in the blood of pa- tients suffering from dengue. No satisfactory evidence of their etiological relation has been presented, and his observations have not yet been con- firmed by other investigators. ECZEMA EPIZOOTICA. Synonym. — Foot and mouth disease. This 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. Up to the present time no satisfactory demonstration has been made of the specific infectious agent ; but Schottelius has recently (1892) described a microorganism which he thinks may bear an etiological relation to the dis- ease. His inoculation experiments do not, however, sustain this view, inas- much 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 in- tense fever, salivation, and great debility. But recovery occurred at the end of five or six days without any aphthous eruption. 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 con- siderable 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 "gfrepfocflfat" 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 COa as well as in atmospheric air. Upon plates of nutrient agar — containing glycerin and formate of soda— at 37° C., 520 BACTERIA IN INFECTIOUS DISEASES NOT PROVED 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. 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 micro- organism 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 pre- sent was " diplococcus pneumonias " (Micrococcus pneumonias crou- posae). The third group included four cases of tubercular empyema ; 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. Xetter, in a series of forty-six cases examined by him, found Micrococcus pneumonias crouposas in fourteen. Koplik (1890) found the same microorganism in seven cases examined by him, and Strep- tococcus pyogenes in two 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 studied 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 pneumonias"' (Micrococcus pneumonias crouposas) was found ; in six Streptococcus pyogenes ; in two Staphylococcus pyogenes aureus ; in two Bacillus endocarditidis griseus (Weichselbaum) ; in one Micrococcus endocar- ditidis rugatus (Weichselbaum) ; in one Bacillus endocarditidis cap- sulatus (Weichselbaum) ; in two cases a bacillus which he did not succeed in cultivating. For further details see the descriptions of microorganisms referred to. TO BE DUE TO SPECIFIC MICROORGANISMS. 521 ERYTHEMA. Cordua (1885) obtained from a series of cases of an erysipelatpid skin affection of the fingers and hands, which he identified as corresponding with eiythema exudativum multiforme of Hebra, a micrococcus resembling Staphylococcus pyogenes albus in its biological characters, but which he de- 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 in- oculation 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). 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 defi- nitely established. 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 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 mici'oorganism ; 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 characterizing 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 522 BACTERIA IN INFECTIOUS DISEASES NOT PROVED from the brain and spinal cord of rabid animals, and states in his article on hydrophobia in " 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. In a paper by Perdrix published in the Annales de Tlnstitut Pasteur, vol. iv., 1890, the following statistics are given for the years 1886-89: Year. Number treated. Teaths. Mortality. 1886 2,671 25 0 94# 1837 1,770 13 0 73 1888 1,622 9 0 55 1889... 1,830 6 0 33 Total - 7,893 53 0.67£ This table includes only those deaths which occurred at least fifteen days after the treatment was terminated. When all deaths are included the fig- ures are as follows : Year. Number treated. Deaths. Mortality. 1886 2 682 36 1 34£ 1887 1 778 21 1 18 1888 1 625 13 0 74 1889 1 834 10 0 54 Total 7,919 79 0.92# In the statistics of the Pasteur Institute the cases have from the com- mencement been classified as follows : A. Persons bitten by animals proved to be rabid by experimental inocu- lations in other animals. B. Persons bitten by animals examined by veterinary surgeons and pro- nounced by them to be rabid. C. Persons bitten by animals suspected of being rabid. The following table gives the results in accordance with this classification : TO BE DUE TO SPECIFIC MICROORGANISMS. 523 A. B. c. Number treated. Died. Mortality. Number treated. Died. Mortality. Number treated. Died. Mortality. 1886... 223 5 2.15* 1,931 24 1.24* 518 7 1.35# 1887.... 357 2 0.56 1,161 15 1.29 260 4 1.54 1888. . . . 403 7 1.74 974 4 0.41 248 1 0.40 1889... 348 4 1.15 1,188 3 0.25 298 3 1.00 Total. 1,341 18 1.84* 5,254 46 0.88* 1,324 15 1.18* At the meeting of the Tenth International Medical Congress in Berlin (1890), Babes reported that in the Pasteur Institute at Bucharest about three hundred persons are inoculated yearly, with a mortality of 0.4 per cent, in cases bitten by dogs, most of which were demonstrated to have been rabid by inoculation experiments made at the Institute. The recent researches of Tizzoni and Schwartz (1892) show that the blood of rabbits which have an artificial immunity against ra- bies contains an antitoxine which has the power of neutralizing the virus of rabies, either in a test tube or in the body of an inoculated animal. Their experiments indicate the possibility of curing rabies in man by subcutaneous inoculations of this antitoxine extracted from the blood serum of immune rabbits. 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 « long and one-third to one /< 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. LEPROSY. No satisfactory experimental demonstration that the Bacillus leprse 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 pre- sence in leprous tissues, its morphology, etc. , the reader is referred to the descriptive account of Bacillus leprae (No. 53, page 394). MALARIA. Klebs and Tommasi-Crudeli, as a result of researches made by them in the vicinity of Rome (1879), announced the discovery of a bacillus which they supposed to be the cause of malarial fevers — their Bacillus mala- ria?. The writer repeated their experiments the following year (1880) in the vicinity of New Orleans, and reported as follows : ' ' Among 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 malariae of 524: BACTERIA IN INFECTIOUS DISEASES NOT PROVED Klebs and Tommasi-Crudeli; but thei-e 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 malariae 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 haematozoon belongs to quite a different class of microorgan- isms. It was first described by Laveran as the Oscillaria malarias, but is more frequently spoken of at present as the Plasmodium malariae. MEASLES. The etiology of measles and of the specific eruptive febrile diseases gene- rally 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 micrococci. In pneumonia occurring during the course of an attack of measles the Mi- crococcus pneumonias crouposae is usually found in the pulmonary exudate. No great importance can be attached to the observations made, with re- ference 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. Recently (1892) Canon and Pielicke, of Berlin, have announced the discovery of a minute bacillus in the blood of patients (fourteen) with measles, which may turn out to be the specific infectious agent in this disease. But this cannot be con- sidered demonstrated by the researches thus far made. (See Bacillus of Canon and Pielicke, No. 486.) MENINGITIS. See Cerebro-spinal meningitis, page 515. NEPHRITIS. The various microorganisms which have occasionally been found in the urine of cases of nephritis are probably not directly related to the renal dis- ease. Numerous observations are on record which show that pathogenic microorganisms present in the blood or tissues may find their way into the urine during the course of the disease due to their presence. In these cases it is probable that the passage of bacteria into the urine depends upon struc- tural changes in the kidneys ; but that these changes are directly due to the bacteria is by no means established. As an example we may mention that TO BE DUE TO SPECIFIC MICROORGANISMS. 525 the bacillus of typhoid fever is occasionally found iu the urine during an attack of this disease. 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 and 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. OSTEOMYELITIS. 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 (1886) and of Lamelongue 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 pyogenes aureus in four only, and in one of these Staphylococcus pyogenes albus was also present ; in three cases Staphylococcus pyogenes albus was obtained in pure cultures ; in two cases it was associated with Streptococcus pyogenes ; and in two cases a strepto-. coccus was found which resembled Streptococcus pyogenes and yet differed from it in some particulars. 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 pneumoniae crouposa3 (" diplococcus pneumonise "), Streptococcus pyogenes, Staphylococcus pyogenes al- bus, Staphylococcus pyogenes aureus, Friedlander's bacillus. The following have also been found occasionally : Staphylococcus tenuis, Bacillus tenuis, Micrococcus tetragenus, Bacillus pyocyanus. According to Zaufal, Micrococcus pneumonice 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. 526 BACTERIA IX INFECTIOUS DISEASES NOT PROVED OZJENA. 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 studied by him, but it was associated with various other species of bacteria, and especially with the pyogenic micrococci and with Ba- cillus fluorescens liquefaciens. He also obtained almost constantly his Bacillus fcetidus ozyenoe (No. Ill), which appears to have been the cause of the foetid odor of the nasal discharge. Marano (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. PAROTITIS. No demonstration of a specific microorganism in mumps has been made, but in non-specific, suppurative parotitis one or other of the pyogenic micrococci appears to be the cause of the inflammation and pus formation. In parotitis occurring as a complication of pneu- monia Micrococcus pneumonise crouposse has been found as the only microorganism in pus from the inflamed gland (Testi, Duplay). PEMPHIGUS. Demme (1886) has cultivated a diplococcus from a case of acute pemphigus which possibly is related to this disease (see Micrococcus of Demme, No. 27, page 319). The same coccus was found by Dahn- hardt in a similar case. PERITONITIS. That peritonitis usually results from the presence of micro- organisms in the cavity of the abdomen seems to be pretty well established by experimental evidence and by bacteriological re- searches 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 microorgan- isms 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 peritonitis may be induced in rabbits and in TO BE DUE TO SPECIFIC MICROORGANISMS. 527 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 following perforation of the bowel it is probable that this bacillus is responsible, in part at least, for the in- tense 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. Weichselbaum has observed two cases of primary peritonitis and pleuritis apparently induced by Micrococcus pneumonise crouposse, 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 recently published (1891) results of A. Frankel's researches are as follows : In thirty-one cases examined pure cultures were obtained in twenty, viz. : Bacillus coli communis, nine times ; strep- tococci, seven times ; Bacillus lactis aerogenes, twice ; Micrococcus pneumonise crouposae, 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. PLEURITIS. See Empyema, page 520. PLEURO-PNEUMONIA OF CATTLE. This is an infectious disease, the etiology of which is still undetermined, notwithstanding the researches of numerous bacteriologists. Various bac- teria have been isolated from the exudate into the pulmonary alveoli, but there is no satisfactory proof that any one of these is the specific cause of the disease. PURPURA H^EMORRHAGICA. See account of bacilli found in purpura hsemorrhagica by Babes (No. 146), Kolb (No. 147), and Tizzoni and Giovannini (No. 145). RHINOSCLEROMA. See 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 528 BACTERIA IX INFECTIOUS DISEASES NOT PROVED 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 during1 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, page 525.) 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). "TEXAS FEVER" OF CATTLE. Billings (1888) has announced the discovery of a bacillus in the blood of cattle suffering from Texas fever, which he supposes 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 septicaemia in cattle similar to the Rinderseuche of German authors. The microorganism which he has described as coming from the blood of the in- fected animals resembles in its morphology Bacillus septicaemias hsemor- rhagicee (No. 61), and, if not identical with this widely distributed species, appears to be very nearly related to it. TYPHUS FEVER. The etiology of typhus fever has not been determined in a definite man- ner. Hlava (1888) has described a " strep tobacillus " which he supposes to be concerned in the etiology of this extremely contagious disease ; but it has 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. Thoinot and Calmette encountered the streptobacillus of Hlava and various other microorganisms in a certain proportion of the cases examined by them ; but no one of these was constantly present. "An interesting organism," which they did not succeed in cultivating, was found in the blood of all cases examined by the investigators last mentioned. 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 vai'ious microorganisms have been isolated from vac- cine vesicles ; but no one of these has been shown to possess the specific pathogenic power of unfiltered lymph from the same source. The experi- ments of Carstens 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 experiments show that various disinfecting agents tested — chlo- rine, 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 virulence depends upon the presence of a living microorgan- ism, however probable this appears, for certain toxalbumins are likewise TO BE DUE TO SPECIFIC MICROORGANISMS. 529 destroyed by a correspondingly low temperature and by the action of vari- ous chemical disinfectants. YELLOW FEVER. In the writer's ' ' Report upon the Prevention of Yellow Fever by Inocu- lation," submitted in March, 1888, the following conclusions are given as the result of his investigations at the date of this report : Conclusions. Facts relating to the endemic and epidemic prevalence of yellow fever, considered in connection with the present state of knowledge concerning the etiology of other infectious diseases, justify the belief that yellow fever is due to a living microorganism capable of development under favorable local and meteorological conditions external to the human body, and of establish- ing new centres of infection when transported to distant localities. Inasmuch as a single attack of yellow fever, however mild, protects, as a rule, from future attacks, there is reason to hope that similar protection would result if a method could be discovered of inducing a mild attack of the dis- ease by inoculation or otherwise. The hypothetical yellow-fever germ, multiplying external to the human body in unsanitary places in tropical regions where the disease is endemic, or during the summer months in the area of ics occasional epidemic preva- lence, establishes infected localities, and susceptible persons contract yellow fever by exposure in these infected areas. We infer, therefore, a priori, that the yellow-fever germ invades the system by the respiratory tract, by the alimentary canal, or from the general surface of the body, and it should be found in the blood and tissues, or in the alimentary canal, or upon the surface. Another possibility presents itself, viz. , that the germ multiplying in un- sanitary localities external to the body produces a volatile poison which contaminates the air, and that an attack is induced by the toxic effects of this potent chemical poison. The more or less prolonged period of incuba- tion— two to five days — in numerous cases in which the attack has been de- veloped after removal from the infected locality, is opposed to this latter hypothesis. In the light of what is known of the etiology of other infectious dis- eases, the hypothesis that the germ really finds entrance to the body of the person attacked and multiplies within it is that which presents itself as most probable, and it hardly seems worth while to consider any other, un- less this is proved by a complete investigation not to be true. Naturally the attention of investigators has first been given to a search for the " germ " in the blood of those attacked and in the blood and tissues of the victims of the malady. The researches made up to the present time have failed to demonstrate the constant presence of any microorganism in the blood and tissues of those attacked. My own researches, recorded in the foregoing report, show that no such microorganism as Dr. Domingos Freire, of Brazil, has described in his pub- lished works, or as he presented to me as his yellow-fever germ at the time of my visit to Brazil, is found, as he asserts, in the blood and tissues of typical cases of yellow fever. There is no satisfactory evidence that the method of inoculation practised by Dr. Domingos Freire has any prophy- lactic value. The claims of Dr. Carmona y Valle, of Mexico, to have discovered the specific cause of yellow fever have likewise no scientific basis, and he has failed to demonstrate the protective value of his proposed method of pro- phylaxis. It is highly important, in the interest of science and of the public health, that further investigations be made by more exact methods which have 530 BACTERIA IX INFECTIOUS DISEASES NOT PROVED been perfected since Drs. Freire and Carmona made their researches, and with which they were evidently not familiar. The failure thus far to find a specific microorganism in the blood or tis- sues makes it desirable that a thorough research should be made with refe-' rence to the microorganisms present in the alimentary canal, for it is possible that in yellow fever, as in cholera, the disease is induced by a microorgan- ism which multiplies in this situation. Additional researches are also re- quired before we can say definitely that there is no germ demonstrable in the blood and tissues. Having exhausted our resoui'ces by the method of direct examination, and by cultures from blood drawn during life, it is highly desirable that various culture media should be inoculated with mate- rial taken, with proper precautions, from the various organs at the earliest possible moment after death. The results of subsequent investigations made by the writer in Cuba during the summers of 1888 and 1889 are given in the following summary statement from the Transactions of the Tenth International Medical Con- gress (Berlin, 1890) : Bacteriological 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, 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 subcutaneously. 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- TO BE DUE TO SPECIFIC MICROORGANISMS. 531 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 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. ' 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 no 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- 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. 532 BACTERIA IN CERTAIN INFECTIOUS DISEASES. 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. XVII. CLASSIFICATION OF PATHOGENIC BACTERIA. A. BACTERIA BELIEVED TO BE THE CAUSE OP INFECTIOUS DISEASES IN MAN. Traumatic infections: Staphylococcus pyogenes aureus (No. 1); Staphylococcus pyogenes albus (No. 2); Streptococcus pyogenes (No. 5). Gonorrhceal infections : Micrococcus gonorrhoeas (No. 6). Croupous pneumonia : Micrococcus pneumonias crouposse (No. 8). Anthrax ("-wool-sorters' disease") : Bacillus anthracis (No. 45). Typhoid fever : Bacillus typhi abdominalis (No. 46). Diphtheria : Bacillus diphtherias (No. 47). Epidemic influenza : Bacillus of influenza (No. 52). Tuberculosis : Bacillus tuberculosis (No. 53). Leprosy : Bacillus lepras (No. 54). Glanders : Bacillus mallei (No. 55). Tetanus : Bacillus tetani (No. 149). Relapsing fever : Spirillum Obermeieri (No. 153). Cholera : Spirillum choleras Asiaticas (No. 155). B. BACTERIA ASSOCIATED WITH DISEASES OF MAN WHICH HAVB BEEN SUPPOSED, ON MORE OR LESS SATISFACTORY EVIDENCE, TO BE THE CAUSE OF THESE DISEASES. Pneumonia : Bacillus of Friedlander (No. 7). Meningitis : Diplococcus intercellularis meningitidis (No. 9) ; Micrococcus pneumonias crouposas (No. 8); Bacillus meningitidis purulentas (No. 131). Biskra button — "Clou de Biskra'': Micrococcus of Heyden- reich (No. 26). Pemphigus acutus : Micrococcus of Demme (No. 27). Bright' s disease : Streptococcus of Manneberg (No. 28); Bacillus of Letzerich (No. 109). 46 534 CLASSIFICATION OF PATHOGENIC BACTERIA. Endocarditis: Staphylococcus pyogenes aureus (No. 1); Micro- coccus pneumonias crouposoa (No. 8); Micrococcus endocarditidis rugatus (No. 29); Bacillus endocarditidis griseus (No. 104); Bacillus endocarditidis capsulatus (No. 105). Syphilis: Bacillus of Lustgarten (No. 56); Bacillus of Eve and Lingard ; Micrococcus of Disse and Taguchi. Rhinoscleroma : Bacillus of rhinoscleroma (?) (No. 58). Erythema nodosum : Bacillus of Demme (No. 107). Green diarrhoea of infants : Bacillus of Lesage (No. 106). Noma : Bacillus nomse (?) (No. 110). Ozcence : Bacillus foetidus ozssn.se, (No. 111). Bronchitis : Bacillus of Lumnitzer (No. 112). Sycosis : Bacillus of Tommasoli (No. 113). Whooping cough : Bacillus of Afanassiew (No. 119). Influenza : Micrococcus of Kirchner (No. 38) ; Micrococcus No. II. of Fischel (No. 39). Measles : Bacillus of Canon. Senile gangrene : Bacillus of Tricomi (No. 128). Cystitis : Bacillus septicus vesicae (No. 132). Egyptian ophthalmia : Bacillus of Kartulis (No. 138). Trachoma : Micrococcus of trachoma (?) (No. 17). Purpura hcemorrhagica : Bacillus of Babes (No. 146); Bacillus of Kolb (No. 147); Bacillus of Tizzoni and Giovannini (No. 145). Cholera infantum : Proteus vulgaris (No. 97). Cholera nostras : Spirillum of Finkler and Prior (No. 156). Peritonitis : Bacillus coli communis (No. 89) ; Staphylococcus pyogenes aureus (No. 1); Streptococcus pyogenes (No. 5). Pleuritis : Micrococcus pneumonias crouposae (No. 8). C. BACTERIA PROVED TO BE THE CAUSE OF INFECTIOUS DISEASES IN THE LOWER ANIMALS. Anthrax : Bacillus anthracis (No. 45). Tuberculosis : Bacillus tuberculosis (No. 53). Glanders : Bacillus mallei (No. 55). Septiccemia in cattle — " Rinderseuche " : Bacillus septicaemias haemorrhagicae (No. 61). Swine plague — " Schweineseuche," Loffler and Schiitz : Bacil- lus septicaemias haemorrhagicae (No. 61). Symptomatic anthrax — "black leg'' in cattle; Bacillus of symptomatic anthrax (No. 352). Farcy in cattle : Bacillus of Nocard (No. 60). Septiccemia in deer — "Wildseuche" : Bacillus septicaemias haem- orrhagicse (No. 61). CLASSIFICATION OP PATHOGENIC BACTERIA. 535 Hog cholera : Bacillus of hog cholera (No. 63). Swine plague, Marseilles : Bacillus of swine plague (No. 65). Septicaemia in ferrets : Bacillus of swine plague, Marseilles (No. 65). " Myko-desmoids " of the horse : Micrococcus botryogenus (No. 19). Septicaemia in geese : Spirillum anserum (No. 154). Bovine mastitis : Micrococcus of bovine mastitis (No. 21); Strep- tococcus of mastitis in cows (No. 31). Mastitis in sheep : Micrococcus of gangrenous mastitis in sheep (No. 30). Pneumonia in horses: Diplococcus of pneumonia in horses (No. 32). Contagious coryza in horses — " Druse der Pferde " •. Strepto- coccus coryzae contagiossB equorum (No. 33). Hog erysipelas — "^rouget," "rothlauf": Bacillus erysipelatos suis (No. 67). Septicaemia in parrots — "gray parrot disease " : Streptococcus perniciosus psittacorum (No. 43). Diphtheria in pigeons : Bacillus diphtherias columbrarum (No. 49). Foivl cholera :, Bacillus septicaemise hsemorrhagicse (No. 61); Ba- cillus gallinarum (No. 77). Cholera in ducks : Bacillus of Cornil and Toupet (No. 62). Grouse disease : Bacillus of grouse disease (No. 76). Septiccemia in fowls: Spirillum Metschnikovi (No. 158). Septiccemia in frogs, etc. : Bacillus hydrophilus fuscus (No. 81). Infectious diseases of bees : Bacillus alvei (No. 140) ; Bacillus of Canestrini (No. 295). Infectious diseases of silkworms: Streptococeus bombycis (No. 24) ; Nosema bombycis (No. 25). D. BACTERIA FROM VARIOUS SOURCES FOUND BY EXPERIMENT TO BE MORE OR LESS PATHOGENIC FOR LOWER ANIMALS. From the pus of abscesses and open wounds : ^ Staphylococcus pyogenes aureus (No. 1). Staphylococcus pyogenes albus (No. 2). Streptococcus pyogenes (No. 5). Staphylococcus pyosepticus (No. 42). Bacillus of Belfanti and Pascarola (No. 64). Bacillus pyogenes fcetidus (No. 72). Bacillus pyocyanus (No. 95). 536 CLASSIFICATION OP PATHOGENIC BACTERIA. Micrococcus of Heydenreich (No. 26). Proteus of Karlinsky (No. 98). From infected animals : Micrococcus of bovine pneumonia (No. 22). Hsematococcus bovis (No. 34). Pseudo-diplococcus pneumonias (No. 36). Bacillus of Ribbert (No. 51). Bacillus enteritidis (No. 75). Bacillus capsulatus (No. 80). Bacillus of Schou (No. 114). Pneumobacillus liquefaciens bovis (No. 120). From the buccal, nasal, or bronchial secretions of man Bacillus of Friedlander (No. 7). Micrococcus pneumonia crouposse (No. 8). Staphylococcus salivarius pyogenes (No. 10). Micrococcus salivarius septicus (No. 15). Micrococcus tetragenus (No. 18). Micrococcus of Manfredi (No. 20). Micrococcus gingivse pyogenes (No. 35). Micrococcus No. II. of Fischel (No. 39). Bacillus of rhinoscleroma (No. 58). Bacillus crassus sputigenus (No. 71). Bacillus smaragdinus foetidus (No. 78). Bacillus tenuis sputigenus (No. 82). Bacillus of Lmnnitzer (No. 112). Bacillus of Afanassiew (No. 119). Bacillus gingivse pyogenes (No. 122). Bacillus dentalis viridans (No. 123). Bacillus pulpse pyogenes (No. 124). From fceces : Bacillus coprogenus parvus (No. 68). Bacillus cavicida (69). Bacillus coli communis (No. 89). Bacillus lactis aerogenes (No. 90). Bacillus leporis lethalis (No. 94). Bacillus of Lesage (No. 106). Bacillus coprogenes fcetidus (No. 116). Bacillus of Gessner (No. 133). Spirillum of Finkler and Prior (No. 156). CLASSIFICATION OF PATHOGENIC BACTERIA. 537 From urine : Streptococcus of Manneberg (No. 28). Bacillus of Letzerich (No. 109). Bacillus septicus vesicse (No. 132). From cadavers and from putrefying material : Diplococcus intercellularis meningitidis (No. 9). Streptococcus septicus liquefaciens (No. 37). Bacillus of Koubasoff (No. 59). Bacillus cavicida Havaniensis (No. 70). Proteus hominis capsulatus (No. 73). Bacillus erysipelatos suis (mouse septicaemia) (No. 67). Bacillus septicaemias ha3morrhagicse (rabbit septicaemia) (No. 61). Proteus capsulatus septicus (No. 74). Bacillus pneumosepticus (No. 79). Bacillus acidiformans (No. 92). Bacillus cuniculicida Havaniensis (93). Bacillus pseudo-tuberculosis (No. 121). Bacillus septicus keratomalacise (No. 125). Bacillus septicus acuminatus (No. 126). Bacillus septicus ulceris gangrsenosi (No. 127). Bacillus albus cadaveris (No. 129). Bacillus meningitidis purulentae (No. 131). Bacillus chromo-aromaticus (No. 134). Bacillus cadaveris (No. 151). Proteus vulgaris (No. 97). Proteus mirabilis (No. 99). Proteus Zenkeri (No. 100). Proteus septicus (No. 101). Proteus lethalis (No. 102). From the soil : Streptococcus septicus (No. 23), Bacillus septicus agrigenus (No. 66). Bacillus cedematis aerobicus (No. 108). Bacillus cedematis maligni (No. 150). Bacillus tetani (No. 149). Bacillus muscoides (No. 401). From various sources : Bacillus oxytocus perniciosus (No. 117) — from milk. Bacillus saprogenes II. (No. 118) — from sweating feet. Bacillus canalis capsulatus (No. 135) — from sewers. 538 CLASSIFICATION OF PATHOGENIC BACTERIA. Bacillus canalis parvus (No. 136) — from sewers. Bacillus indigogenus (No. 137) — from leaves of the indigo plant. Bacillus of Utpadel (No. 139)— from dust. Bacillus of Okada (No. 144)— from dust. Spirillum tyrogenum (No. 157)— from cheese. Micrococcus endocarditidis rugatus (No. 29) — cardiac valves. Bacillus endocarditidis griseus (No. 104) — cardiac valves. Bacillus endocarditidis capsulatus (No. 105) — cardiac valves. PART FOURTH. SAPROPHYTES. 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 AR- TICLES OF FOOD. VIII. NON-PATHOGENIC MICROCOCCI. IX. NON-PATHOGENIC BACILLI. X. NON-PATHO- GENIC SPIRILLA. XI. LEPTOTRICHE^E AND CLADOTRICHE^E. XII. ADDITIONAL SPE- CIES OF BACTERIA NOT CLASSIFIED. XIII. BACTERIOLOGICAL DIAGNOSIS. I. BACTERIA IN THE AIR. THE saprophy tic 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- 542 BACTERIA IX 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 rot 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. TyndalFs 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 surfaces 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 plants 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. 186. — Penicillum glau- cum; m, mycelium, from which is given off a branching pedicle bearing spores. X 150. BACTERIA IX THE AIR. are borne upon projecting pedicles by which they are removed from the moist material upon which and in which the mycelium develops (Fig. 186), 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. 187. 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. 187. 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 544: 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 the 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 most 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. 545 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. 188. 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 546 BACTERIA IX THE AIR. manj' 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. 188, 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 ob- tained interesting results ; but his method of ensemencements frac- 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. 189. 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. 547 FIG. 189. filters, c and 6. 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. 190 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 Fio. 190. 548 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. 191. Two sand filters, c, 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. 192), 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. FIG. 191. BACTERIA IN THE AIR. 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 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 of this water, containing the sugar 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 47 FIG. 192. FIG. 193. 550 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. 193). 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 are 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 the number of living germs in a cubic metre of air averaged two hundred and fifty, on the sea coast the average was one hundred, in the court- yard of the University of Rostock four hundred and fifty. The num- ber was materially reduced after a rainfall and increased when a dry land wind prevailed. Frankland found that fewer germs were present in the air in winter than in summer, and that when the earth was covered with snow the number was greatly reduced, as also during a light fall of snow ; the air of towns was found to be more rich in germs than the BACTERIA IN THE AIR. 551 air of the country ; the lower strata of the atmosphere contained more than the air of elevated localities. Von Freudenreich also found that the air of the country contained fewer germs than that of the city. Thus in the city of Berne a cubic metre of air often contained as many as two thousand four hundred germs, while the maximum in country air was three hundred. His re- sults corresponded with those of Miquel in showing that the number of atmospheric organisms is greater in the morning and the evening, between the hours of 6 and 8, than during the rest of the day. Neu- mann, whose researches were made in the Moabite Hospital, found the greatest number of bacteria in the air in the morning after the patients able to sit up had left their beds and the wards had been swept. The number of germs was then from eighty to one hundred and forty in ten litres of air, while in the evening the number fell to four to ten germs in ten litres. Miquel has given the following summary of results obtained in his extended experiments, made in Paris during the years 1881, 1882, and 1883 : Average for 1880 Number of Germs in a Cubic Metre of Air. Air of Laboratory, Montsouri. Air of Park, Mont souri. 215 348 550 71 62 51 1881 1882. . Rue de Rivoli, average for one year, 750 ; summit of Pantheon, 28 ; Hotel-Dieu, 1880, average for four months, male ward 6,300, female ward 5,120 ; La Piete Hospital, average of fifteen months, 11,100. It must be remembered that the figures given relate both to bac- teria and to the spores of mould fungi, and that the latter are com- monly the most numerous when the experiment is made in the open air. Petri has shown that when gelatin plates are exposed in the air the relative number of spores of mould fungi deposited upon them is less than is obtained in aspiration experiments. The number of colonies which develop on exposed plates does not represent the full number of bacteria deposited, for these colonies very frequently have their origin in a dust particle to which several bacteria are attached, or in a little mass of organic material contain- ing a considerable number. It is generally conceded that sea air and country air are more wholesome than the air of cities, and especially of crowded apart- ments, in which the number of bacteria has been shown to be very much greater. But it would be a mistake to ascribe the sanitary value of sea, country, and mountain air to the relatively small num- 553 BACTERIA IX THE AIR. ber of bacteria present in such air. There are other important fac- tors to be considered, and we have no satisfactory evidence that the number of saprophytic bacteria present in the air has an important bearing upon the health of those who respire it. We do know that the confined air of crowded apartments, and especially of factories in which a large quantity of dust is suspended in the air, predisposes those breathing such air to pulmonary diseases and lowers the gen- eral standard of health. But it has not been proved that this is due to the presence of bacteria. Infectious diseases may, under certain circumstances, be communicated by way of the respiratory passages as a result of breathing air containing in suspension pathogenic bac- teria ; but there is reason to believe that this occurs less frequently than is generally supposed. Kruger has shown that the dust of a hospital ward in which pa- tients with pulmonary consumption expectorated occasionally upon the floor contained tubercle bacilli. This was proved by wiping up the dust on a sterilized sponge, washing this out in bouillon, and in- jecting this into the cavity of the abdomen of guinea-pigs. Two animals out of sixteen injected became tuberculous. In pulmonic anthrax, which occasionally occurs in persons engaged in sorting wool — "wool-sorters' disease" — infection occurs as a result of the respiration of air containing the spores of the anthrax bacillus. Among the non-pathogenic saprophytes found in the air certain aerobic micrococci appear to be the most abundant, and, as a rule, bacilli are not found in great numbers or variety. In some localities various species of sarcinsB are especially abundant. The following is a partial list of the species which have been shown by the researches of various bacteriologists to be occasionally present in the air. But, as heretofore remarked, their presence is to be regarded as acci- dental, and so far as we know there is no bacterial flora properly be- longing to the atmosphere : Micrococcus ureae (Pasteur), Diplococcus roseus (Bumm), Diplococcus citreus conglomerates (Bumm), Micrococcus radiatus (Fliigge), Micrococcus flavus desidens (Fliigge), Micrococcus flavus liquefaciens (Fliigge), Micro- coccus tetragen us versatilis (Stern berg), Micrococcus pyogenes aureus (Rosen- bach), Micrococcus pyogenes citreus (Passet), Micrococcus cinnabareus (Fliigge), Micrococcus flavus tardigradus (Fliigge), Micrococcus versicolor (Fliigge), Micrococcus viticulosus (Katz), Micrococcus candidans (Fliigge), Pediococcus cerevisiae (Balcke), Sarcina lutea (SchrOter), Sarcina rosea (Schroter), Sarcina aurantiaca, Sarcina alba, Sarcina Candida (Reinke), Bacillus tumescens (Zopf), Bacillus subtilis (Ehrenberg), Bacillus multipedi- culosus (Fliigge), Bacillus mesentericus fuscus (Fliigge), Bacillus mesenteri- cus ruber (G-lobig), Bacillus inflatus (A. Koch), Bacillus mesentericus vul- gatus, Bacillus prodigiosus, Bacillus aerophilus (Liborius), Bacillus pestifer (Frankland), Spirillum aureuni (Weasel), Spirillum flavescens (Weibel), Spi- rillum flavum (Weibel), Bacillus Havaniensis (Sternberg). In the recent researches of Welz, made in the vicinity of Freiburg, twenty-three different micrococci and twenty-two bacilli were obtained from the air. II. BACTERIA IN WATER. THE water of the ocean, of lakes, ponds, and running streams necessarily contains bacteria, as they are constantly being carried into it by currents of air passing over the neighboring land surfaces, and by rain water which washes suspended microorganisms from the atmosphere ; and, as such water contains more or less organic material in solution, many of the saprophytic bacteria multiply in it abundantly. It is only in the water of springs and wells which comes from the deeper strata of the soil that they are absent. The number and variety of species present in water from any given source will depend upon conditions relating to the amount of organic pabulum, the temperature, the depth of the water, the fact of its being in motion or at rest, its pollution from various sources, etc. The comparatively pure water of lakes and running streams contains a considerable number of bacteria which find their normal habitat in such waters and which multiply abundantly in them, notwith- standing the small quantity of organic matter and salts which they contain. The water of stagnant, shallow pools, and of sluggish streams into which sewage is discharged, contains a far greater number and a greater variety of species. The study of these bacteria in water has received much attention on account of the sanitary questions involved, relating to the use of water from various sources for drinking purposes. In the present section we shall first give an account of the methods of bacteriologi- cal water analysis, and then a condensed statement of results ob- tained in the very numerous investigations which have been made. A very important point to be kept in view is the fact that a great increase in the number of bacteria present, in samples of water col- lected for investigation, is likely to occur if these samples are kept for some time. A water which, for example, contains only two hundred to three hundred bacteria per cubic centimetre when the ex- amination is made at once, may contain several thousand at the end of twenty-four hours, and at the end of the second or third day twenty thousand or more may be present in the same quantity. 554 BACTERIA IN WATER. Later, on account of the exhaustion of organic pabulum, the num- ber is again reduced as the bacteria present gradually lose their vitality. Under these circumstances it is evident that an estimate of the number of bacteria present in water from a given source can have no value, unless a sample is 'tested by bacteriological methods within a short time after it has been collected. Not more than an hour or two should be allowed to elapse, especially in warm weather. By placing the water upon ice the time may be extended somewhat, but Wolffhugel has shown that the number of germs is gradually diminished when water is preserved in this way, and it will be safest to make an immediate examination when this is practicable. The collection may be made in a sterilized Erlenmeyer flask pro- vided with a cotton air filter, or in a bottle having a ground-glass stopper which has been wrapped in tissue paper and sterilized for an hour or more at 150° C. in the hot-air oven. Or the small flasks with a long neck may be used, as first recommended by Pasteur. These are prepared as follows : The bulb is first gently heated, and the ex- tremity of the tube dipped into distilled water, which mounts into FIG. 194. the bulb as it cools ; the water is then made to boil, and when all but a drop or two has escaped and the bulb is filled with steam the extremity of the tube is hermetically sealed. When the steam has condensed by the cooling of the bulb a partial vacuum is formed, and the tube is ready for use at any time. It is filled with water by breaking off the sealed extremity under the surface of the water of which a sample is desired. This is done with sterilized forceps, and care must be taken that the exterior of the tube is properly sterilized before the collection is made. The end is immediately sealed in the flame of a lamp. A difficulty with these vacuum tubes is that they are so completely filled with water that this cannot be readily drawn from them again in small quantities. The writer therefore prefers to make the collection in a tube shaped as shown in Fig. 194, in which a partial vacuum is formed just before the collection by heating the air in the bulb. The water mounts into the tube as the air in the bulb cools, and is readily forced out again for making cultures by applying gentle heat to the bulb. As a lamp is needed to seal the end of the tube in either case, there is 110 special advantage in having a vacuum formed in advance, and, as stated, the vacuum tubes are So BACTERIA IN WATER. 555 nearly filled with water that it is not so simple a matter to obtain the contents for our culture experiments without undue exposure to at- mospheric germs. In practice small glass bottles with ground-glass stoppers will be found most convenient, and, when properly steril- ized, are unobjectionable. They should be filled at a little distance below the surface, as there is often a deposit of dust upon the surface of standing water, and sometimes a delicate film made up of aerobic bac- teria. When water is to be obtained from a pump or a hydrant it should be allowed to flow for some time before the collection is made. To collect water at various depths the apparatus shown in Fig. 195 is recommended by Lepsius. An iron frame supports an inverted flask, A, filled with sterilized mercury and containing about three hundred cubic centimetres. The flask B is intended to receive the mercury when, at the desired depth, it is al- lowed to flow through the capillary tube b. This is sealed at the extremity and bent as shown in the figure. By pulling upon the cord c this tube is broken, and as the mercury flows from the flask this is filled with water through the tube a. The extremity of the broken tube b is closed by the mercury in the flask B when A is full of water, and the apparatus can be brought to the surface with only such water as was collected at the depth from which a sample was desired. The bacteriological analysis is made by adding a definite quantity of the water under investigation to liquefied gelatin or agar-gelatin, and making a plate or Esmarch roll tube, which is put aside for the devel- opment of colonies. Miquel and others have preferred to use liquid cultures and the method of fractional cultivation described in the previous section. The use of a solid culture medium has, however, such obvious advantages that we do not consider it necessary to do more than refer to the other method as one which, when applied with skill and patience, may give sufficiently accurate results. FIG. 195. 556 BACTERIA IN WATER. The amount of water which should be added to the usual quan- tity of liquefied flesh-peptone-gelatin in a test tube, in order that the colonies which develop may be well separated from each other and easily counted, can only be determined by experiment. If the water is from an impure source a single drop may be too much, and it will be necessary to dilute it with distilled water recently sterilized. But for ordinary potable water it will usually be best, in a first experi- ment, to make two trials, one with one cubic centimetre and one with one-half cubic centimetre added to the liquefied nutrient gelatin. The water in the collecting bottle should be shaken, to distribute the bacteria which may have settled to the bottom, before drawing off by means of a sterilized pipette the amount used for the experiment, and the germs present in it are to be distributed through the liquefied gelatin by gently moving the tube to and fro. Koch's method of preparing a gelatin plate is illustrated in Fig. 196. A glass dish, containing ice water and covered with a large Fio. 196. plate of glass, is supported upon a levelling tripod. By means of a spirit level this is adjusted to a horizontal position, so that when the liquefied gelatin is poured upon the smaller sterilized glass plate, seen in the centre of the large plate of glass, it will not flow, but may be evenly distributed over the surface by means of a sterilized glass rod. The glass cover resting against the side of the apparatus is placed over the gelatin plate while it is cooling, to protect it from atmo- spheric germs, and when the gelatin is hard the plate is transferred to a shallow glass dish, which is kept at a temperature of about 20° C. for several days for the development of colonies. It is difficult to count colonies when more than five thousand develop upon a plate of the usual size, and for this reason it will be best to repeat the ex- periment with a smaller quantity of water from the same source, if this is at hand, rather than to attempt to count an overcrowded plate. Before pouring the gelatin upon the plate the lip of the test tube containing it should be sterilized by passing it through a flame. The liquefied gelatin should ba carefully distributed to cover a rect- BACTERIA IN WATER. 557 angular surface and leaving a margin of about one centimetre around the edge of the plate. The Koch's dish in which the gelatin plate is placed for the development of colonies should be carefully sterilized by heat or by washing it out with a sublimate solution. A circular piece of filtering paper, saturated with sublimate solution or distilled water, is placed at the bottom of the lower dish to keep the air in a moist condition and prevent drying of the gelatin. Usually two or three plates made at the same time are placed one above the other on glass supports made for this purpose. If many liquefying organisms are present it will be necessary to count the colonies before these run together — usually on the second day ; but in the absence of liquefy- ing colonies it is best to wait until the third, or even the fifth day, as the number of visible colonies and the ease of counting them will be greater than at an earlier date. The development of a few scattered liquefying colonies which threaten to spoil the plate may be arrested by taking up the liquefied gelatin from each with a bit of filtering paper, and then, by means of a camel's-hair brush, applying a solu- tion of potassium permanganate to the margin of the colony. The growth of colonies of mould fungi, which have developed from spores from the atmosphere falling upon the plate while it is exposed, can be checked by the application of collodion containing bichloride of mercury. Counting of the colonies is a simple matter when they are few in number ; when they are numerous it is customary to place the plate over a dark background, and to place above it a glass plate divided into square centimetres by lines ruled with a diamond. By means of a lens of low power the colonies in a certain number of squares are counted and the average taken. This multiplied by the number of square centimetres in the gelatin-covered surface gives approximately the entire number of colonies which have developed from the amount of water used in the experiment. Instead of using Koch's original plate method, as above described, the shallow, covered glass dishes recommended by Petri may be employed. These are from one to one and one-half centimetres high and from ten to fifteen centimetres in diameter. The liquefied gel- atin is poured into the lower dish and the cover at once placed over it. The gelatin does not dry out very soon, but, if necessary, several of these Petri's dishes may be placed in a larger jar, which serves as a moist chamber. The roll tubes of Esmarch may also ba used, and have the ad- vantage that accidental colonies from air-borne germs are excluded. The counting of colonies is not quite as easy, but by the use of a mounted lens especially designed for the purpose it is attended with no great difficulty. The surface of the tube is divided into squares 558 BACTERIA IN WATER. by colored lines, and the number of colonies in several squares is counted in order to obtain an average and estimate the entire number. Water which contains numerous liquefying bacteria had better be examined by the use of nutrient agar instead of gelatin; and in very warm weather it will be necessary to use an agar medium, as ten-per-cent gelatin is likely to melt if the temperature goes above 22° C. A difficulty in the use of agar for plates consists in the lia- bility of the film to slip from the glass. This may be remedied to some extent by adding a few drops of a concentrated solution of gum acacia to the liquefied agar medium. Petri's dishes are well adapted for the use of the agar medium, as the objection referred to does not apply to them. The gelatin-agar medium, containing 5 per cent of gelatin and 0. 75 per cent of agar, may also be used with advan- tage in the bacteriological analysis of water. Much stress was at one time laid upon the enumeration cf liquefying colonies, upon the supposition that the liquefying bacteria were especially harmful as compared with the non-liquefying, and that a water containing many liquefying colonies was to be looked upon with suspicion. We now know, however, that there are many common and harmless saprophytes which cause the liquefaction of gelatin, and that some of the most dangerous pathogenic bacteria do not liquefy gelatin. This distinction has therefore no special value, and the question for bacteriologists to-day is not how large is the comparative number of liquefying colonies, but what species are represented by the colonies present, liquefying and non-liquefying, and what are the special pathogenic properties of each. The answer to these questions, in the case of any particular water supply, calls for special knowledge and great patience and care in the isolation in pure cultures, and careful study of the various species present. It is now generally recognized that a mere enumeration of the number of colonies which develop from a water under investigation is not a sufficient indication upon which to found an opinion as to its potability. An excessive number of bacteria is an indication that the water contains a large amount of the organic material which serves as pabulum for these microorganisms. But the chemists are able to determine the amount of organic matter present in water with greater precision ; and, as we have seen, the number of bacteria may increase many-fold in water which is kept standing in the labo- ratory for two or three days in a well-corked bottle. As a matter of fact, the enumeration of bacteria in water, although it has given us results of scientific interest, has not materially added to the methods previously applied for estimating the sanitary value of water ob- tained from various sources for drinking purposes. But the bacte- BACTERIA IX WATER. riological examination may prove to be of great value if it succeeds in demonstrating the presence of certain pathogenic bacteria and in thus preventing the use of a dangerous water. We do not mean to say, however, that an enumeration of the bacteria present in drink- ing water has no practical value. An excessive number indicates an excessive amount of organic pabulum, which may have come from a dangerous source; and the dangerous pathogenic bacteria are not only more likely to be present in such water, but they can more readily multiply in it, while in a pure water they would fail to in- crease in number, and, as has been shown by experiment, would die out within a short time. The number of bacteria present in rain water, or in snow which has recently fallen, varies greatly at different times. Naturally the number is greater when the surface of the earth is dry and the at- mosphere loaded with dust by currents of wind passing over it, and less when the surface is moist and the atmosphere has been purified by recent rains. In snow from the surface of a glacier in Norway, Schmelck found two bacteria and two spores of mould fungi per cubic centimetre of water from the melted snow. Ganowski, in experiments made with freshly fallen snow collected in the vicinity of Kiew, obtained the fol- lowing results : February 3d, 1888 : temperature of the air, 7.2° C. ; snowfall, 0. 1 millimetre ; number of bacteria in 1 cubic centimetre of water from melted snow, 34 in one sample and 38 in another. February 20th, 1888 : temperature, 11.1° C. ; snowfall, 1.1 milli- metres ; number of bacteria in one sample, 203, in another 384. Miquel obtained from rain water collected at Montsouri during a rainy season 4. 3 germs per cubic centimetre ; in rain water collected in the centre of the city of Paris, 10 per cubic centimetre. Hail has also been shown to contain bacteria in considerable num- bers. Bujwid found in hailstones which fell at Warsaw 21,000 bacteria in 1 cubic centimetre ; but this is exceptional, and is supposed to be due to the fact that surface water had been carried into the air by the storm and frozen. Fontin examined hail which fell in St. Petersburg, and obtained an average of 729 bacteria per cubic centi- metre of water from the melted hail. River water has been carefully examined by numerous bacterio- logists in various localities and at different seasons of the year. We give below some of the results reported : Water of the Seine at Choisy, before reaching Paris, 300 ; at Bercy, 1,200 ; at Saint-Denis, after receiving the sewer water from the city, 200,000 germs per cubic centimetre (Miquel). Water of the Spree beyond Kopenick, 82,000 ; two hundred steps below the mouth of the Wuhle, 118,000 ; in Berlin above the mouth 560 BACTERIA IX WATER. of the Panke, 940,000; below the mouth of the Panke, 1,800,000 (Koch). Water of the Main above the city of Wiirzburg, in the month of February, 520 ; below the city, 15,500 (Rosenberg). Water of the Potomac, at Washington, in 1886 : January, 3,774 ; February, 2,536 ; March, 1,210 ; April, 1,521 ; May, 1,064 ; June, 348 ; July, 255 ; August, 254 ; September, 178 ; October, 75 ; No- vember, 116 ; December, 967 (Theobald Smith). The Thames, in the autumn of 1885, in the vicinity of London Bridge two hours after high water, contained 45,000 germs per cubic centimetre ; the water of the Lea at Lea Bridge, 4,200,000 (Bisch- off). The Neva inside the city of St. Petersburg, in September, 1883, contained 1,500 in one sample and 1,040 in another ; in November (20th), 6,500 (Poehl). The water of the Oder, collected within the limits of the city of Stettin, was found by Link to contain from 5,240 to 15,000 bacteria per cubic centimetre ; that of the Limmat, at Zurich, 346 in one specimen and 508 in another (Cramer). Lake ivater, as a rule, contains fewer bacteria than river water. Wolffhugel, in researches extending from July, 1884, to July, 1885, obtained from the water of the Tegeler Lake an average of 396 bacteria per cubic centimetre. Cramer obtained an average of 168 per cubic centimetre during the months of October, December, and January, 1884, from the water of Lake Zurich ; in June of the same year the average of 42 examinations gave 71 per cubic centimetre. In Lake Geneva, Fol and Dunant obtained from water collected some distance from the shore an average of 38 bacteria per cubic centi- metre. Ice which is usually collected from lakes and rivers contains a greater or less number of bacteria, according to the depth and purity of the water. The ice used in Berlin, collected from the surface of lakes and rivers in the vicinity of the city, contains from a few hun- dred to 25,000 bacteria to the cubic centimetre (Friinkel). In the ex- periments of Heyroth samples of ice from the same source gave less than 100 per cubic centimetre in three, from 100 to 500 in eight, from 500 to 1,000 in six, from 1,000 to 5,000 in seven, and 14,400 in one. Prudden obtained from Hudson River ice, put up six miles below the city of Albany, an average of 398 bacteria per cubic centimetre from transparent ice, and in the superficial "snow ice" 9,187. Ice collected lower down the river contained an average of 189 in the transparent and 3,693 in the snow ice. Ice from the Dora at Turin was found by Bordoni-Uffreduzzi to contain from 120 to 3,546 bacteria per cubic centimetre. BACTERIA IN WATER. 561 Hydrant water, as supplied to cities, has received the attention of numerous investigators. The water supply of Berlin was ex- amined by Plagge and Proskauer at intervals of a week from June, 1885, to April, 1886. Their tabulated results show considerable variations. We give the figures for a single day, June 30th, 1885 : Stralauer works, water of the Spree, unfiltered 4,400, filtered 53 ; Tegeler works, water of the lake, unfiltered 880, filtered 44 ; high re- servoir at Charlottenberg, 71 ; 75 W. Wilhelmstrasse, 121 ; Fried- richstrasse, 41-42 S. W., 160 ; Schmidstrasse, 165 E., 51 ; Friedrich- strasse, 126 N., 151 ; Weinmeisterstrasse, 15 C., 63. Wells which are supplied by water from deep strata contain few bacteria, unless contaminated by surface water in which they are usually very abundant. Roth examined the water of sixteen surface wells in Belgard, which has a very porous subsoil, and found from 4,500 to 5,000 bacteria in three, from 7,800 to 15,000 in six, from 18,000 to 35,000 in six, and 130,000 per cubic centimetre in one. Forty-seven wells in Stettin, the water of which was examined by Link, gave the following results : Less than 100 in six, 100 to 500 in twenty-one, and in the remainder (sixteen) from 1,000 to 18,000. Sixty-four wells in Mainz examined by Egger, and 53 in Gotha by Becker, gave more favorable results ; the number of wells in the former city, in which less than 100 colonies developed from 1 cubic centimetre, was 34, and in the latter the same (34). Bolton examined the water of 13 wells in Gottingen, and found but 1 in which the number of colonies from 1 cubic centimetre was less than 100 ; in 12 the number varied from 180 to 4,940. The water of deep wells and springs may be entirely free from bacteria, or nearly so. Egger found in the water of an artesian well at Mainz 4 bacteria per cubic centimetre, and the same number was found by Hueppe in the deep well at the Wiesbaden slaughter-house. The artesian well at the gasworks of Kiel was found by Brennig to contain from 6 to 30 bacteria per cubic centimetre. In a spring at Batiolettes, Fol and Dunant found 57 bacteria per cubic centimetre. Furbringer obtained from springs at Jena 156 from one, 51 from another, 32 from another, and 109 from another. The water supplied to Danzig from the Prangenaur Spring was found in several experi- ments to be free from bacteria (Freimuth). In a summary of results obtained in various German cities Tie • mann and Gartner find that sixty-nine per cent of the wells from which samples of water were examined contained less than 500 bac- teria per cubic centimetre. The water of sewers is naturally rich in bacteria. Miquel found that at Clichy the sewer water contained 6,000,000 bacteria per cubic centimetre. Bischoff found in water from London sewers 7,500.000, 562 BACTERIA IN WATER. and numerous observations show that the number of bacteria in river water is greatly increased in the vicinity of and below the mouths of city sewers. We conclude from the experimental data recorded that water containing less than 100 bacteria to the cubic centimetre is presum- ably from a deep source and uncontaminated by surface drainage, and that it will usually be safe to recommend such water for drink- ing purposes, unless it contains injurious mineral substances. Water that contains more than 500 bacteria to the cubic centimetre, although it may in many cases be harmless, is to be looked upon with some suspicion, and water containing 1,000 or more bacteria is presumably contaminated by sewage or surface drainage and should be rejected or filtered before it is used for drinking purposes. But, as heretofore stated, the danger does not depend directly upon the number of bacteria present, but upon contamination with pathogenic species which are liable to be present in surface water and sewage. In swallowing a glassful of pure spring water a number of bacteria from the buccal cavity are washed away and carried into the stomach, which, if enumerated, would doubtless far exceed in numbers those found in the most impure river water. The number of bacteria does not depand alone upon the amount of organic pabulum contained in a water, and cannot be depended upon in forming an estimate of this ; for, as has been shown by Bolton, certain water bacteria multiply abundantly in water con- taining comparatively little organic matter, while other species fail to grow unless the quantity is greater. In a water containing con- siderable nutrient material the water bacteria may be restrained in their development by other species present until the amount of pabu- lum is reduced so that these no longer thrive, when the common water bacteria will take the precedence, and an enumeration may show a greater number of colonies than at first. But, in general, water rich in organic material contains a greater number of bacteria and a greater variety of species than that which is comparatively pure. That certain bacteria may multiply in water which has been carefully sterilized has been shown by Bolton and others. Two com- mon water bacteria — Micrococcus aquatilis and Bacillus erythrospo- rus — multiplied abundantly in doubly distilled water, and when this water was again sterilized and re-inoculated with one of these species the same abundant increase occurred. This was repeated six times with the same result (Bolton). Computing the number of these water bacteria in ten cubic centimetres of distilled water at twenty millions, and estimating their specific gravity at one, and the diameter of the individual cells at one /<, the total weight of the entire BACTERIA IN WATER. 563 number, according to Bolton, would be less than one-hundredth of a milligramme, and at least three-fourths of this must consist of water. The organic material represented by this number of bacteria would therefore be so minute that it might be supplied by dust par- ticles accidentally falling into the distilled water. Rosenberg has shown that while many of the species which he obtained in pure cultures from the water of the river Main multiplied in sterilized distilled water, other species quickly died out in such water. The growth of certain bacteria depends not only upon the quantity of nutritive material present, but upon its quality, the con- ditions in this regard being widely different for different species. In view of the facts heretofore stated bacteriologists are now giv- ing more attention to a careful study of the kinds of bacteria pre- sent in their examinations of water. Rosenberg, in his examinations of the water of the Main in the vicinity of Wiirzburg (1886), found that before the river reached the city the water contained more micrococci than bacilli, but that after receiving the sewage of the city the number of bacilli was greatly in excess. Adametz (1888) has described eighty-seven species obtained by him from water in the vicinity of Vienna ; Maschek found fifty-five different species in the drinking water used at Leitmeritz; and Tils (1890) has described fifty-nine species obtained by him from the city water supply at Freiburg. Among the pathogenic bacteria which are liable to find their way into water used for drinking purposes, the most important, from a sanitary point of view, are the bacillus of typhoid fever and the spirillum of Asiatic cholera. Both of these microorganisms are pre- sent in great numbers in the excreta of persons suffering from the specific forms of disease to which they give rise, and are consequently liable to contaminate wells and streams which receive surface water, when such excreta are thrown upon the surface or into sewers, etc. Epidemics of these diseases have frequently been traced to the use of such contaminated water, and in a few instances the presence of these specific disease germs in water has been demonstrated by bac- teriological methods. Laboratory experiments indicate, however, that an increase of these pathogenic bacteria in drinking water is not likely to occur, except under special conditions, and that they die out after a time, being at a disadvantage in the struggle for exist- ence constantly going on among the numerous species which have their normal habitat in water. Bolton, Frankland, and others have shown that the anthrax ba- cillus, not containing spores, dies out in hydrant water within five or six days. In the experiments of Kraus the anthrax bacillus added to well water, not sterilized, at a temperature of 10.5° C., was still 50-4 BACTERIA IN WATER. present in a living condition on the second day, but no colonies de- veloped after the third day ; the typhoid bacillus died out between the fifth and seventh days ; the cholera spirillum was no longer found on the second day. In the meantime the common water bacteria had increased in numbers enormously. Similar results have been reported by Hochstetter and others. Hueppe, in ten experiments in which the typhoid bacillus was added to well water of a bad quality, found that in two no development of this bacillus occurred after the fifth day, while a few colonies developed in the other experiments as late as the tenth day. In these experiments the temperature was comparatively low (10.5° C.). At a higher temperature the experi- ments of Wolffhugel and Riedel show that an increase may take place. At the room temperature (about 20° C. ) the typhoid bacillus added to distilled water, to well water, and to Berlin hydrant water was still present, in some instances, at the end of thirty-two days. And it was found that in some cases a decrease in the number occurred, then a notable increase, and finally a second diminution. Koch found the cholera spirillum in a water tank at Calcutta during a period of fourteen days, and in his experiments showed that it preserved its vitality in well water for thirty days, in Berlin sewer water for six to seven days, and in the same mixed with faeces for twenty-seven hours only. In the experiments of Nicati and Rietsch the cholera spirillum preserved its vitality in distilled water for twenty days, in sewer water (of Marseilles) thirty-eight days, in water of the harbor for eighty-one days. The numerous experiments recorded by the observers named, and by Bolton, Hueppe, Hoch- stetter, Maschek, Kraus, and others, show that while the cholera spirillum may sometimes quickly die out in distilled water, in other experiments it preserves its vitality for several weeks (Maschek), and that it lives still longer in water of bad quality, such as is found in sewers, harbors, etc. Bolton found that for its multiplication a water should contain at least 40 parts in. 100,000 of organic material, while the typhoid bacillus grew when the proportion was considerably less than this— 6.7 parts in 100,000. Russell has recently (1891) studied the bacterial flora of the Gulf of Naples, and of the mud at the bottom of this gulf, col- lected at various depths up to eleven hundred metres. His inves- tigations show that sea water does not contain as many bacteria as an equal volume of fresh water ; that bacteria are found in about equal numbers in water from the surface and in that from various depths ; that the mud at the bottom constantly contains large num- bers of bacteria ; that some of the species isolated grow best in a culture medium containing sea water. At a depth of 50 metres the water contained 121 bacteria per cubic BACTERIA IN WATER. 565 centimetre, and the mud from the bottom 245,000 ; at 100 metres the water contained 10 and the mud 200,000 per cubic centimetre ; at 500 metres the water contained 22 and the mud 12,500 per cubic centimetre ; at 1,100 metres the mud contained 24,000. The following new species were obtained by Russell from the source mentioned : Bacillus thalassophilus, Cladothrix intricata, Bacillus granulosus, Bacillus limosus, Spirillum marinum, Bacillus litoralis, Bacillus halophilus. The bacterial flora of fresh and sea water is very extensive, as will be seen by the following list of species which have been described by various bacteriologists who have given their attention to its study : NON-PATHOGENIC MICROCOCCI. Micrococcus aurantiacus (Colin), Micrococcus luteus (Cohn), Micrococcus violaceus (Cohn), Micrococcus flavus liquefaciens (Fliigge), Micrococcus fla- vus desideiis (Fliigge), Micrococcus radiatus (Fliigge), Micrococcus cinnaba- reus (Fliigge), Micrococcus flavus tardigradus (Fliigge), Micrococcus versi- color (Fliigge), Micrococcus agilis (Ali-Cohen) , Micrococcus fuscus (Maschek), Diplococcus luteus (Adametz), Pediococcus albus (Lindner), Micrococcus cerasinus siccus (List), Micrococcus citreus (List), Micrococcus aquatilis (Bolton), Micrococcus fervidosus (Adametz), Micrococcus plumosus (Brauti- gam), Micrococcus viticulosus (Katz), Micrococcus cremoides (Zimmermann), Micrococcus carneus (Zimmermann), Mici'ococcus concentricus (Zimmer- mann), Micrococcus rosettaceus (Zimmermann), Micrococcus ureae (Pasteur), Weisser Streptococcus (Maschek), Wurmformiger Streptococcus (Maschek), Micrococcus aerogenes (Miller), Sarcina alba, Sarcina Candida (Reinke), Sarcina lutea. PATHOGENIC MICROCOCCI. Staphylococcus pyogenes aureus (Rosenbach), Micrococcus of Heydeii- reich — " Micrococcus Biskra." NON-PATHOGENIC BACILLI. Bacillus arborescens (Frankland), Bacillus viscosus (Frankland), Bacil- lus aquatilis (Frankland), Bacillus liquidus (Frankland), Bacillus nubilis (Frankland), Bacillus vermicularis (Frankland), Bacillus aurantiacus (Frankland), Bacillus cceruleus (Smith), Bacillus glaucus (Maschek), Bacil- lus albus putidus (Maschek), Bacillus fluorescens liquefaciens, Bacillus flup- rescens nivalis (Schmolck), Bacillus lividus (Plagge and Prpskauer), Bacil- lus rubidus (Eisenberg), Bacillus sulfureum (Holschewnikoff), Bacillus violaceus, Bacillus gasoformans (Eisenberg), Bacillus liquefaciens (Eisen- berg), Bacillus phosphorescens indicus (Fischer), Bacillus phosphorescens indigenus (Fischer), Bacillus phosphorescens gelidus (Katz), Bacillus sma- ragdino-phosphoresceiis (Katz), Bacillus argenteo-phosphorescens Nos. I., II., and III. (Katz), Bacillus cyaneo-phosphorescens (Katz), Bacillus ar- genteo-phosphorescens liquefaciens (Katz), Bacillus ramosus, Bacillus sub- tilis (Ehrenberg), Proteus sulfureus (Linden born), Bacillus aureus (Ada- metz), Bacillus brunneus (Adametz), Bacillus flavocoriaceus (Adametz), Bacillus fluorescens noii-liquefaciens, Bacillus latericeus (Adametz), Bacillus stolonatus (Adametz), Bacillus berolinensis indicus (Classen), Bacillus ery- throsporus (Eidam), Bacillus luteus (List), Bacillus aquatilis sulcatus Nos. 1, 2, 3, 4, and 5 (Weichselbaum), Bacillus albus (Eisenberg), Bacillus multi- ped iculosus( Fliigge), Bacillus Ziirnianum (List), Bacillus fulvus (Zimmer- mann), Bacillus helvolus (Zimmermann), Bacillus ochraceus (Zimmer- 48 566 BACTERIA IN WATER. mann), Bacillus plicatus, Bacillus devorans (Zimmermann), Bacillus gracilis (Zimmermann), Bacillus guttatus (Zimmermann), Bacillus implexus (Zim- mermann), Bacillus punctatus (Zimmermann), Bacillus radiatus aquatilis (Zimmermann), Bacillus vermiculosus (Zimmermann), Bacillus constrictus (Zimmermann), Bacillus fluorescens aureus (Zimmermann), Bacillus fluo- rescens longus (Zimmermann), Bacillus fluorescens tenuis (Zimmermann), Bacillus fuscus (Zimmermann), Bacillus rubefaciens (Zimmermann), Bacil- lus subflavus (Zimmermann), Bacillus janthinus (Zopf), Bacillus mycoides (Fliigge), Bacillus tremelloides (Tils), Bacillus cuticularis (Tils), Bacillus filiformis (Tils), Bacillus ubiquitus (Jordan), Bacillus circulans (Jordan), Bacillus superficialis (Jordan), Bacillus reticularis (Jordan), Bacillus ru- bescens (Jordan), Bacillus hyalinus (Jordan), Bacillus cloacae (Jordan), Bacillus delicatulus (Jordan), Bacillus violaceus laurentius (Jordan). PATHOGENIC BACILLI. bilis (Hauser), Bacillus canalis capsulatus (Mori), Bacillus canalis parvus (Mori), Spirillum cholerae Asiaticse (" Comma bacillus," Koch), Bacillus coli commums (Escherich), Bacillus hydrophilus _ fuscus (Sanarelli), Bacillus venenosus (Vaughan), Bacillus yenenosus bre vis (Vaughan), Bacillus vene- nosus invisibilis (Vaughan), Bacillus venenosus liquefaciens (Vaughan). 3USU11 III. BACTERIA IN THE SOIL. SURFACE soil, and especially that which is rich in organic matter, contains very numerous bacteria of many different species. Some of these are of special interest on account of their pathogenic power. Thus the bacillus of malignant oedema and the bacillus of tetanus have been shown to be widely distributed species, which have been obtained by investigators in various parts of the world by inoculating susceptible animals — guinea-pigs or mice — with a little rich surface soil. Other species are interesting because of their action in. nitrifi- cation and in the destructive decomposition of organic material by which it is fitted for assimilation by the higher plants. Many of the bacteria present in the soil are strictly anaerobic, and in attempts to estimate the number and kind of microorganisms present in a given sample this fact must be kept in view. The simplest method of studying the bacteria in the soil consists in introducing a small quantity into liquefied gelatin in test tubes, and, after carefully crushing it with a sterilized glass rod and thor- oughly mixing it with the gelatin, making roll tubes in the usual way. Some of these should be put up for anaerobic cultures — i.e., the tube should be filled with an atmosphere of hydrogen according to Frankel's method. If the object in view is to estimate the num- ber of bacteria in a given sample of soil the difficulty is encountered that, however finely crushed, the little masses of earth are likely to contain numerous bacteria, and we cannot safely assume that each colony originates from a single germ. Thoroughly washing a small quantity of soil, by agitation, in a considerable quantity of distilled water, and then adding a definite quantity of the water to nutrient gelatin and making roll tubes or plates, as in water analysis, sug- gests itself as a simple method ; but Frankel has shown that it is far from being reliable when the object is to estimate the number of bacteria. He obtained more uniform and accurate results by intro- ducing the earth at once into liquefied gelatin and crushing it as thoroughly as possible with a strong platinum wire, after which as thorough a mixture as possible was effected by tilting the tube up 568 BACTERIA IN THE SOIL. and down. But for the purpose of obtaining pure cultures from sin- gle colonies of the various species present, we should prefer to wash the earth in distilled water and to allow the sediment to settle before taking a portion of the water to add to the nutrient medium. In some experiments made in 1881 Koch ascertained that in soil which had not been disturbed but few bacteria were to be found at the depth of a metre; and this fact has since been established by the extended researches of Frankel, who devised a special boring instru- ment for obtaining samples of earth from different depths. Miquel, in 1879, estimated the number of bacteria in one gramme of earth collected in the park of Montsouri, Paris, at a depth of twenty centi- metres, at 700,000; and in a cultivated field which had been treated with manure, at 900,000. The following results were obtained by Adametz : One gramme of earth from a sandy soil contained at the surface 380,000, at a depth of twenty to twenty-five centimetres 400,000 ; the same quantity of clayey soil contained at the surface 500,000, at a depth of twenty to twenty-five centimetres 460,000. In experiments made by Beumer (1886) and by Maggiora (1887) considerably greater numbers were found, but the last-named ob- server, in some instances at least, kept the earth for some time after collecting it, which may have materially influenced the result. Beumer obtained from a specimen of sandy humus taken from a depth of three metres 45,000,000 to the gramme ; at four metres, 10,000,000; at five metres, 8,000,000; at six metres, 5,000,000. These specimens were obtained from the vicinity of hospitals at Greifswald. In a churchyard, at a depth of four metres, the num- ber in one experiment was 1,152,000, and in another 1,278,000. Frankel has given special attention to the examination of undis- turbed soil not in the immediate vicinity of dwellings. In samples from a fruit orchard near Potsdam he found that the superficial layers contained from 50,000 to 350,000 germs per cubic centimetre. The greatest number was not immediately upon the surface, but at from one-quarter to one-half metre below the surface. The num- ber was found to be greater in summer than in winter, the maximum being in July and August. At a depth of three-quarters of a metre to a metre and a half there was a very great and abrupt diminution in the number of germs. From 200,000 atone-half metre the number fell to 2,000 at a depth of a metre, from 250,000 at three-quarters of a metre to 200 at one metre, etc. , and at a depth of one and one-half metres, in some instances, no more living germs were obtained. In other experiments a few colonies developed from earth obtained at a depth of three or four metres, but these were slow in making their appearance, and often several days, or even Aveeks, elapsed before they became visible in Esmarch roll tubes. In experiments with sur- BACTERIA IX THE SOIL. 569 face soil, on the contran*, a multitude of colonies developed within twenty-four to forty-eight hours, and, as many liquefying bacteria were present, it was necessary to make the enumeration on the first or second day, at which time, no doubt, many of the bacteria present had not yet formed visible colonies. The results obtained have, therefore, only a relative value. The most important fact developed by Frankel's researches is that in virgin soil there is a dividing line at a depth of from three-quarters to one and one-half metres, below which very few bacteria are found, and that, consequently, the " ground- water region " is free from micro- organisms, or nearly so, notwithstanding the immense numbers pre- sent in the superficial layers. The extended researches of Maggiora, made in the vicinity of Turin, led him to the following conclusions : 1. The number of germs in desert and forest soil§ is much smaller, other conditions being equal, than in cultivated lands, and in these it is less than in inhabited localities. 2. In desert soils the number of germs bears a relation (a) to the geologi- cal epoch to which the lands belong, and, within certain limits, to the heignt above the level of the sea — the older the soil and the greater the altitude, other things being equal, the fewer the germs ; (6) to the compactness and aeration of the soil — the more compact and impermeable to air the smaller the number of germs capable of developing in gelatin ; (c) to the nature of the soil — sandy soils contain fewer germs than soils rich in clay and in humus. 3. In cultivated lands the number of germs augments with the activity of cultivation and the strength of the fertilizers used. 4. In inhabited localities the number of germs in the superficial layers is very great. In the deep layers it usually diminishes rapidly, as is the case in all other soils. As to the kinds of bacteria present, and their biological characters and functions in preparing organic material for assimilation by the plants whose roots penetrate the soil, we have yet much to learn. Frankel remarks that the species most frequently encountered in the deeper strata of the soil were three bacilli which also abound in the superficial layers — viz., the " hay bacillus," the "wurzel bacillus," and the "hirnbacillus." In all eleven bacilli were isolated and cul- tivated. Micrococci were only found four times, and spirilla not at all. Mould fungi were more abundant, and especially one previously obtained from the air by Hesse and called by him "brauner Schim- melpilz." Anaerobic bacilli, contrary to expectation, were not ob- tained in FrankeFs researches, and no pathogenic species were found in the deeper layers of the soil. We have already referred to the fact that the bacillus of malignant oedema and the bacillus of tetanus, two pathogenic, anaerobic species, are common in rich surface soil in various parts of the world. 570 BACTERIA IN THE SOIL. The results obtained in the researches referred to, in which nutri- ent gelatin was used as a culture medium, are no doubt very in- complete, not only on account of the liquefaction of the gelatin by common liquefying bacilli before other species present have formed visible colonies, but also because this is not a favorable culture me- dium for some of the species present in the soil. Thus Prankland has succeeded in isolating a nitrifying ferment which he calls " Bacillo- coccus," which grows abundantly in bouillon, but fails to grow in nutrient gelatin. Winogradski has also obtained in pure cultures a nitrifying ferment from the soil in the vicinity of Zurich, which he has called " Nitromonas." Comparatively few micrococci are found in the soil, while in the air they are usually found to be more abundant than bacilli. This is perhaps due to the fact that the bacilli are more promptly destroyed by desiccation and the action of sunlight. Several bacteriologists have made investigations relating to the duration of vitality of pathogenic bacteria in the soil. Frankel found that in Berlin the bacillus of anthrax, in Esmarch roll tubes, when buried m the soil at a depth of two metres, only occasionally gave evidence of growth, and at three metres no development occurred. The comparatively low temperature at this depth was no doubt an important factor in influencing the result. The cholera spirillum in the months of August, September, and October grew at a depth of three metres, but in the remaining months of the year failed to grow at two, while growth occurred at one and one-half metres. The bacillus of typhoid fever grew at three metres during the greater portion of the year. Giaxa has made extended and interesting experiments with the cholera spirillum, cultures of which he added to different kinds of soil (garden earth, clay, sand) and placed at different depths below the surface — one-quarter, one-half, and one metre. Some of the earth was sterilized and some was not. In the unsterilized earth he found the cholera spirillum in considerable numbers at the end of twenty- four hours at the greatest depth tested (one metre), but at the end of forty- eight hours it had disappeared in five experiments out of seven — the lowest temperature at this depth was 20° C. In the sterilized soil the result was different ; the cholera spirillum was present in enormous numbers at the end of four days at a depth of a metre, and was still found in smaller numbers at the end of twelve days, but had disappeared at the end of twenty-one days. These results indicate that the presence of common saprophytes in the soil is prejudicial to the development of the cholera spirillum, and that under ordinary circumstances it succumbs in the struggle for existence with these more hardy microorganisms. BACTERIA IN THE SOIL. 571 The recently published researches of Proskauer (1891) confirm those of Frankel and others as to the rapid diminution in the number of bacteria in the deeper layers of the soil. They also agree with those of Gartner in showing that in the soil of churchyards the number of bacteria diminishes greatly in the soil beneath the layer containing coffins. In general the influence of dead bodies upon the bacteria in the soil in the vicinity of coffins was very slight ; in the subsoil of the graveyard there were not many more bacteria than in similar soil outside of this. Reimers had previously shown that samples of earth from two graves, in one of which the body had been buried for thirty-five years and in the other for one and one- half years, gave similar results when examined by bacteriological methods. Manfredi has recently (1892) published the results of his extended investigations relating to the dust in the streets of Naples. The number of bacteria varied greatly in different parts of the city. In streets where the traffic was least and hygienic conditions the best the average number was 10,000,000 per gramme. In dirty and busy thoroughfares the average was 1,000,000,000, and in certain localities the number was even five times as great as this. Injections into guinea-pigs gave a positive result in seventy-three per cent of the animals experimented upon. Among the known pathogenic bacteria obtained in this way were the pus cocci (in eight), the Bacillus tuber- culosis (in three), the bacillus of malignant oedema, and the tetanus bacillus. In the recently published memoir of Fiilles (1891) the following species are described as having been found by him in the soil at Freiburg, Germany: MICROCOCCI. (a) Non-liquefying. — Micrococcus aurantiacus (Colin), Micrococcus can- didus (Cohn), Micrococcus luteus (Cohn), Micrococcus candicans (Flugge), Micrococcus versi color (Flugge), Micrococcus cirftiabareus (Fliigge), Micro- coccus cereus albus (Passet), Micrococcus fervitosus (Adametz), Rother coc- cus (Maschek). (6) Liquefying. — Micrococcus flavus liquefaciens (Flugge), Micrococcus vus desidens (Flugge), Diplococc flavus desidens (Flugge), Diplococcus luteus (Adametz), Sarcina lutea. NON-PATHOGENIC BACILLI. (a) Non-liquefying.— Bacillus fluorescens putidus (Flugge), Bacillus mus- coides (Liborius), Bacillus scissus (Frankland), Bacillus candicans, Bacillus diffusus (Frankland), Bacillus filiformis (Tils), Bacillus luteus (Fliigge), Fluorescent water bacillus (Eisenberg), Bacillus viridis pallescens (Frick), Bluish-green fluorescent bacillus (Adametz), Bacillus stolonatus (Adametz), Bacillus Ziirnianum (List), Bacillus aerogenes (Miller), Bacillus No. 1 and Bacillus No. 2 (Fiilles). (6) Liquefying. — Bacillus ramosus liquefaciens (Fliigge), Bacillus liqui- dus (Frankland), Bacillus ramosus — "wurzel bacillus," Bacillus subtilis 572 BACTERIA IN THE SOIL. (Ehrenberg), Bacillus mesentericus fuscus (Fliigge), Bacillus mesentericus vulgatus (Fliigge), Bacillus fluorescens liquefaciens (Fliigge), Lemon-yellow bacillus (Maschek), Green yellow bacillus (Eisenberg), Gas-forming bacillus (Eisenberg), Gray bacillus (Maschek), Bacillus prodigiosus (Ehrenberg), Proteus mirabilis (Hauser), Proteus vulgaris (Hauser), Bacillus mesentericus vulgatus, Bacillus cuticularis (Tils), " Weisser bacillus" (Eisenberg). (c) Pathogenic. — Bacillus cedematis maligni (Koch). In addition to the above the following species have been described by other authors: Bacillus liquefaciens magnus (Liideritz), Bacillus radiatus (Liideritz), Bacillus solidus (Liideritz), Bacillus mycoides roseus (Scholl), Bacillus viscpsus (Frankland), Bacillus candicans (Frankland), Bacillus poliformis (Liborius), Clostridiutn fcetidum (Liborius). Pathogenic species. — Staphylococcus pyogenes aureus (Eosenbach), Ba- cillus tetani(Nicolaier), Streptococcus septicus (Nicolaier), Pseudo-oedema ba- cillus (Liborius), Bacillus septicus agrigenus (Nicolaier), Bacillus of Utpadel. IV. BACTERIA OF THE SURFACE OF THE BODY AND OF EXPOSED MUCOUS MEMBRANES. GREAT numbers of bacteria of various species multiply upon the surface of the human body, where they find the necessary pabulum in the excretions from the skin and the exfoliated epithelium. Evi- dently the number will be largely influenced by the clothing worn, the atmospheric conditions as to heat and moisture, personal habits, etc. The writer has frequently inoculated culture media with a drop of sterilized fluid which had been placed upon the surface of the body of patients in hospitals and of healthy persons. By friction with a platinum needle at the point where the drop of fluid is applied the surface is washed and a little epithelium detached. Cultures may always be obtained by inoculating nutrient media from a drop of fluid applied in this way. Micrococci of various species, including the pus cocci, are very commonly encountered ; sarcinaB and various bacilli are also frequently met with. Even the hands, which by reason of their exposure and frequent ablutions are freer from exfoliated epi- thelium than portions of the body covered with clothing, have con- stantly attached to their surface a considerable number of bacteria. This is shown by the experiments of Kummel and Forster, of Fiir- bringer and others, with reference to the disinfection of the hands. Forster found that after the most careful cleaning of the hands with soap, water, and a brush, contact of the fingers with nutrient gelatin always resulted in the development of a greater or less number of colonies. Bordoni-Uffreduzzi, in his researches relating to the bacteria of the skin, obtained in pure cultures five different species of micrococci and two bacilli. Pure cultures of his Bacterium graveolens, which was usually found between the toes, gave off a disagreeable odor like that observed from this locality in certain individuals. In his re- searches made in Havana the writer frequently encountered in cul- tures from the surface, associated with various micrococci, his Micro- coccus tetragenus versatilis. Fiirbringer found quite frequently in the spaces beneath the fin- 574 BACTERIA OF THE SURFACE OF THE BODY ger nails Staphylococcus pyogenes aureus associated with various other microorganisms. A similar result had previously been reported by Bockhart. In his examinations of water from various sources Miquel found that "wash- water" from the floating laundries on the Seine con- tained more bacteria than water from any other source, even than the water of the Paris sewers. His enumeration gave twenty-six million germs per cubic centimetre. Hohein has enumerated the colonies developing from undercloth- ing worn for various lengths of time and made of different kinds of material. A piece of the goods to be tested was sewed fast to the underclothing, so as to come in immediate contact with the body ; at the end of a given time a fragment one-quarter of a centimetre square was cut up as fine as possible and distributed in nutrient gelatin. Plates were made and the colonies counted at the end of five or six days. In an experiment in which sterilized woven goods were worn next to the skin of the upper arm the following results were obtained : Linen goods, at the end of one day 28, two days 4,180 colonies ; cot- ton goods, end of one day 105, end of two days 1,870 ; woollen goods, end of one day 606, end of two days 6,799. When the material had been in contact with the skin for four days the colonies which devel- oped were so numerous that they could not be counted. Maggiora isolated twenty-two species of bacteria from his cultures inoculated with epidermis from the foot. None of these proved to be pathogenic for mice, rabbits, or guinea-pigs. Several gave off a strong odor of trimethylamin, similar to that of sweating feet. The following species have been found upon the surface of the body : Non-pathogenic. — Diplococcus albicans tardus (Unna and Tommasoli), Diplococcus citreus liquefaciens (Unna and Tommasoli), Diplococcus flavus liquefaciens tardus (Unna and Tommasoli). Staphylococcus viridis flaves- cens (Gruttmanti), Bacillus graveolens (Bordoni-Uffreduzzi), Bacillus epider- midis (Bprdoni), Ascobacillus citreus (Unna and Tommasoli), Bacillus fluo- rescens liquefaciens minutissimus (Unna and Tommasoli), Bacillus aureus (Unna and Tommasoli), Bacillus ovatus minutissimus (Unna and Tomma- soli), Bacillus albicans pateriformis (Unna and Tommasoli), Bacillus spini- ferus (Unna and Tommasoli), Bacillus of Scheurlen, Micrococcus tetragenus versatilis (Sternberg), Bacillus Havaniensis liquefaciens (Stern berg). Pathogenic. — Staphylococcus pyogenes albus, Staphylococcus pyogenes aureus, Streptococcus pyogenes, Diplococcus of Demme, Bacillus of Uemme, Bacillus of Schimmelbusch, Bacillus of Tommasoli, Bacillus saprogenes II. (Rosenbach), Bacillus parvus ovatus (Loffler). SURFACE OF MUCOUS MEMBRANES. Cultures made from the conjunctives, of healthy persons usually show the presence of various micrococci, and sometimes of bacilli. AXD OF EXPOSED MUCOUS MEMBRANES. 575 In diseased conditions these are more numerous than in health, but the pus cocci are not infrequently found in healthy eyes. As bacteria are constantly present in the air, they are necessarily deposited upon the moist mucous membrane of the nose during in- spiration. Indeed, it would appear as if an important function of this extended mucous membrane is to purify the air from suspended particles, and it has been shown by experiment that expired air is practically free from bacteria. The greater number of those con- tained in inspired air are deposited upon the mucous membrane of the anterior and posterior nares. In culture experiments made by Von Besser, Wright, and others the nasal mucus was found to con- tain a great variety of bacteria; among others the pus cocci were frequently found by both of the observers mentioned. In eighty-one cases Von Besser found the "diplococcus pneumonise" fourteen times, Staphylococcus pyogenes aureus fourteen times, Streptococ- cus pyogenes seven times, and Friedlander's bacillus twice. Twenty- eight of the cases examined were convalescents in hospital ; among these the pathogenic species mentioned were found less frequently than in other individuals. The following non-pathogenic species were isolated : Micrococcus liquefaciens albus in twenty-two cases, Micrococcus albus in nine cases, Micrococcus cumulatus tenuis in fourteen cases, Micrococcus flavus liquefaciens in three cases, Bacil- lus striatus albus in ten cases, etc. Paulsen (1890) made thirty-one cultures in nutrient gelatin from sixteen persons and thirty-three in nutrient agar from twenty-two persons, with the following result : Eleven remained sterile, nine- teen showed not more than ten colonies, sixteen less than one hun- dred, twelve more than one hundred, and in six the number was so great that they could not be counted. Micrococci were more nu- merous than bacilli ; of these a " sulphur-yellow coccus" in tetrads was found in eight individuals. Various species of liquefying cocci, resembling the pus cocci, were isolated, but the conclusion was reached that none of these were identical with the staphyloccoci of pus, which Von Besser and Wright both found in a considerable proportion of the culture experiments made by them. Very extended researches have been made with reference to the bacteria present in the human mouth, which show that numerous species are constantly present in the buccal secretions and upon the surface of the moist mucous membrane. Some of these are occa- sional and accidental, while others appear to have their normal habi- tat in the mouth, where the conditions as to temperature, moisture, and presence of organic pabulum are extremely favorable for their development. A minute drop of saliva spread upon a glass slide, dried, and stained with one of the aniline colors, will always be 576 BACTERIA OF THE SURFACE OF THE BODY found to contain an immense number of bacteria of various forms. Some of these are attached to epithelial cells and some scattered about singly or in groups. Among those seen in a single specimen we will usually find cocci in tetrads, in chains, and in irregular groups, bacilli of various dimensions, and occasionally spirilla. According to Prof. Miller, of Berlin, the following species "almost inva- riably occur in every mouth : Leptothrix innominata, Bacillus buccalis maximus, Leptothrix buccalis maxima, lodococcus vagina- tus, Spirillum sputigenum, SpirochaBte dentium. All of these fail to grow in ordinary culture media. Miller has made extended at- tempts to obtain cultures by varying the medium used and attempt- ing to imitate as nearly as possible the natural medium in which they are found ; but his attempts have been unsuccessful, or nearly so — " only line cultures afforded a limited growth, but the colonies never developed more than fifteen to twenty cells, and a transference to a second plate proved futile, no further growth taking place/' Up to the year 1885 Miller had isolated twenty -two different spe- cies of bacteria from the human mouth. Ten of these were cocci, five short bacilli, six long bacilli, and one a spirillum. Later the same author cultivated eight additional species. Vignal has iso- lated and described seventeen species obtained by him in pure cul- tures from the healthy human mouth ; most of these are bacilli, and Miller, who found micrococci to be more numerous, supposes the difference in results to be due to the fact that many of the cocci do not grow in nutrient gelatin, which was the medium employed by Vignal. In the researches of the last-named author the follow- ing species were obtained most frequently, in the order given : 1. Bacterium termo. 2. Bacillus e (Bacillus ulna ?). 3. Potato ba- cillus. 4. Coccus a. 5. Bacillus b. 6. Bacillus d. 7. Bacillus c (Bacillus alvei ?). 8. Bacillus subtilis. 9. Staphylococcus pyogenes albus. 10. Staphylococcus pyogenes aureus. Among the species above enumerated we find two of the most common pus cocci, Staphylococcus albus and aureus, but no mention is made of another important pathogenic micrococcus which is fre- quently found in the healthy human mouth, viz. , the micrococcus of sputum septicaemia, first named by the writer Micrococcus Pasteuri. This does not grow at ordinary temperatures, and consequently would not be obtained in gelatin plate cultures. Very different re- sults have been reported by different observers as to the frequency with which these pathogenic cocci are found in the buccal cavity. Black found in the saliva of ten healthy individuals the Staphy- lococcus pyogenes aureus seven times, Staphylococcus pyogenes al- bus four times, and Streptococcus pypgenes three times. On the other hand, Netter found Staphj'lococcus aureus only seven times in AND OF EXPOSED MUCOUS MEMBRANES. 577 one hundred and twenty-seven individuals examined. Miller also has rarely found the pus cocci in the mouths of healthy persons. Streptococcus pyogenes was not found by Vignal in his extended researches. The experiments of the writer, of Vulpian, Frankel, iSTetter, Claxton, and others show that the micrococcus which in 1885 I named Micrococcus Pasteuri, and which is identical with the ' ' diplococcus pneumonise " of German authors, is frequently present in the healthy human mouth— now called Micrococcus pneumonise crouposse. Netter examined the saliva of one hundred and sixty-five healthy individuals and obtained it in fifteen per cent of the number examined. Another pathogenic micrococcus which is frequently present in the mouths of healthy persons is the Micrococcus tetragenus of Koch. The following pathogenic bacteria have also been isolated and de- scribed : Bacillus crassus sputigenus (Kreibohm), Bacillus salivarius septicus (Biondi). The Streptococcus septo-pyaemicus of Biondi is described as having characters identical with those of the Strepto- coccus pyogenes of Rosenbach. Two other pathogenic species de- scribed by Biondi were each found in a single case only. Miller has described the following pathogenic species isolated and studied by him : Micrococcus gingivse pyogenes, Bacterium gingivse pyo- genes, Bacillus dentalis viridans, Bacillus pulpse pyogenes. Vignal has tested a considerable number of microorganisms, ob- tained by him in his cultures from the healthy human mouth, with reference to their peptonizing action upon various kinds of food, with the idea that some of them may have an important physiological function of this kind. Out of nineteen species he found ten which, after a longer or shorter time, dissolved fibrin, nine which dissolved gluten, ten which dissolved casein, and five which dissolved albu- min ; nine changed lactose into lactic acid, seven inverted cane sugar, seven caused the fermentation of glucose, and seven coagulated milk. Recently (1891) Sanarelli has shown that normal saliva has the power of destroying the vitality of a limited number of certain patho- genic bacteria, including the following species : Staphylococcus pyo- genes aureus, Streptococcus pyogenes, Micrococcus tetragenus, Bacillus typhi abdominalis, Spirillum choleras Asiaticse. When to ten cubic centimetres of saliva, sterilized by filtration through porce- lain, the above-mentioned pathogenic bacteria were added in small numbers by means of a platinum needle carried over from a pure culture, no development oscurred, and at the end of twenty-four hours the bacteria introduced were incapable of growth in a suitable medium. But when this amount of filtered saliva was inoculated with a large platinum loop — an ose — a certain number of the bacteria 578 BACTTCRIA OF THE SURFACE OF THE BODY survived, and at the end of three or four days an abundant develop- opment occurred. At first, however, the number of living cells was considerably diminished. In saliva to which one ose of a culture of Staphylococcus aureus was added thirteen thousand eight hundred and forty colonies developed in a plate made immediately after inocu- lation, while a plate made at the end of twenty-four hours contained but one hundred and thirty-two colonies, and one at the end of forty- eight hours had but eight colonies. Subsequently multiplication occurred, and a plate made on the ninth day after inoculation con- tained so many colonies that they could not be counted. The diphtheria bacillus was not destroyed in filtered saliva, but did not multiply in it. On the other hand, it proved to be a very favorable medium for the development of Micrococcus pneumonise crouposse. . Mucus from the surface of the meatus urinarius of man and woman, or from the vagina, will always be found to contain various bacteria ; but the bladder, the uterus, and Fallopian tubes in healthy individuals are free from microorganisms. Winter has isolated twenty-seven different species from vaginal and cervical mucus, and reports that he found Staphylococcus pyogenes albus in one-half of the cases examined. A streptococcus was also encountered which resembled Streptococcus pyogenes, although not positively identified with it. Samschin, on the other hand, failed to obtain the pus cocci in vaginal mucus from healthy women. Donderlein, Von Ott, and others have carefully examined the lochial discharge with reference to the presence of bacteria. The first-named author found that in healthy women the lochial discharge obtained from the uterus was free from germs, but when collected from the* vagina various microorganisms were obtained. In one case in which some fever existed Staphylococcus pyogenes aureus was found in the vagina, while the discharge from the uterus was free from germs. In five cases of puerperal fever Streptococcus pyogenes was obtained in the lochial discharge from the uterus. The results of Von Ott correspond with those of Donderlein. Czerniewski, in the lochia of fifty-seven healthy women, found the Streptococcus pyogenes but once, while in the lochial discharge of fatal cases of puerperal fever it was always present. Steffeck (1892) has examined the vaginal secretion of twenty-nine pregnant females who had not been subjected to digital examina- tion, and found Staphylococcus pyogenes albus in nine, Staphylo- coccus pyogenes aureus in three, and Streptococcus pyogenes in one. These results indicate that puerperal septicaemia from self-infection may occur in exceptional cases. In seventeen of the twenty-nine cases examined none of these pyogenic micrococci were found. AND OF EXPOSED MUCOUS MEMBRANES. 579' The following species have been obtained from the nasal and buccal secretions : FROM THE NOSE. Non-pathogenic. — Micrococcus nasalis (Hajek), Diplococcus coryzae (Hajek), Micrococcus albus liquefaciens (Von Besser), Micrococcus cumu- latus tenuis (Von Besser), Micrococcus tetragenus subflavus (Von Besser), Diplococcus fluorescens foetidus (Klamann), Micrococcus totidus (Klamanu), Vibrio nasalis (Weibel), Bacillus striatus flavus (Von Besser), Bacillus striatus albus (Von Besser). Pathogenic. — Staphylococcus pyogenes aureus, Staphylococcus pyogenes albus, Streptococcus pyogenes, Bacillus of Friedlander, Bacillus of rhino- scleroma (?), Bacillus foetidus ozaenae (Hajek), Bacillus mallei (Loffler), Ba- cillus smaragdinus foetidus (Reimannj. FROM THE MOUTH. Non-pathogenic. — Micrococcus roseus (Eisenberg), Micrococcus A, B, C, D, E of Podbielskij, Sarcinapulmonum (Hauser), Sarcina lutea, Micrococcus candicaiis (Fliigge), Bacillus of Miller, Bacillus virescens (Frick), Vibrio rugula, Vibrio lingualis (Weibel), Pseudo-diphtheria bacillus (Von Hoff- mann), Bacillus mesentericus vulgatus, Bacillus subtilis, Bacillus a, b, c, d, e, f, g, h, i, and y of Vignal, Bacillus subtilis similis, Bacillus radiciformis (Eisenberg), Bacillus luteus, Bacillus fluorescens non-liquefaciens, Bacillus ruber, Bacillus viridiflavus, Proteus Zenkeri, Bacillus G, H, I, J, K, L, M, N, and Vibrio O and P of Podbielskij, Vibrio viridans (Miller), Micrococcus nexifer (Miller), lodococcus magnus (Miller), Ascococcus buccaljs (Miller), Bacillus fuscans (Miller). Pathogenic. — Staphylococcus pyogenes albus, Staphylococcus pyogenes aureus, Staphylococcus salivarius septicus(Biondi), Streptococcus pyogenes, Micrococcus salivarius septicus (Biondi), Micrococcus tetragenus (Gaffky), Micrococcus gingivae pyogenes (Miller), Streptococcus septo-pysemicus (Bi- ondi), Streptococcus articulorum (Loffler), Micrococcus of Manfredi, Micro- coccus pneumoniae crouposae — ' ' Micrococcus Pasteuri " (Sternberg) ; Bacillus diphtherias (Loffler), Bacillus tuberculosis (Koch), Bacillus of Friedlander, Bacillus bronchitidse putridae (Lumnitzer), Bacillus septicaemias haemorrha- gicae, Bacillus gingivae pyogenes (Miller), Bacillus pulpse pyogenes (Miller), Bacillus dentalis viridans (Miller), Bacillus crassus sputigenus (Kreibohm), Bacillus saprogenes No. 1 (Rosenbach), Bacillus pneumoniae agilis* (Schou), Bacillus pneumoniae of Klein, Bacillus pneumosepticus (Babes). V. BACTERIA OF THE STOMACH AND INTESTINE. As the secretions of the mouth contain numerous bacteria, these must constantly find their way to the stomach, but conditions are not favorable for their development when the stomach is in a healthy state and its secretions normal. Under certain circumstances, how- ever, there may be an abundant development in the stomach of spe- cies which give rise to various fermentations, and no doubt dyspep- tic symptoms are frequently due to this cause. In the present section we are, however, only concerned with the bacteria of the healthy stomach. Most of these, we think, are to be considered as only temporarily and accidentally present in this viscus as the result of the swallowing of the buccal secretions and of food and drink con- taining them. The experiments of Straus and Wiirtz and of others show that normal gastric juice possesses decided germicidal power, which is due to the free hydrochloric acid contained in it. Hamburger (1890) found that gastric juice containing free acid is almost always free from living microorganisms, and that it quickly kills the cholera spirillum and the typhoid bacillus, but has no effect upon anthrax spores. Straus and Wiirtz found that the cholera spirillum is killed by two hours' exposure in gastric juice obtained from dogs, the typhoid bacillus in two to three hours, anthrax bacilli in fifteen to twenty minutes, and the tubercle bacillus in from eighteen to thirty- six hours. The experiments of Kurlow and Wagner, made with gastric juice obtained from the stomach of healthy men by means of a stomach sound, gave the following results : Anthrax bacilli with- out spores failed to grow after exposure to the action of human gas- tric juice for half an hour, but spores were not destroyed in twenty- four hours ; the typhoid bacillus was killed in one hour ; the cholera spirillum, the bacillus of glanders, and Bacillus pyocyanus were all destroyed at the end of half an hour ; the pus cocci showed greater resisting power. Certain bacteria have a greater resisting power for acids than any of those above mentioned, and some of them may consequently pass through the healthy stomach to the intestine BACTERIA OF THE STOMACH AND INTESTINE. 581 in a living condition, but there is good reason to believe that the spirillum of cholera or the bacillus of anthrax would not. On the other hand, the tubercle bacillus and the spores of other bacilli can, no doubt, pass through the stomach to the intestine without losing their vitality. Of nineteen species isolated by Vignal in his cultures from the healthy human mouth, the greater number resisted the action of the gastric juice for more than an hour, and six species which did not form spores were found to retain their vitality in gastric juice for more than twenty-four hours. In making a bacteriological analysis of the contents of the healthy stomach the more resistant microorganisms and those which form spores will naturally be found in greater or less numbers, inasmuch as some of them are likely to be present in food and water ingested. Van Puteren (1888) obtained a variety of microorganisms in very considerable numbers from the stomachs of infants fed upon un- sterilized cow's milk, but in healthy nursing infants the number was much smaller, especially when the mouth was washed out with dis- tilled water immediately before and after nursing. In 18 per cent of the cases no microorganisms were found under these circum- stances, and in 41 per cent the number fell below one thousand per cubic centimetre. Among the nursing infants examined (eighty- five) the following species were most numerous : Monilia Candida, Bacillus lactis aerogenes, a non-liquefying coccus, Staphylococcus pyogenes aureus, Bacillus subtilis. In infants fed upon cow's milk (eleven) Bacillus lactis aerogenes was present in 45.4 per cent of the cases, and Staphylococcus pyogenes aureus in 27.2 per cent, non- liquefying cocci in 54.4 per cent, liquefying cocci in 72. 7 per cent, Bacillus subtilis in 36.3 per cent, and Bacillus butyricus (Hueppe) in all of the cases ; next to these Bacillus flavescens liquefaciens was the most abundant. The author named reaches the conclusion that no species is constant and that the presence of those found de- pends upon accidental circumstances. Abelous (1889) found in his own stomach, washed out while fast- ing, a considerable number of species of bacteria, viz. : Sarcina ventriculi, Bacillus pyocyanus, Bacillus lactis aerogenes, Bacillus subtilis, Bacillus mycoides, Bacillus amylobacter, Vibrio rugula, and eight other undescribed bacilli and one coccus. All of these microorganisms were able to resist the action of hydrochloric acid in the proportion of 1.7 grammes in 1,000 grammes of water. Several were found to be facultative anaerobics. The action of the bacteria isolated by him was tested by Abelous upon various alimentary substances. The time required to effect changes, such as the digestion of fibrin, the changing of starch 49 582 BACTERIA OF THE STOMACH AND INTESTINE. into glucose, etc., was found to be so long that there was no reason to suppose that any one of the microorganisms tested was con- cerned in ordinary stomach digestion. In the intestine conditions are favorable for the development of many species of saprophytic bacteria, and the smallest quantity of excrementitious material from the bowels, spread upon a glass slide and stained with one of the aniline colors, will be found to contain a multitude of microorganisms of this class, of various forms. Among these are certain species which have their normal habitat in the intestine, and which may always be obtained in cultures from this source, while others, having been present in food or water in- gested, and having escaped destruction in the acid juices of the stomach, are accidentally and temporarily present. These latter may or may not increase in the organic pabulum which abounds in the intestine, according as the conditions are favorable or otherwise. The strictly aerobic bacteria could not multiply because of the ab- sence of oxygen, and the species encountered are for the most part anaerobics or facultative anaerobics. The Bacillus coli communis of Escherich, which is the most constant and abundant species found in the intestine of man and of certain of the lower animals, is a facul- tative anaerobic, which grows readily in the ordinary culture media, either in the presence of oxygen or in an atmosphere of hydrogen. But certain other bacteria of the intestine are strictly anaerobic and do not grow readily in the media commonly employed by bacteri- ologists. Escherich has shown that in new-born infants the meconium is free from bacteria. At the end of twelve to eighteen hours after birth bacteria appear in the alvine discharges, and the number is already considerable at the expiration of the first twenty -four hours of independent existence. The species first found are cocci and yeast cells which no doubt come from the atmosphere, having been de- posited upon the moist mucous membrane of the mouth and swal- lowed with the buccal secretions. When the meconium is replaced by "milk faeces" these contain in large numbers the Bacillus coli communis, heretofore spoken of as the most common species found in the intestine of adults. Another species associated with this, but not so abundant, is the Bacillus lactis aerogenes of Escherich. Other bacilli and cocci are found occasionally in smaller numbers. These bacilli do not liquefy gelatin, and, as a rule, the microor- ganisms found in the alvine discharges of healthy persons are non- liquefying bacteria. Escherich's researches led him to the conclu- sion that the Bacillus lactis aerogenes is constantly present in the small intestine of milk-fed children as the most prominent species, and that its multiplication there is favored by the presence of milk BACTERIA OF THE STOMACH AND INTESTINE. 583 sugar, and that Bacillus coli communis finds the most favorable conditions for its growth in the large intestine. Brieger, in 1884, isolated from faeces and carefully studied two bacilli, one of which has since been called by his name. This is a non-liquefying bacillus which is very pathogenic for guinea-pigs, and which in its morphology and characters of growth closely re- sembles the Bacillus coli communis of Escherich. Indeed, a num- ber of non-liquefying bacilli, differing but slightly in their morpho- logical and biological characters, have been obtained by various investigators from the alimentary canal of man and the lower ani- mals, and it is still a question whether they are to be regarded as distinct species or as varieties of the "colon bacillus " of Escherich. The bacillus obtained by Emmerich from cholera cadavers in Na- ples belongs to this group, and, if not identical with the colon bacil- lus, resembles it so closely that its differentiation is extremely diffi- cult. Brieger's bacillus forms propionic acid in solutions containing grape sugar. A second bacillus obtained by him from the same source resembles the " pneumococcus " of Friedlander ; this causes the fermentation of saccharine solutions, with production of ethyl alcohol. Bienstock (1883) isolated four species of bacilli from normal faeces, two of which are comparatively large and resemble Bacillus sub- tilis in their morphology and in the formation of spores. A third species is described as an extremely slender pathogenic bacillus, re- sembling the bacillus of mouse septicaemia. The fourth species is an actively motile bacillus which forms end spores, causing the rods to have the form of a drumstick. This is said to cause the decomposi- tion of albumin, with production of ammonia and carbon dioxide. Later researches do not sustain Bienstock's conclusion that the ba- cilli described by him are the principal forms found in normal faeces. Among the species encountered by Escherich, in addition to those . mentioned above (Bacillus coli communis and Bacillus lactis aero- genes), are the following : Proteus vulgaris, found three times in meconium, and constantly in the faeces of dogs fed upon flesh ; Strep- tococcus coli gracilis, found in meconium, but not during the period of nursing, is constantly present in the intestine when a flesh diet is employed. The intestine of carnivorous and omnivorous animals contains a greater number of bacteria than that of the herbivora, and in the large intestine they are far more numerous than in the small intes- tine (De Giaxa). Sucksdorf has enumerated the colonies developing from one milligramme of faeces from individuals on mixed diet. He obtained an average of 380,000 from a series of observations in which the maximum was 2,300,000 and the minimum 25J300. 584 BACTERIA OF THE STOMACH AND INTESTINE. The following species have been isolated from faeces and the con- tents of the intestine of cadavers : ^ Non-pathogenic. — Streptococcus coli gracilis (Escherich), Micrococcus aerogenes (Miller), Micrococcus tetragenus versatilis (Sternberg), Micrococ- cus ovalis (Escherich), "Yellow liquefying1 staphyiococcus " (Escherich), " Porzellancoccus " (Escherich), Bacillus subtilis, Bacillus aerogenes (Miller), Bacterium aerogenes (Miller), Bacillus lactis erythrogenes (Hueppe), Clostri- dium fcetidum (Liborius), Bacillus nauscoides(Liborius), Bacillus putriflcus coli (Bienstock), Bacillus subtilis similis I. and II. (Bienstock), Bacillus Zopfii, Bacillus liquefaciens communis (Stern berg), Bacillus in testinus lique- faciens (Sternberg), Bacillus intestinus motilis (Sternberg), Bacillus fluores- cens liquefaciens (Fliigge), "Colorless fluorescent liquefying bacillus'* (Escherich), "Yellow liquefying bacillus" (Escherich), Bacillus mesenteri- cus vulgatus, Bacilli of Booker, A to T, first series ; a to s, second series ; Bacilli of Jeffries A to Z, and «, /?. Pathogenic. — Staphyiococcus pyogenes aureus. Bacillus typhi abdo- minalis, Bacillus septicaemise haemorrhagicae, Bacillus of Belfanti and Pas- carola, Bacillus enteritidis (Gartner), Bacillus of Lesage, Bacillus pseuclo- murisepticus (Bienstock), Bacillus coli communis (Escherich), Bacillus lactis aerogenes (Escherich), Bacillus cavicida (Brieger), Bacillus of Emmerich, Bacillus coprogenesfcetidus(Schottelius), Bacillus of Utpadel, Bacillus leporis lethalis (Sternberg), Bacillus acidiformans (Sternberg), Bacillus cuniculicida Havaniensis (Sternberg), Bacillus cadaveris (Sternberg), Bacillus cavicida Havaniensis (Sternberg), Proteus vulgaris (Hauser), Bacillus tuberculosis^ Spirillum cholerse Asiaticse, Spirillum of Finkler and Prior. A . t. VI. BACTERIA OF CADAVERS AND OF PUTREFYING MATERIAL FROM VARIOUS SOURCES. THE putrefactive changes which occur so promptly in cadavers, when temperature conditions are favorable, result chiefly from post- mortem invasion of the tissues by bacteria contained in the alimen- tary canal. But it is probable that under certain circumstances microorganisms from the intestine may find their way into the cir- culation during the last hours of life, and that the very prompt putre- factive changes in certain infectious diseases in which the intestine is more or less involved are due to this fact. The writer has made numerous experiments in which a portion of liver or kidney re- moved from the cadaver at an autopsy made soon after death — one to six hours — has been enveloped in an antiseptic wrapping and kept for forty-eight hours at a temperature of 25° to 30° C. In every in- stance there has been an abundant development of bacteria, although as a rule none were obtained from the same material immediately after the removal of the organ from the body. This shows that a few scattered bacteria were present. The same result was obtained in cases of sudden death from accident, as from portions of liver or kidney removed from the bodies of persons dying of yellow fever, tuberculosis, and other diseases. Numerous researches show that the blood of healthy men and animals is free from bacteria, and that saprophytic bacteria injected into a vein soon disappear from the circulation ; and recent experi- ments show that blood serum has decided germicidal power. But in spite of this fact the experiments of Wyssokowitsch show that cer- tain bacteria injected into the circulation may be deposited in the liver, the spleen, and the marrow of the bones, and there retain their vitality for a considerable time. The spores of Bacillus subtilis were found by the observer named to preserve their vitality in the liver or spleen of animals into which they had been injected, for a period of two or three months. In the writer's experiments the microorgan- isms which first developed in fragments of liver preserved in an an- tiseptic wrapping were certain large anaerobic bacilli, and especially 586 BACTERIA OF CADAVERS AND OF my Bacillus cadaveris, together with the Bacillus coli communis of Escherich, my Bacillus hepaticus fortuitus, and other non-lique- fying bacilli of the "colon group." These bacteria did not give rise to a putrefactive odor, and the fragment of liver when cut into had a fresh appearance and a very acid reaction. Later, putrefactive changes occurred and Proteus FIG. 197.— Smear preparation from liver of yellow-fever cadaver, kept forty-eight hours in an antiseptic wrapping, x 1,000. From a photomicrograph. (Sternberg.) vulgaris and other putrefactive bacteria obtained the precedence. Evidently all of these species must have been present in the liver at the time it was removed from the cadaver, although in such small numbers that they were rarely seen in smear preparations or ob- tained in cultures from the fresh liver tissue. The appearance of a smear preparation from the interior of a fragment preserved for forty-eight hours in an antiseptic wrapping is shown in Fig. 197. The horribly offensive gases which are given off from dead ani- mals in a state of putrefaction appear to be due to certain large an- aerobic bacilli which are found in such material, and which have not yet been thoroughly studied owing to the difficulty of cultivating them in arti- VS ficial media ; among them is a large bacillus with c-ff* round ends which forms an oval spore at one ex- ^P ^. ^ tremity of the rather long rod. This the writer ^ ^Sfc has described under the name of Bacillus cada- FIQ. we. veris grandis, Fig. 198. In the interior of a putrefying mass of this kind only those bacteria are found which are able to grow in the absence of oxygen, but aerobic saprophytes may multiply upon the surface of PUTREFYING MATERIAL FROM VARIOUS SOURCES. 587 such a mass, or in organic liquids to which the air has free access. Among the most common putrefactive bacteria are the Proteus vul- garis, Proteus mirabilis, and Proteus Zenkeri of Hauser. Formerly the minute motile bacteria found in putrefying animal infusions, etc. , were commonly spoken of as belonging to the species " Bacterium termo," but recent researches show that several different species were included 'Under this name by those whose researches were made be- fore the introduction of Koch's method for isolating and differentiat- ing microorganisms of this class by the use of solid' culture media. The different species of Proteus are all facultative anaerobics. They are more or less pathogenic, and according to Hauser produce a chem- ical poison which, when injected into small animals, causes death with all of the symptoms of putrid intoxication. The bacillus of mouse septicaemia, which was first obtained by Koch from a putrefying meat infusion, is also pathogenic, as are the writer's Bacillus cadaveris and various other anaerobic bacteria found in putrefying material. Some account of the various products of putrefaction and the microorganisms concerned in their production will be found in Sec- tion IV., Part Second, of the present volume. VII. BACTERIA IN ARTICLES OF FOOD. Milk always contains bacteria, unless drawn with special precau- tions into a sterilized flask. In the healthy udder of the cow it is sterile, but in tuberculous cows, when the milk glands are involved, tubercle bacilli may find their way into the milk in considerable numbers. Ag ordinarily obtained and preserved, milk is greatly ex- posed to bacterial contamination from various sources ; desquamated cuticle from the external surface of the udder and from the hands of the milker, and floating particles from the air of the stable, fall into it at the very moment it is drawn, and it is subsequently contaminated by bacteria from the air, and from water used in washing the recep- tacles in which it is placed or added to it by the thrifty milkman. As it furnishes an excellent nutrient medium for many of the bacteria which are thus introduced into it, under favorable conditions of tem- perature it' quickly undergoes changes due to the multiplication in it of one or more of these microorganisms. The acid fermentation and coagulation of the casein which so constantly occurs is completely prevented by sterilizing fresh milk in flasks provided with a close- fitting cork or cotton air filter. Numerous researches have been made with reference to the microorganisms found in milk and the various fermentations to which they give rise. Naturally a great variety of species will be found in an extended research, but all are accidentally present, and only those demand special attention which produce the various fermentations of this fluid commonly encoun- tered, or which have special pathogenic properties. Several different bacteria produce an acid fermentation and con- sequent coagulation of milk, but the usual agent in producing this fermentation is the Bacillus acidi lactici, which is identical with the " ferment lactique " of Pasteur. When a pure culture of this bacillus is introduced into sterilized milk kept at a temperature of 25° to 30° C. , coagulation occurs in from fifteen to twenty-four hours. A uniform, gelatinous mass is produced which does not subsequently become dissolved (Adametz). Various other bacteria produce a similar change, including a number of common water bacteria, several spe- BACTERIA IN ARTICLES OF FOOD. 589 cies of sarcina, Staphylococcus pyogenes aureus, and other pus cocci. Usually coagulation is due to the combined action of several bacteria, among which Bacillus acidi lactici is apt to be the most prominent. Other bacteria produce coagulation without the lactic acid fer- mentation. This appears to be due to the formation of a soluble ferment which acts like rennet, causing the coagulation of milk which has a neutral or slightly alkaline reaction. The coagu- lated casein in this case is subsequently redissolved. The bacteria which produce this change for the most part form spores, while the lactic acid ferments do not. If, therefore, milk is heated nearly to the boiling point the acid-forming bacteria will be destroyed and the spores of the other species surviving will give rise to coagulation without the production of lactic acid. Among the more common microorganisms of this group are the Bacillus butyricus (Hueppe), Bacillus mesentericus vulgatus, Loffler's " white milk-bacillus," and the bacilli described by Duclaux under the generic name of Tyrothrix. Other fermentations are produced by certain chromogenic bacteria, and these, as a rule, are not as harmless from a sanitary point of view as those above referred to. Blue milk is produced by the presence of Bacillus cyanogenus, yellow milk by Bacillus synxanthus (Schroter) and by a species obtained by List from the faeces of a sheep and another found by Adametz in cheese. The well-known Bacillus prodigiosus produces its characteristic red pigment when present in milk, and a bluish-red color is caused by Bacterium lactis erythrogenes (Hueppe). Viscous fermentation in milk is produced by several different bac- teria, among others by a micrococcus studied by Schmidt-Muhlheim, and a short bacillus isolated by Adametz — Bacillus lactis viscosus. Milk which has undergone this change is unwholesome as food ; it is recognized by the long filaments which are produced when it is touched with any object and this is slowly withdrawn. The Caucasian milk ferment, Bacillus Caucasicus, produces a special fermentation, which has been referred to in Section IV. , Part Second (page 132). Various pathogenic bacteria have occasionally been found in milk in addition to the tubercle bacillus already referred to. Thus Adametz found Staphylococcus pyogenes aureus in two samples which had been submitted to him for examination, one of which had given rise to vomiting and diarrhoea. Wyssokowitsch cultivated from milk which had been standing some time a pathogenic bacillus, named by him Bacillus oxytocus perniciosus. The special microorganism which produces the poisonous pto- maine called by Vaughan tyrotoxicon has not yet been isolated ; nor do we know the exact cause of scarlet fever, although there is evi- 590 BACTERIA IN ARTICLES OF FOOD. dence that this disease has been spread by the use of contaminated milk, as have also diphtheria and typhoid fever, which diseases are due to bacilli now well known. As the cholera spirillum grows readily in milk, this disease could 110 doubt also be transmitted in the same way. Recently (1892) Sedgwick and Batchelder have examined a large number of specimens of milk obtained in Boston and vicinity, for the purpose of determining the number of bacteria present. They found, as an average of several trials, that milk obtained in a clean stable, from a well-kept cow, by milking in the usual way into a sterilized bottle, contained 530 bacteria per cubic centimetre. . " When, however, the milkman used the ordinary milk pail of flaring form, seated himself with more or less disturbance of the bedding, and vigorously shook the udder over the pail during the usual process of milking," the numbers were very much higher — on an average 30,500 per cubic centimetre immediately after milking. The average of fifteen samples taken from the tables of persons living in the suburbs of Boston was 69,143 per cubic centimetre. The average of fifty-seven samples of Boston milk, obtained directly from the milk wagons and plated at once, was 2,355,500 per cubic centimetre. The average of sixteen samples from groceries in the city of Boston was 4,577,000 per cubic centimetre. Prof. Renk found in the milk supply of Halle from 0,000,000 to 30,000,000 bacteria per cubic centimetre — a number considerably exceeding that usually found in the sewage of American cities (Sedg- wick). In fresh butter of good quality but few microorganisms are found, but in " cheesy butter" having a disagreeable odor Kreuger has found a great number of bacteria. Among these the most numerous were an oval coccus, Micrococcus acidi lactici (Kreuger), a slender bacillus closely resembling, and possibly identical with, the Bacillus fluorescens, and the Bacillus acidi lactici of Hueppe. Duclaux (1887) has isolated from different kinds of cheese no less than eleven different species of bacteria, which he believes are concerned in the " ripening process." Seven of these are aerobic and four anaerobic species. Adametz (1889) has also isolated and studied a number of species to which he attributes the ripening of cheese. 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. 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 BACTERIA IN ARTICLES OF FOOD. 591 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. 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 592 BACTERIA IN ARTICLES OF FOOD. 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 mesen- tericus vulgatus, which appears to have been the cause of the fer- mentation, which was produced in bread having a slightly alkaline reaction by inoculating it with a pure culture of this bacillus. The infected bread has a brownish color, a peculiar odor, and becomes sticky and viscid. Uffelmann (1890) has also studied the bacteria in spoiled rye bread, and obtained, in addition to common mould fungi, Bacillus mesente- ricus vulgaris and Bacillus liodermus. Bernheim (1888) has examined various grains used as food, with reference to the presence of bacteria, and claims to have demon- strated their presence by staining thin sections, and also by cultures, in corn, wheat, rye, barley, and peas. He supposes that they find their way from the earth through the roots and stems of plants. This appears to be very doubtful, in view of the researches of other observers, and further researches are necessary before we can accept the fact as demonstrated that they are usually present in healthy kernels of the grains mentioned. VIII. NON-PATHOGENIC MICROCOCCI. MANY of the saprophytic micrococci and bacilli have already been described in the sections devoted to pathogenic bacteria (Part Third). We propose at present to give an account of the morphological and biological characters which distinguish those microorganisms which have not been shown to possess pathogenic power. But it must be re- membered that in many instances the bacteria described in this and the following sections have not been tested at all, or only very im- perfectly tested, with reference to this point ; and no doubt some of them, if tested upon the various animals usually employed in experi- ments of this kind, would prove to be more or less pathogenic. On the other hand, many of the saprophytes heretofore described as pathogenic only produce marked morbid phenomena in susceptible animals when they are injected beneath the skin, into a serous cavity, or into the circulation in considerable quantities. The experiments of Buchner show that very many of the common saprophytes usually classed as non-pathogenic give rise to a local abscess when sterilized cultures are injected subcutaneously into rabbits or guinea-pigs. In short, there is no well-defined dividing line between the pathogenic and non-pathogenic bacteria, and some of those now described as non-pathogenic, as the result of more extended experiments, will no doubt eventually be transferred to the list of pathogenic bacteria. 159. MICROCOCCUS FLAVUS LIQUEFACIENS (Flugge). Found in the air and in water. Morphology. — Tolerably large micrococci, in pairs or in irregular groups. Biological Characters . — Anaerobic, liquefying, chromogenic micrococ- cus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms small, yellow colonies, which under a low power are seen to be spherical or oval, with a finely granular surface and a yellowish- brown color; lines radiate from the centre through a zone of transparent liquefied gelatin to the sharply defined border, and later the colonies, which have a diameter of four to six millimetres, resemble a wagon wheel. In gelatin stick cultures smooth, spherical, yellow colonies form upon the sur- face ; these become confluent and form a yellow layer, which by the slow liquefaction of the gelatin becomes depressed ; at the end of five days the gelatin is liquefied to a depth of about two millimetres and a yellowish, flocculent deposit is seen at the bottom of the yellowish-white fluid. Upon potato a deep-yellow layer with irregular margins is quickly developed. 594 NON-PATHOGENIC MICROCOCCI. 160. MICROCOCCUS FLAVUS DESIDENS (Fltigge). Found in air and water. Morphology. — Small micrococci, usually in pairs, but sometimes seen in groups of three or in short chains. Biological Characters. — An aerobic, liquefying, chromogenic micro- coccus. Grows slowly in the usual culture media at the room temperature. Upon gelatin plates the deep colonies appear as white or yellow points; under a low power they are seen as oval, yellowish-brown, finely granular discs. The superficial colonies are circular, with irregular margins, and are not elevated above the level of the gelatin ; by the fourth day they may attain a diameter of five to ten millimetres ; they have a brownish-yellow color ; the gelatin is gradually liquefied, and the colony which sinks below the surface is surrounded by a ring of liquefied gelatin from one to four millimetres broad. In gelatin stick cultures a slimy, yellowish- brown layer of limited extent is formed upon the surface, and a confluent, porcelain- white mass along the line of puncture ; at the end of eight days liquefac- tion has occurred under the superficial layer to a depth of three to four mil- limetres, forming a cylinder filled with a thick fluid, to the bottom of which the surface growth gradually sinks. Upon potato a slimy, yellowish-brown layer with irregular outlines is slowly developed. 161. MICROCOCCUS AGILIS (Ali-Cohen). Found in water. Morphology. — Micrococci, one n in diameter, usually in pairs, oc- casionally in tetrads or in chains ; have extremely slender flagella, which are four to five IJL in length. Biological Characters. — An aerobic, liquefying (very slowly), motile, chromogenic micrococcus. Grows at the room temperature in the usual cul- ture media — not in the incubator at 37° C. This micrococcus is distinguished by its active movements and by the presence of a longflagellum. which may be demonstrated by Loffler's method of staining. In gelatin stick cultures growth occurs along the line of inoculation, and liquefaction commences at the end of three to four weeks ; sometimes only a dry, funnel-shaped cavity is formed. Upon agar and upon potato a pink layer is slowly developed. 162. MICROCOCCUS FUSCUS (Maschek). Found in water. Morphology. — Micrococci, which are often elliptical, or even in the form of short rods (bacilli ?). Biological Characters. — An aerobic, liquefying, chromogenic micrococ- cus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms spherical colonies which under a low power present the appearance of being finely cleft and vary in color from pale-brown to black; liquefaction quickly occurs. In gelatin stick cultures but scanty growth occurs along the line of puncture ; upon the surface a sepia-brown layer is formed and the gelatin is quickly liquefied. Gelatin cultures have a strong putrefactive odor. Upon potato a slimy, brown layer is formed, which be- comes almost black. 163. DIPLOCOCCUS CITREUS CONGLOMERATUS (Bumm). Obtained from gonorrhceal pus and from the air — in dust. Morphology. — Diplococci, consisting of two hemispherical elements sepa- rated by a narrow cleft, and closely resembling the Micrococcus gonorrhoeas ; about 1.5 jj. in diameter; frequently in tetrads, usually united in conglomerate masses. Biological Characters. — An aerobic and facultative anaerobic, liquefy- NON-PATHOGENIC MICROCOCCI. 595 ing, chromogenic micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms lemon-yellow colonies, which throw out tongue-like projections upon the surface of the gelatin and have wave-like margins ; the surface is at first moist and shining, later cleft and scaly In gelatin stick cultures development occurs along the line of punc- ture and on the surface; liquefaction, beginning near the surface, progresses slowly ; a yellowish layer floats upon the surface, and later settles to the bottom of the tube. 164. DIPLOCOCCUS CITREUS LIQUEFACIENS (Unna). Found on the skin of persons suffering from eczema seborrhoeicum. Morphology. — Small, oval cocci, in pairs or in tetrads, often in irregular groups or in short chains ; the diameter of a single element in a pair is from 0.4 to 0.1 >«. Biological Characters. — An aerobic, liquefying, chromogenic micrococ- cus. Grows in the usual culture media at the room temperature. Upon gelatin plates, at the end of four days, the superficial colonies are grayish- white, flat, circular discs the size of a mustard seed ; at the end of eight days they are grayish-yellow, opaque, and about one to two millimetres in dia- meter; at the end of two weeks they are lemon -yellow, concave, and begin to sink into a shallow funnel of liquefied gelatin ; under a low power they are seen to be finely granular. The deep colonies appear at first as white points; under the microscope they are seen to be spherical or oval, brownish- yellow, and have sharply defined outlines. In gelatin stick cultures, at the end of six days, a thin, shining, yellowish layer has formed on the surface; at the end of two weeks the gelatin is softened and a thick, yellowish- white, flocculent deposit is seen, "while upon the surface of the liquefied medium is an irregular, plate-shaped, deep lemon-yellow layer ; at the end of three weeks the liquefaction has extended to a depth of about six millimetres ; the liquefied gelatin is opaque and of a yellow color. Upon the surface of agar a yellowish-brown layer with irregular margins is quickly developed; upon potato, at the end of two weeks, a grayish-yellow layer. 165. DIPLOCOCCUS FLAVUS LIQUEFACIENS TARDUS (Unna). Found upon the skin of individuals suffering from eczema seborrhoeicum. Morphology. — Biscuit-formed diplococci, resembling the " gonococcus" ; each element in a pair is from 0.5 to 0.8 /^ in diameter. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, chromogenic micrococcus. Grows in the usual culture media at the room temperature— very slowly upon gelatin and potato, more rapidly on agar. Upon gelatin plates, at the end of eight days, the superficial colonies are very small, circular, shining, pale grayish-yellow discs; at the end of three weeks they are as large as a hempseed and of a chrome-yellow color; later they become greenish-yellow and float in a circular zone of transparent, liquefied gelatin. The deep colonies are at first punctiform ; later they are small, opaque spheres of an olive brownish-yellow color. In gelatin stick cultures a thin, yellowish-white, slimy layer is slowly developed upon the surface ; at the end of three weeks this is from three to four millimetres in diameter and irregular in outline ; as it becomes older the color is dark- yellow or greenish-yellow ; a thin, yellowish growth develops along the line of puncture ; at the end of four weeks the surface is depressed without being really liquefied ; in eight weeks about half of the gelatin in the tube is lique- fied and ti'anspareiit. The surface growth first floats upon the liquefied me- dium ; later it settles to the bottom as a thick, flocculent, yellow deposit, and the gelatin acquires a yellow color. Upon the surface of agar a thick, slimy, yellowish white layer with wavy margins is developed ; later this has a greenish-yellow color. Upon potato a sulphur-yellow layer is formed. 500 NON-PATHOGENIC MICROCOCCI. 166. DIPLOCOCCUS FLUORESCENS FCETIDUS (Klamann). Obtained from the posterior nares. Morphology. — Diplococci, about 1.4 n in diameter (the pair), often ar- ranged in chains containing from six to ten elements. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, chromogenic micrococcus. Grows in the usual culture media at the room temperature — better at 37° C. Upon gelatin plates the superficial colonies are at first gray or brownish circular masses, which soon sink be- low the surface of the liquefying gelatin and later form a crater-like de- pression, in the centre of wnich is seen a brownish-gray sediment, while the surrounding gelatin has a grass-green or violet color. In gelatin stick cultures a circular, shallow, pale-gray, saucer-shaped cavity forms at the surface, and a purse-like pouch along the line of puncture ; a shining, iri- descent film floats upon the surface of the liquefied gelatin, and a greenish- gray sediment accumulates at the bottom ; finally the gelatin is completely liquefied and has a green color above, while a violet-colored film floats upon the surface. Upon agar a granular, brownish-gray layer is quickly devel- oped. Upon potato a finely granular layer, which after a time acquires a dark, bluish-green color, while the potato around it is colored blue. The color is changed to red by acids. 167. DIPLOCOCCUS LUTEUS (Adametz). Found in water, Morphology. — Micrococci, usually in pairs, of 1.2 to 1.3 fit in diameter ; sometimes observed in chains of eight to ten elements or in irregular groups. Biological Characters. — An aerobic, liquefying, motile, chromogenic micrococcus. Grows in the usual culture media at the room temperature. This micrococcus is described by Adametz as actively motile. Upon gelatin plates, at the end of three days, circular, pale-yellow, viscous colonies are developed, which have a diameter of about one millimetre. Under a low power they are seen to be granular and brownish-yellow in the centre, while the margins are pale-yellow ; at the end of six days the colonies are of an in- tense yellow color and about three millimetres in diameter. In gelatin stick cultures growth occurs rapidly upon the surface only, at first as a cir- cular, lemon-yellow layer marked with concentric circles; at the end of about ten days the gelatin at the surface acquires an intense brownish-red color, which extends downward in a cloud-like manner, gradually dimin- ishing in intensity ; liquefaction commences at the end of several weeks. Upon the surface of agar a viscid yellow layer is formed along the impf- strich, and the medium acquires a brownish-red color. Upon potato a dirty- yellow layer, which subsequently has a brownish color, is developed ; this gives off the characteristic odor of penicillum cultures. In milk coagula- tion of the casein is produced in about five days. 168. DIPLOCOCCUS ROSEUS (Bumm). Found in the air. Morphology.— Diplococci resembling the " gonococcus, " the elements of a pair being hemispherical and separated by a tolerably broad cleft; the diameter, measured from pole to pole, is from 1 to 1.5 n. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, chromogenic micrococcus. Grows in nutrient gelatin at the room temperature. Upon gelatin plates slightly elevated, pink colonies are de- veloped, which under the microscope are seen to be finely granular and ir- regular in outline. In gelatin stick cultures an abundant development oc- curs upon the surface and along the line of puncture ; this has a pink color ; the gelatin is slowly liquefied after a considerable time. NON-PATHOGEXIC MICROCOCCI. 597 169. MICROCOCCUS CREMOIDES (Zimmermann). Found in water. Morphology. — Micrococci, about 0.8 n in diameter, arranged in grape-like masses. Biological Characters. — An aerobic, liquefying, chromogenic micrococ- cus. Grows in the usual culture media at the room temperature. Upon gelatin plates the deep colonies are small and yellowish- white in color; under a low power they are seen to be spherical, granular, and yellow or brownish-gray. Superficial colonies have an irregular, ' ' gnawed " margin, and cause a saucer-like liquefaction of the gelatin, at the bottom of which a yellowish- white mass, arranged in concentric rings, may be seen ; around the margin delicate outgrowths into the unliquefied gelatin may be seen. In gelatin stick cultures liquefaction occurs along the line of puncture in three or four days; an air bubble is usually seen near the surface, and below this an accumulation of a yellowish-white color; the liquefied gelatin below this is transparent for some distance, and the bottom of the narrow channel is again filled with a yellowish-white mass of micrococci ; at the end of a week the liquefied channel measures about eleven millimetres at the surface and a yellowish- white film floats upon the liquefied gelatin. Upon the surface of agar a yellowish- white layer with irregular margins and a lustre like that of amber is developed. Upon potato a tolerably abundant, cream-colored layer extends over the surface. 170. MICROCOCCUS ROSEUS (Eisenberg). Found in sputum of a patient with influenza. Morphology. — Micrococci of 0.8 to 1 n in diameter, solitary or in irregular groups. Biological Characters. — An aerobic and facultative anaerobic, lique- fying, chromogenic micrococcus. Grows in the usual culture media at the room temperature, and at 37° C. without production of color. Upon gelatin plates, at the end of three to four days, minute pink colonies are formed ; later liquefaction commences about the colonies and progresses slowly. In gelatin stick cultures development occurs slowly both upon the surface and along the line of puncture ; the growth is at first colorless ; after three to four days a small, round, pink layer is formed, which is depressed in the centre ; at the end of a week the color resembles that of a red azalea blossom, and liquefaction commences ; at the end of three weeks the gelatin is about- half -liquefied and a pink sediment is seen. Upon the surface of agar, at the room temperature, a soft, dark-pink layer is formed along the impfstrich in two days ; at 37° C. a similar development occurs in twenty-four hours, but without color. Upon potato, at the end of three to four days, a cherry- red streak is seen along1 the impfstrich ; this gradually becomes darker and covers the entire surface ; the growth then resembles that of Bacillus pro- digiosus. 171. MICROCOCCUS AURANTIACUS (Cohn). Found in water. Morphology. — Spherical or slightly oval cocci, 1.3 to 1.5 >« in diameter, solitary, in pairs, or in irregular groups. Biological Characters. — An aerobic, non-liquefying, chromogenic mi- crococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates spherical or elliptical colonies of an orange-yellow color and smooth, shining surface. In gelatin stick cultures a small, button-like, yellow growth develops upon the surface, and after a considerable time mi- nute yellow colonies are seen along the line of puncture. Upon agar an orange-yellow layer is formed, and upon potato a slimy, yellow growth. 50 598 NON-PATHOGENIC MICROCOCCI. 172. MICROCOCCUS CERASINUS SICCUS (List). Found in water. Morphology. — Micrococci, from 0.25 to 0.32 /* in diameter, solitary or in pairs. Biological Characters. — An aerobic, non-liquefying, chromogenic mi- crococcus. Grows best at 37 C. Does not grow well in nutrient gelatin. Upon the surface of agar a dry, cherry-red layer is quickly developed. Upon potato the surface is quickly covered with a cherry-red layer. The pigment is not soluble in water, alcohol, or ether, and is not changed by acids or alkalies. 173. MICROCOCCUS VERSICOLOR (Fliigge). Found in water. Morphology. — Micrococci, in pairs or in irregular groups. Biological Characters. — Anaerobic, non-liquefying, chromogenic micro- coccus. Grows in the usual culture media at the room temperature. Upon gelatin plates the deep colonies are at first white points; later yellow, opaque, finely granular spheres. The superficial colonies are irregular in outline, slimy, and have a pear]y lustre; they may attain a diameter of two to ten millimetres. In gelatin stick cultures small, spherical, yellow colonies develop along the line of puncture, and a layer with irregular, "gnawed " margins and a pearly lustre upon the surface. Upon agar a slimy, opaque layer of a yellowish-brown color. Upon potato a slimy layer is quickly de- veloped. 174. MICROCOCCUS OF DANTEC. Obtained by Dantec (1891) from salted codfish which had undei'gone changes characterized by a red color and an offensive odor. Morphology. — Micrococci, from three to five n in diameter, often marked by a line of commencing binary division. Biological Characters. — An aerobic, non-liquefying, chromogenic micro- coccus. Forms a red pigment. In gelatin plates small, disc-shaped colo- nies of a red color are slowly developed ; these rarely measure more than a millimetre in diameter. In gelatin stick cultures development is slow ; along the line of puncture the growth has a yellowish color ; on the surface it is of a pale-red, and later of deeper-red color. Upon agar the development is more rapid than upon gelatin. It grows upon dried codfish without pro- duction of pigment, except when it is associated with other microorganisms — especially a liquefying coccus which is often found with it. Not pathogenic. 175. MICROCOCCUS CARNEUS (Zimmermann). Found in water. Morphology. — Micrococci, having a diameter of about 0.8 y«, united in irregular, grape-like masses. Biological Characters. — An aerobic, non-liquefying, chromogenic mi- crococcus. Grows best at the room temperature ; very scanty develop- ment at 30° to 33° C. Upon gelatin plates the deep colonies are small, spherical, and grayish-white in color. Superficial colonies are but slightly elevated, circular in outline, and of a grayish-red to pale-red color ; under the microscope they are seen as circular discs with a more opaque, reddish- gray centre surrounded by a somewhat more transparent zone, and this by a second, still paler zone ; in older cultures the distinct zones are no longer to be distinguished, but the reddish-brown color fades out from the centre towards the margin of the colonies. In gelatin stick cultures, at the end of five days, a thin, circular, pale-pink layer, with irregular outlines and about NON-PATHOGENIC MICROCOCCI. 599 3.5 millimetres in diameter, is developed upon the surface, and a finely granular, white growth along the line of puncture. Upon the surface of gelatin a flesh-red layer, which later acquires a violet hue, is formed along the impfstrich. Upon agar the growth is similar but more abundant, and the margins are coarsely toothed. Upon potato an abundant red layer is de- veloped. 176. MICROCOCCUS CINNABAREUS (Flligge). Found in air and in water. Morphology. — Large, spherical cocci, frequently associated in pairs or in tetrads. Biological Characters. — An aerobic, non-liquefying, chromogenic mi- crococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates the deep colonies are first seen as minute points at the end of four days ; under a low power they are seen to be oval or lenticular, with a well-defined contour and of a dark reddish-brown color. The super- ficial colonies, at the end of four days, are from 0. 5 to 1 millimetre in dia- meter and brick- red ; at the end of eight days they project from the gelatin in button-shape and are cinnabar-red. In gelatin stick cultures isolated white colonies are seen along the line of puncture at the end of four to five days, and upon the surface a button-like mass of moderate dimensions is de- veloped, which is first pink and later cinnabar-red. Upon potato a cinnabar- red layer is slowly developed. 177. MICROCOCCUS CEREUS ALBTis (Passet). Obtained by Passet (1885) in the pus of acute abscesses (two cases out of thirty-three examined), and by Tils (1890) from the Freiburg water supply. Morphology. — Large cocci, *1. 16 n in diameter, solitary or associated in irregular groups. Biological Characters. — An aerobic, non-liquefying micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms superficial colonies, which attain a diameter of one to two mil- limetres and resemble drops of stearin or white wax. In gelatin stick cul- tures grows upon the. surface as a grayish- white layer with irregular, thick- ened margins, resembling a drop of stearin; scanty growth along the line of puncture. Upon potato a dirty-white layer of moderate thickness is de- veloped. 178. MICROCOCCUS CEREUS FLAVus (Passet). Obtained by Passet (1885), in a single case out of thirty-three examined, from the pus of an acute abscess. Morphology. — Micrococci of irregular dimensions, associated in irregular groups and occasionally in chains. Biological Characters. — An aerobic, non-liquefying, chromogenic micro- coccus. Grows in the usual culture media at the room temperature. Upon gelatin plates lemon-yellow colonies are developed, which attain a diameter of one to two millimetres. In gelatin stick cultures the, growth around the •point of inoculation resembles a drop of stearin or wax with elevated mar- gins and has a yellow color ; a scanty yellow streak is developed along the line of puncture. Upon potato a citron-yellow layer is formed. 179. MICROCOCCUS CITREUS. Synonym. — Cremefarbiger micrococcus (List). Found in water. Morphology. — Large, spherical cocci, from 1.5 to 2.2/*in diameter, soli- .ary, in pairs, or in chains of eight or more elements. Biological Characters. — Anaerobic, non-liquefying, chromogenic micro- 600 NON-PATHOGENIC MICROCOCCI. coccus. Grows in the usual culture media at the room temperature — better at 37° C. Upon gelatin plates forms upon the surface dirty pale-yellow or cream-colored colonies, which after several days have a diameter of 0.5 to 0.8 centimetre and are about 0.5 millimetre thick; these have usually irre- gular outlines and a moist, shining appearance. In gelatin stick cultures very scanty growth occurs along the line of puncture. Upon agar a pale- yellow layer is formed. Upon potato, at 37° C., an abundant growth occurs, forming a yellow layer. 180. MICROCOCCUS FERVIDOSUS (Adametz). Found in water. Morphology, — Small, round cocci, 0.6 n in diameter, in pairs or in ir- regular groups. Biological Characters. — An aerobic, non-liquefying micrococcus. Grows in the usual media at the room temperature. Upon gelatin plates, at the end of four to five days, the deep colonies appear as white points, which under a low power have a pale-yellow color and resemble dewdrops; upon the sur- face transparent, yellow colonies with irregular, jagged edges are devel- oped ; later these are granular in the centre and have a brownish color, while the marginal zone is yellowish and slightly wrinkled. In gelatin stick cultures a thin, circular layer with finely toothed margins forms upon the surface, and a granular growth along the line of puncture. In glycerin- gelatin numerous gas bubbles of various sizes are developed in the medium. Upon agar a circular, milk-white, slimy layer is formed, which later has a pearly lustre. Upon potato a dirty- white layer with irregular margins. 181. MICROCOCCUS FLAVUS TARDIGRADUS (Fliigge). 'Found in the air and in water. Morphology. — Large, spherical cocci, usually associated in irregular groups ; sometimes have peculiar dark poles. Biological Characters. — An aerobic, non-liquefying, chromogenic mi- crococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates the deep colonies are spherical or oval, dark chrome- yellow, and from 0.4 to 0.6 millimetre in diameter; under a low power they appear to have a dark olive-green color. The superficial colonies are from 0.5 to 1 millimetre in diameter, have a smooth, varnished-looking sur- face, and are slightly elevated above the surface of the gelatin at the mid- dle ; under a low power the centre is grayish-yellow and the margin paler. In gelatin stick cultures, at the end of eight days, a row of small, spherical, isolated yellow colonies is developed along the line of puncture. 182. MICROCOCCUS LUTEUS (Cohn). Found in water. Morphology. — Oval cocci, from 1 to 1.2 /* in diameter, associated in zo- oglcea masses ; the intercellular substance is easily soluble in water. Biological Characters. — An aerobic, non-liquefying, chromogenic mi- crococcus. The yellow pigment produced is not soluble in water, ether, or alcohol, and is not changed by acids or alkalies. Grows in the usual cul- ture media at the room temperature. Upon gelatin plates sulphur-yellow, superficial colonies are developed, which have irregular outlines and may attain a diameter of 4 millimetres and a thickness of 0. 5 millimetre ; under a low power they are seen to be granular. In gelatin stick cultures a yel- low layer forms about the point of puncture and a granular growth along the line of inoculation. Upon agar a yellow, slimy layer. Upon potato an intensely yellow layer with irregular margins, which after a time has a wrinkled surface. NON-PATHOGENIC MICROCOCCI. 601 183. MICROCOCCTJS VIOLACEUS (Cohn). Found in water. Morphology. — Elliptical cocci, frequently united in chains. Biological Characters. — An aerobic, non -liquefy ing, chromogenic mi- crococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms superficial colonies of hemispherical form and violet color. In gelatin stick cultures scanty growth along the line of puncture, and upon the surface a hemispherical mass of violet-blue color. Upon agar a violet -blue layer. Upon potato a violet-colored streak is formed along the impfstrich. 184. STAPHYLOCOCCUS VIRIDIS FLAVESCENS (Guttmann). Found in the vesicles of varicella. Morphology. — Micrococci of irregular dimensions, solitary, in pairs, or in irregular groups; does not differ in morphology from Staphylococcus pyogenes aureus. Biological Characters. — An aerobic, non-liquefying, chromogenic mi- crococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates, at the end of two days, small, greenish-yellow colo- nies become visible ; under a low power these are seen to be spherical and slightly granular upon the surface — less so at a later date. In gelatin stick cultures growth occurs both upon the surface and along the line of punc- ture, of a grayish-yellow color. Upon agar, at the end of twenty- four hours at 37° C., a greenish-yellow growth occurs along the line of puncture. An abundant development occurs upon potato in twenty-four hours at 37° C. 185. MICROCOCCUS OCHROLEUCUS (Prove). Found in urine of man. Morphology. — Micrococci, from 0.5 to 0.8 /* in diameter, solitary, in pairs, or in short chains. Biological Characters. — An aerobic, non- liquefy ing, chromogenic micro- coccus. The pigment is soluble in alcohol, insoluble in water, and is decol- orized by acids. Grows in the usual culture media at the room temperature. Upon gelatin plates, at the end of twenty-four hours, small, colorless colo- nies are developed, surrounded by a somewhat elevated and wavy border ; later branching offshoots are given off from the margin and the centre ac- quires a sulphur-yellow color. In gelatin stick cultures a thin, colorless, superficial layer is quickly developed ; this in three or four days acquires a sulphur-yellow color. The growth upon potato is scanty and is scarcely visible before the fifth day. Old gelatin cultures give off a peculiar odor. 186. MICROCOCCUS ACIDI LACTICI LIQUEFACIENS (Kreuger). Found in " cheesy butter." Morphology. — Oval cocci, from 1 to 1.5 ft in diameter, frequently associ- ated in pairs or in tetrads. Biological Characters.— An aerobic smd facultative anaerobic, liquefy- ing micrococcus. Grows best at the room temperature. Upon gelatin plates small, white colonies are developed at the end of three days, which under the microscope are seen to have deeply cleft margins ; the gelatin about the colonies is gradually liquefied. In gelatin stick cultures a white, granular growth occurs along the line of puncture, and a funnel-shaped liquefaction of the gelatin occurs by the third day ; liquefaction progresses rapidly, and a dirty-white, slightly wrinkled layer forms upon the surface, while the gelatin below is clouded. In milk coagulation occurs in three days at 20° to 25° C., and lactic acid is formed; a clear layer of serum is seen above the homogeneous coagulated mass of casein, which is not subse- quently peptonized. 602 NON-PATHOGENIC MICROCOCCI. 187. MICROCOCCUS AEROGENES (Miller). Found in the alimentary canal. Morphology. — Large oval cocci. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing micrococcus. Grows in the usual culture media at the room tempera- ture. Upon gelatin plates forms spherical colonies of dark color and smooth contour. In gelatin stick cultures development occurs along the line of puncture, with a brownish-yellow color, and a flat, button-like, grayish- white, soft mass is formed upon the surface ; slight liquefaction occurs after some days. Upon the surface of agar a yellowish-white, pap-like layer is formed. The growth upon potato is similar to that upon agar. Possesses a great resistance against the action of acids. 188. MICROCOCCUS ALBUS LIQUEFACIENS (Von BeSSer). Very common in the nasal mucus of healthy persons. Morphology. — Micrococci, about twice as large as Staphylococcus pyo- genes albus, spherical or elliptical ; in irregular groups or in chains. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing micrococcus. Grows in the usual culture media at the room tempera- ture. In gelatin stick cultures produces a stocking-shaped pouch of lique- fied gelatin, and after a time complete liquefaction— sometimes only pai'tial and very tardy liquefaction. Upon agar plates white, shining colonies with an elevation at the centre and of the margin, with a depression be- tween, 0.5 centimetre or more in diameter; under a low power the centre appears brownish and is surrounded by a dark zone, this py a more trans- parent, grayish-brown zone, and finally by an opaque marginal ring. Upon potato a shining, white layer is developed. 189. MICROCOCCUS FCETIDUS (Klamann). Found in the posterior nares of man. Morphology. — Micrococci of irregular dimensions, solitary, in pairs, in short chains, or in irregular groups; the diplococci measure 1.4/* in dia- meter. Biological Characters. — An aerobic, liquefying micrococcus. Grows at the room temperature in the usual culture media — not so well in the incu- bating oven. Upon gelatin plates oval or spherical white colonies are slowly developed. In gelatin stick cultures a milk-white, shining, elevated mass, with a knobby surface, is developed about the point of puncture ; later this presents a central prominence surrounded by concentric circles and acquires a brownish color ; liquefaction occurs slowly and the cultures develop a disagreeable odor like that of ozaena. Upon the surface of agar a whitish, irregular layer is slowly developed and extends over the entire surface. Upon potato a slimy, irregular growth, of a pale reddish-gray color and a knobby surface, which gives off an intense and disagreeable odor like that of oza3na. 190. MICROCOCCUS RADIATUS (Fliigge). Found in the air and in water. Morphology. — Micrococci, from 0.8 to 1 /* in diameter, solitary, in short chains, or in irregular groups. Biological Characters. — An aerobic, liquefying micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates, at the end of two days, white colonies with a yellowish-green shimmer, about one millimetre in diameter, are developed ; under a low power these appear yellowish-brown and have starfish-like outgrowths ; at the end of four days a delicate, regularly -arranged, radiating aureole surrounds the colonies, out- NON-PATHOGENIC MICROCOCCI. 603 side of which a second and finally a third similar " Strahlenkranz " is often developed; liquefaction progresses slowly about the colonies. In gelatin stick -cultures feathery outgrowths occur at intervals along the line of puncture, radiating horizontally into the gelatin ; liquefaction commences near the surface as a pointed funnel and gradually extends downward. Upon potato a yellowish-brown layer is quickly developed. 191. DIPLOCOCCUS ALBICANS AMPLUS. Synonym. — Gray- white micrococcus (Bumm). Found in mucus from the healthy vagina. Morphology. — Diplococci resembling the " gonococcus " inform, but con- siderably larger, from 2 to 2.8 ft in diameter; the diplococci are usually soli- tary, but sometimes are in groups of three or four. "Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing micrococcus. Grows at the room temperature in the usual culture me- dia. Upon gelatin plates slightly elevated, grayish-white colonies are formed. In gelatin stick cultures growth occurs upon the surface and along the line of puncture as a grayish-white stripe ; after a time liquefaction com- mences under the surface growth. 192. MICROCOCCUS CANDICANS (Flugge). Very common in the air and in water. Morphology. — Spherical cocci, from 1 to 1.2 M- in diameter, associated in irregular groups. Biological Characters. — An aerobic, non-liquefying micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates, at the end of two days, the deep colonies are spherical and white or yellow- ish in color, of from 0.4 to 0.5 millimetre in diameter ; under the micro- scope they are seen to be finely granular, dark-brown spheres. Upon the surface milk-white, shining colonies with irregular outlines, which under the microscope are seen to be finely granular and to have jagged margins. In gelatin stick cultures a confluent white growth forms along the line of puncture, and a button-like mass upon the surface. Upon potato a slimy, white layer is quickly developed. 193. MICROCOCCUS CANDIDUS (Cohn). Found in water. Morphology. — Small, perfectly spherical cocci, from 0.5 to 0.7 n in diame- ter, united in zoo'gloea masses — the intercellular zooglcea substance is soluble in water. Biological Characters. — An aerobic, non-liquefying micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms snow-white colonies with irregular outlines, which under the micro- scope are seen to be slightly granular. In gelatin stick cultures a flat, milk- white layer is formed about the point of puncture; very scanty development along the line of inoculation. Upon agar the same as on gelatin. 194. MICROCOCCUS ACIDI LACTICI (Marpmann). Found in cow's milk. Morphology. — Large cocci, solitary or in pairs. Biological Characters. — An aerobic, non-liquefying micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms at the end of twenty-four hours punctiform, yellowish-white, lustre- less colonies. In gelatin stick cultures a thin, yellowish layer forms upon the surface, which is thickest in the middle, thin and transparent at the 604 NON-PATHOGENIC MICROCOCCI. margin, and without lustre. In miflc, at the end of twelve hours, a red color is developed, which disappears at the end of twenty-four hours, when coagulation has occurred as a result of the formation of lactic acid. 195. MICROCOCCUS LACTIS VISCOSUS. Synonym. — Micrococcus of bitter milk (Conn). Found in cream which had a bitter taste. Morphology. — Micrococci of moderate dimensions, frequently united in pairs ; in agar cultures forms short chains. Biological Characters. — Anaerobic and facultative anaerobic, liquefying micrococcus. Grows in the usual culture media at the room temperature — more rapidly at 35° C. Upon gelatin plates forms small, spherical colonies, which, as liquefaction commences, spread out upon the surface as a thin, granular mass. In gelatin stick cultures liquefaction commences at the sur- face, forming a shallow cavity, and rapidly progresses until the gelatin is entirely liquefied ; the liquefied gelatin is extremely viscid. Upon agar a shining, homogeneous white layer is developed. Upon potato white, shining masses, which are more or less separated from each other. In bouillon an abundant development occurs and a thin film is formed upon the surface ; the bouillon becomes very viscous. In milk growth is rapid and the milk acquires a bitter taste; at 35° C. coagulation occurs in twenty-four hours and the milk has an acid reaction ; the coagulum is soft and soon commences to dissolve from the peptonizing action of the ferment, but solution is not com- plete. Cultures in gelatin and bouillon are especially viscid, and may be drawn out into threads which are scarcely visible and are as much as three metres long. The acid formed by the growth of this micrococcus in milk is butyric. 196. SPHJEROCOCCUS ACIDI LACTICI (Marpmann). Found in fresh cow's milk. Morphology. — Very small, oval cocci, in pairs or in short chains. Biological Characters. — An aerobic, non-liquefying micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms porcelain-white colonies, the size of a pin's head, upon the surface of the gelatin. In gelatin stick cultures growth is scanty along the line of puncture ; upon the surface a layer is developed which has sloping, toothed margins, and at the end of six weeks acquires a pale-yellow color. Milk ac- quires a reddish color, and is coagulated at the end of twenty-four hours, with formation of lactic acid. 197. MICROCOCCUS AQUATILIS (Bolton). Very common in water. Morphology. — Small cocci, associated in irregular groups. Biological Characters. — An aerobic, non-liquefying micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms circular, porcelain- white, slightly elevated colonies; under a low power the deep colonies are seen to be mulberry-like in form, with roughly toothed contour and a pale-yellowish color; the superficial colonies are cir- cular and are surrounded by a narrow, homogeneous marginal zone, while the interior is peculiarly marked, resembling a schematic drawing of a sec- tion of a liver acinus. In gelatin stick cultures growth occurs both on the surface and along the line of puncture, of a white color. Upon the surface of agar a white layer is formed. 198. MICROCOCCUS CONCENTRICUS (Zimmermann). Found in water. Morphology. — Micrococci, 0.9 /* in diameter, associated in irregular masses. NON-PATHOGENIC MICROCOCCI. 605 Biological Characters. — Anaerobic, non-liquefyingmicrococcus. Grows in the usual culture media — best at the room temperature. Upon gelatin plates the deep colonies appear as small, bluish-gray points; the superficial colonies are at first small, bluish-gray discs, which later are irregular in out- line ; at the end of five days they are about three millimetres broad and con- sist of a central grayish- white disc surrounded by a bluish-gray ring with ir- regular outlines. Under a low power the deep colonies are seen to be spherical and granular, of a pale-brown or yellowish-green color, and in the interior usually several concentric rings are observed; the superficial colonies show in the interior a darker disc with irregular margins and marked by fine ra- diating fissures ; around this is an irregular marginal zone of a pale-brown color and granular in appearance, and this is enclosed in a white, shining border. In gelatin stick cultures a thin, bluish-gray layer forms upon the surface, which consists of a number of concentric rings of growth arranged around the point of puncture as a centre. Upon agar a broad, smooth, shin- ing layer with toothed margins and of bluish-gray white color. Oil potato a thin, slimy, yellowish-gray layer. 199. MICROCOCCUS CUMULATUS TENUis (Von Besser). Very common in nasal mucus of man. Morphology. — Large oval cocci, associated in masses. Biological Characters. — Anaerobic and facultative anaerobic, non-lique- fying micrococcus. Grows in the usual culture media at the room tempera- ture. In gelatin stick cultures grows along the line of puncture as a delicate white stripe composed of small colonies ; upon the surface as a flat, trans- parent layer with slightly elevated margins. Upon agar plates as thick, transparent drops about 0.2 millimetre in diameter; under a low power these are seen to have a large brown nucleus surrounded by a grayish-brown zone having wrinkled margins ; later the colonies attain a diameter of 0. 5 centimetre, and appear as flat, transparent discs with a large central nucleus. Scarcely any growth upon potato. In bouillon a considerable deposit accu- mulates at the bottom of the tube, and the liquid is nearly transparent above. 200. MICROCOCCUS PLUMOSUS (Brautigam). Found in water. Morphology. — Micrococci, 0.8 n in diameter, associated in zoogloea. Biological Characters. — Anaerobic, non-liquefying micrococcvis. Grows in the usual culture media at the room temperature. Upon gelatin plates yellowish- white colonies are developed, which send out tongue-like processes and the margins of which are abruptly thickened. In gelatin stick cultures, from along the line of puncture, at certain points, long, delicate, white off- shoots are given off into the gelatin, which resemble needle- like crystals; similar offshoots from the layer upon the surface of the gelatin are also seen ; these consist of cocci in masses, arranged like chains of pearls. Upon potato an irregular, yellowish- white layer with tongue-like offshoots. 201. MICROCOCCUS ROSETTACEUS (Zimmermaiin). Found in water. Morphology. — Spherical or elliptical cocci, from 0.7 to 1 ft in diameter, associated in grape-like masses. Biological Characters. — An aerobic, non-liquefying micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates the deep colonies are small, grayish-white, and usually spherical in form ; under a low power they are sometimes seen to be lenticular or mussel - shaped. The superficial colonies are rather broad, shining, yellowish-gray drops, with more or less irregular outlines. In gelatin stick cultures a thin, 606 NON-PATHOGENIC MICROCOCCI. gray, rosette-like layer of irregularly circular contour develops upon the surface; very scanty growth along the line of puncture. Upon aoar a smooth, shining, gray layer with finely toothed margins. Upon potato a yellowish-gray layer is quickly formed. 202. MICROCOCCUS URE.E (Pasteur). Found in the air and in ammoniacal urine. Morphology.— Micrococci, from 0.8 to 1 /f in diameter, solitary, in pairs, in tetrads, or in short chains ; also in zoogloea masses. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying micrococcus. Grows in the usual culture media at the room tem- perature—better at 30° to 35° C. Upon gelatin plates forms, at the end of twenty-four hours, small, white, pearly, shining colonies of smooth surface and sharply defined outline ; at the end of ten days these are large, flat colo- nies resembling a drop of stearin. In gelatin stick cultures development oc- curs along the line of puncture in form of a thin, tenacious thread. Old cul- tures have a paste- like odor. According to Von Jaksch, a very favorable medium for the growth of this micrococcus is made by adding to one litre of water, magnesia sulphate one-sixteenth gramme, potassium hypophosphite one-eighth gramme, potas- sium sodium tartrate five grammes, urea five grammes. In urine and solu- tions containing urea carbonate of ammonia is formed during the develop- ment of this micrococcus — ammoniacal fermentation. 203. MICROCOCCUS URE.E LIQUEFACIENS (Flugge). Found in ammoniacal urine. Morphology. — Spherical cocci, from 1.25 to 2 n in diameter, solitary, in chains of three to ten elements, or in irregular groups. Biological Characters. — An aerobic and facultative anaerobic, liquefying micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms, at the end of twenty-four hours, small, white, punctiform colonies, which under a low power appear as well-defined, dark- gray spheres; after they reach the surface of the gelatin the colonies become considerably larger, have a yellowish-brown color and a central nucleus consisting of the original deep colony ; the surface of the superficial colo- nies is granular, the outline becomes gradually wavy ; liquefaction of the gelatin around the colonies occurs gradually. In gelatin stick cultures a confluent, white growth develops along the line of puncture, and liquefac- tion quickly occurs and extends to the walls of the tube; finally one-half or more of the gelatin is liquefied and has a whitish, clouded appearance^ while a thick, yellowish- white deposit is seen at the bottom. 204. MICROCOCCUS VITICULOSUS (Katz). Found in the air and in water. Morphology. — Oval micrococci, from 1 to 1.2 n in diameter; forms thick zoogloea masses. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying micrococcus. Grows rapidly in the usual culture media at the room temperature. Upon gelatin plates the deep colonies are seen to con- sist of hair-like branches given off from a centre, and which for some dis- tance form a delicate network ; under a low power these branches are seen to be made up of zoogloea masses of various dimensions united in chaplets. The superficial colonies extend rapidly as a thin, jelly-like, clouded white layer, from which fine threads are given off into the deeper layers of the gelatin. In gelatin stick cultures growth occurs along the line of puncture as a delicate network of threads ; upon the surface a feathery growth occurs along the line of inoculation. Upon potato a dry, dirty-white layer is. quickly developed. NON-PATHOGENIC MICROCOCCI. 607 205. DIPLOCOCCUS ALBICANS TARDissiMUS (Eisenberg). Synonym. — Milk-white micrococcus (Bumm). Found in secretions from the vagina and cervix, especially in the vaginal secretions of puerperal women. Morphology. — Diplococci resembling the " gonococcus, " and consisting of two biscuit-shaped halves separated by a cleft which is not as broad as that seen in Micrococcus gonorrhcese; the diameter, from pole to pole, averages about 1.25 fi. In unstained preparations the cleft is not seen and the diplococci appear as spherical, highly refractive bodies. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying micrococcus. Grows very slowly at the room temperature in the usual culture media. Upon gelatin plates forms extremely small, puncti- form colonies, which under a low power are seen to be spherical, opaque, and brownish-green in color; at the end of two weeks they may attain a dia- meter of two millimetres. In gelatin stick cultures small, isolated, gray- ish-white colonies are developed, after some days, along the line of puncture, and a thin, whitish, stearin-like layer with irregularly dentate margins is slowly developed upon the surface. Upon the surface of agar a thin, moist, grayish-white layer with dentate margins is slowly developed. 206. DIPLOCOCCUS ALBICANS TAKDUS (Unna). Found upon the surface of the body in individuals having eczema se- borrhoeicum. Morphology. — Diplococci consisting of two oval elements, with the long diameters in parallel planes, from 0.7 to 0.8 /* long and 0.6 /* broad; often associated in short chains or in irregular groups. Biological Characters. — An aerobic, non-liquefying micrococcus. Grows slowly in the usual culture media at the room temperature. Upon gelatin plates the deep colonies are usually oval, dark-yellow, and at the end of eight days are as large as a mustard seed. The superficial colonies are cir- cular in outline, with well-defined margins, elevated, grayish-yellow, and at the end of eight days one to two millimetres in diameter ; under a low power they are seen to be granular, grayish-yellow, with shining margins ; at the end of five weeks they are gray and present two or three zones of dif- ferent dimensions, with an elevated circular centre and finally with thin, slimy, dentate margins ; under the microscope finely granular projections are seen upon the sm-face. In gelatin stick cultures, at the end of three weeks, a thin, waxy-looking, yellowish-white layer with finely dentate mar- gins develops upon the surface and a scanty growth has occurred along the line of puncture. At the end of five weeks this superficial layer may have a diameter of one centimetre. Upon the surface of agar, at the end of five weeks, a yellowish-gray streak with irregular, dentate margins and a dull lustre is formed along the line of inoculation. 207. STAPHYLOCOCCUS ALBUS LIQUEFACIENS. Synonym. — White liquefying staphylococcus (Escherich). Found occasionally in the alvine discharges of healthy infants. Morphology. — Micrococci of from 0.8 to 1.2 M in diameter, occasionally oval in form and three V- in long diameter ; associated in irregular groups — considerably larger than Staphylococcus pyogenes albus. Biological Characters. — An aerobic, liquefying micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms spherical, white colonies, which after some time cause a gradual liquefaction of the surrounding gelatin. In gelatin stick cultures a scanty development is seen along the line of puncture at the end of three to four days, and gradual liquefaction of the gelatin occurs in funnel form ; the liquefied gelatin is viscid, of syrupy consistence, and slightly clouded; £he 608 NON-PATHOGENIC MICROCOCCI. surface is covered by a white layer of micrococci ; development usually ceases before complete liquefaction has occurred. Upon agar and upon blood serum a white layer is developed, which presents nothing characteris- tic ; blood serum is not liquefied. Upon potato a very scanty, thin, color- less layer, which later appears as a collection of white, button-like masses. 208. MICROCOCCUS OVALIS (Escherich). Found frequently in meconium and faeces of milk-fed infants. Morphology. — Micrococci of from 0.2 to 0.3 n in diameter, frequently seen as oval cells 0.6 to 0.7 n long and 0.3 /* broad, in the middle of which a commencing line of division may sometimes be seen ; sometimes in short chains. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms very small colonies which are in no way characteristic. In gelatin stick cultures small, white colonies are developed along the line of puncture, and no development occurs upon the surface, or a scanty, colorless ring of growth surrounds the point of punc- ture. Upon potato a tolerably abundant growth occurs, consisting of a small, white layer. In milk it causes an acid reaction, and coagulation after several days. 209. DIPLOCOCCUS CORYZ^E (Hajek). Found in nasal mucus in acute nasal catarrh. Morphology. — Large diplococci, flattened along the line of contact; re- semble short bacilli with round ends. Biological Characters. — Anaerobic, non-liquefying micrococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms white, glass-like, slightly elevated colonies. In gelatin stick cultures the growth resembles that of Friedliinder's bacillus at first, but the super- ficial growth is flatter after some days. Upon the surface of agar forms a diffuse layer. 210. MICROCOCCUS FINLAYENSIS (Sternberg). Obtained by Finlay in cultures from the liver and spleen of a yellow- fever cadaver. Morphology. — Micrococci, from 0.5 to 0.7/* in diameter, solitary, in paii*s, or occasionally in groups of four; also in irregular masses. Like other staphylococci, the cocci are seen, in properly stained preparations, to be made up of two hemispherical portions. Biological Characters. — An aerobic, liquefying, chromogenic micro- coccus. Grows slowly at the room temperature in the usual culture media. In gelatin stick cultures growth occurs along the line of puncture and lique- faction near the surface; the cup shaped cavity which is slowly formed is lined with a very viscid, opaque, pale- yellow layer of cocci. Upon the sur- face of agar a viscid layer having a pale-yellow color is formed. Not path- ogenic for rabbits or guinea-pigs. 211. MICROCOCCUS OP FREIRE. Presented to the writer, at the time of his visit to Brazil (1887), by Dr. Domingos Freire, as his yellow-fever germ — so-called Cryptococcus xantho- geuicus. Morphology. — Micrococci, from 0.5 to 0.8 n in diameter, solitary, in pairs, or in irregular agglomerations ; like other staphylococci, groups of four and chains of three or four elements are occasionally formed. NON-PATHOGENIC MICROCOCCI. 609 Stains with the aniline colors usually employed and by Gram's method. Biological Characters. — An aerobic, liquefying staphylococcus. Grows in the usual culture media at the room temperature. Is killed by exposure to a temperature of 60° C. for ten minutes. Vitality not destroyed by long exposure to a freezing temperature. Development occurs at a comparatively low temperature — 10° to 15° C. Preserves its vitality for several months in cultures. In gelatin stick cultures development occurs along the line of puncture and liquefaction in cup shape near the surface ; the cocci accumu- late at the bottom of the cup as a milk-white deposit; later the gelatin may be completely liquefied at the upper part of the tube for half an inch or more, and the non -liquefied gelatin forms a horizontal floor upon which a milk-white deposit is seen. In agar stick cultures an irregular, white, opaque growth is seen along the line of puncture, and a soft, milk-white layer, with irregular outlines, forms upon the surface. Upon potato a milk-white layer, from three to five millimetres wide, is developed along the line of in- oculation at the end of forty-eight hours at 37° C. Does not coagulate milk. FIG. 199. FIG. 200. FIG. 199.— Micrococcus of Freire, from a gelatin culture, x 1,000. From a photomicrograph. (Sternberg.) Fm. 200.— Culture of Freire's micrococcus in nutrient gelatin, end of eight dayg at 28° C. From a photograph. (Sternberg.) In the writer's experiments this micrococcus has not proved to be patho- genic for guinea-pigs. According to Freire, it is not pathogenic for these animals in the winter months, but in summer, at Rio de Janeiro, it is fatal to guinea-pigs and to small birds. 212. STREPTOCOCCUS COLI GRACILIS (Escherich). Found in the faeces of healthy children on flesh diet— not during the period of nursing (Escherich). Morphology.— Micrococci, from 0.2 to 0.4 n in diameter, usually in S- shaped chains containing from six to twenty elements. In agar cultures the chains are shorter, and upon potato they are rarely seen; the elements in a chain are often elongated and show indications of commencing transverse division. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing streptococcus. Grows rapidly in the usual culture media at the room temperature. Upon gelatin plates the colonies are at first small, spherical, and well defined, later bulging and lying at the bottom of a funnel of Hque- 610 NON-PATHOGENIC MICROCOCCI. fied gelatin ; by reflected light the colonies are of a whitish lustre. In gela- tin stick cultures development occurs along the line of puncture, and lique- faction commences on the second day along the entire line, forming a tube of rather uniform dimensions ; the liquefied gelatin is slightly clouded, and a very slight deposit of a white color is seen at j^jjP the bottom of the tube ;. by the eighth to tenth day liquefac- MB» ^& tioii is complete ; in older cultures a finely granular, whitish >* • deposit is seen, while the gelatin above is quite transparent »** and has an acid reaction. Upon agar a very scanty super- ^^ f ficial growth occurs. Blood serum is not liquefied, but vW» / small, shining scales are developed upon the surface. Upon FIG. 201.— Str. young potato a thin layer of white, button like colonies de- coli graciiis,from velops upon the surface; no growth occurs on old potato, a liquefied geia- Milk is coagulated and acquires an acid reaction after a con- tin culture, x siderable time. 970. (Escherich.) 213. STREPTOCOCCUS ACIDI LACTICI (Grotenf eld) . Found in coagulated milk in Finnland. Morphology. — Spherical or oval cocci, from 0.5 to 1 jj. long and 0.3 to 0.6 H thick, associated in long chains. Biological Characters. — An anaerobic (not strict), non-liquefying strep- tococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms spherical, white colonies — best when the plate is cov- ered with a layer of gelatin to exclude the air. In gelatin stick cultures an abundant development along the line of puncture only. In milk causes co- agulation of the casein and an acid reaction. 214. STREPTOCOCCUS GIGANTEUS URETHRA (Lustgarten). Found in the normal human urethra. Morphology. — Spherical cocci, 0.8 to 1 n in diameter, associated in chains which often contain many hundred elements, and are often united in thick, tangled masses. Biological Characters. — An aerobic micrococcus. Grows slowly in nu- trient agar at 37° C. ; does not grow at the room temperature. Upon agar plates elongated, drop-like colonies are slowly formed, which are not ele- vated above the surface ; these are easily overlooked in reflected light, and in transmitted light are iridescent; the colonies are often shaped like a clover leaf ; they never become confluent, and attain their greatest development by the eighth day ; transplantation to other agar tubes is rarely successful. In streak cultures upon agar development occurs for the most part in the con- densation water as a flocculeiit deposit. 215. STREPTOCOCCUS ALBUS. Synonym.— Weisser Streptococcus (Maschek). Found in Freiburg water by Tils. Morphology. — Streptococci, which show independent movements when undergoing binary division (Tils). Biological Characters. — An aerobic, liquefying streptococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates superficial colonies are developed, which are flat, circular in outline, and bounded by a white margin ; saucer-shaped liquefaction quickly occurs, and under a low power a pale-brown cloud is seen in the centre. Upon agar the disc-shaped colonies have a darker interior. In gelatin stick cultures the development is chiefly upon the surface ; liquefaction progresses rapidly, and a white deposit is formed. Upon potato a slimy, white layer is quickly developed. NON-PATHOGENIC MICROCOCCI. 611 216. STREPTOCOCCUS VERMIFORMIS. Synonym. — Wurmformiger Streptococcus (Maschek). Found in Freiburg water by Tils. Morphology.— Streptococci, which show slow, vermiform, progressive movements ; the juxtaposition of sevei'al cocci in a chain gives the appearance of bacilli, which are again united in chains (Tils). Biological Characters. — An aerobic, liquefying streptococcus. Grows in the usual culture media at the room temperature. Upon gelatin plates forms yellowish-white colonies, which sink into the gelatin as liquefaction occurs; about the more transparent interior a darker liquefied ring is seen, which is bounded by a white border ; under a low power the interior is seen to be finely granular and pale-brown in color ; the margin has a radiate ap- pearance. In gelatin stick cultures growth occurs upon the surface and along the line of puncture, causing rapid liquefaction of the gelatin; a dirty- yellow deposit accumulates at the Dottom of the tube. Upon potato a dirty- yellow layer is slowly developed ; later this becomes somewhat darker. 217. STREPTOCOCCUS «REVis (Von Lingelsheim). Obtained from normal human saliva, and differentiated from Streptococ- cus pyogenes by Von Lingelsheim (1891). Morphology. — Micrococci, solitary, in pairs, or in short chains— seldom as many as eight to ten elements. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying streptococcus. Grows in the usual culture media at the room temperature —more rapidly than Streptococcus pyogenes. Bouillon is uni- formly clouded by the growth of this streptococcus, while the streptococci which form long chains leave the bouillon transparent and form conglo- merate masses. In blood serum of the ox the growth of this streptococcus exactly resembles that of Streptococcus pyogenes, long chains being formed which are associated in conglomerate masses. Upon gelatin plates, at the end of twenty -four hours, forms punctiform colonies, which under the mi- croscope are usually seen to be spherical in form with an even contour. Upon the surface ofagar a thin, homogeneous, yellowish-gray layer is de- veloped along the impf strich — not composed of separate colonies ; along the line of puncture in nutrient agar the colonies are largest below and are fre- quently flattened or irregular in corm. In gelatin stick cultures the devel- opment at first resembles that of "Streptococcus longus " (Streptococcus pyogenes) , but after three days a funnel-like cavity forms near the surface of the gelatin, which finally extends downward for a distance of four to five millimetres, while below this, usually separated by a space in which there is no evidence of growth, colonies the size of a pin's head are developed along the line of puncture in rows. Upon, potato, at 37° C., an abundant develop- ment occurs within forty-eight hours, in the form of a confluent, white layer, easily stripped from the surface. STREPTOCOCCUS CADAVERIS (Sternberg). Possibly identical with Streptococcus brevis, described above. Found by the writer in the liver of a yellow-fever cadaver. Morphology. — Micrococci, in pairs, or in short chains which are usually made up of cocci in pairs, or oval elements which show, more or less dis- tinctly, commencing binary division; diameter about 0.5 //. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying streptococcus. Grows in the usual culture media at the room temperature. Grows readily in a decidedly acid medium. In. gelatin stick cultures opaque, spherical colonies are formed along the line of puncture — larger and more opaque than in similar cultures of Streptococcus pyogenes 612 NON-PATHOGENIC MICROCOCCI. made at the same time ; later the isolated colonies at the bottom of the line of puncture are coarsely granular and irregular in outline ; no growth upon the surface of gelatin. Upon the surface of agar, in Havana, a white mass FIG. 202.— Streptococcus cadaveris, from a culture in agua coco, x 1,000. From a photo- micrograph. (Sternberg.) formed about the point of puncture ; later, in Baltimore, a thin, translucent layer developed upon the surface of agar cultures. Very thin, white growth upon potato at the end of twelve days. In bouillon and agua coco it forms FIG. 203.— Streptococcus Havaniensis, from acid vomit of yellow-fever patient, kept twenty four hours in a collecting tube, x 1,000. From a photomicrograph. (Sternberg.) little flocculi made up of chains. In old agar cultures it is very small and not in chains. In veal broth, long chains, in which the cocci are larger than usual; the elements vary considerably in size in the same chain. 218. STREPTOCOCCUS HAVANIENSIS (Sternberg). Found in acid vomit of a yellow-fever patient in military hospital, Ha- vana. NON-PATHOGENIC MICROCOCCI. 613 Morphology. — Micrococci, from 0.6 to 0.9 n in diameter, associated in long chains which are made up of cocci in pairs or of oval elements showing commencing transverse division. Biological Characters not determined. 219. STREPTOCOCCUS LIQUEFACIENS (Sternberg). Obtained from liver of yellow-fever cadavers, also from contents of in- testine. Morphology. — Spherical or short oval micrococci, from 0.4 to 0.6 n in diameter, solitary, in pairs, or in short chains. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing streptococcus. Grows in the usual culture media at the room tempera- ture. In gelatin stick cultures liquefaction occurs rapidly along the entire- FIG. 205. FIG. 204.— Streptrococcus liquefaciens, from anaerobic culture in nutrient gelatin, x 1,000.. From a photomicrograph. (Sternberg.) FIG. 205.— Streptococcus liquefaciens ; culture in nutrient gelatin, end of seven days at 22° C. (Sternberg.) line of puncture, and by the end of a week the gelatin is completely lique- fied ; it is but slightly opalescent, and a scanty white deposit forms at the bottom of the tube. In agar stick cultures a scanty growth occurs at the point of puncture, and closely crowded colonies are developed along the line of inoculation. Upon potato a thin and limited, dry, white layer is formed along the line of inoculation in four to five days. Not pathogenic for rabbits or guinea-pigs. 220. MICROCOCCUS TETRAGENUS VERSATILIS (Sternberg). Synonym. — Micrococcus tetragenus febris flavae (Finlay). Obtained by Dr. Finlay, of Havana, from his ' ' mosquito cultures "- from the excrement of mosquitoes which he had allowed to draw blood from yellow-fever patients ; by the writer from cultures from the surface of the body of patients in hospital in Havana, and from the air. Morphology. — Micrococci, in tetrads, or in irregular groups containing three or more united elements ; varies greatly in the size and grouping of the ele- ments, which are sometimes less than 0.5/< in diameter and may measure 1.5 p. This micrococcus would probably be classed as a sarcina by somebac- 51 614 NON-PATHOGENIC MICROCOCCI. •teriologists, but division in three directions, forming packets, has not been •observed. Stains with the usual aniline colors and by Gram's method. Biological Characters.— An aerobic, liquefying, chromogenic micro- coccus. Grows in the usual culture media at the room temperature. Colo- nies in gelatin roll tubes are spherical, opaque, at first of a pale-yellow and later of a lemon-yellow color ; . liquefaction around the colony usually does not commence for several days and progresses slowly. In gelatin stick cul- tures very scanty development occurs along the line of puncture, and the gelatin is liquefied in cup shape by the surface growth ; at the bottom of the liquefied gelatin a viscid, pale-yellow mass accumulates. Upon the surface of agar a thick, viscid, yellow layer is formed along the impfstrich, which Fio. 206. Fia. 207. FIG. 206.— Micrococcus tetragenus versatilia, from a single colony in nutrient gelatin. X 1,000. From a photomicrograph. (Steinberg.) FIG. 207.— Micrococcus tetragenus versatilis; culture in nutrient gelatin, end of two weeks at 22° C. (Sternberg.) gradually extends over the entire surface ; the color varies from cream-yellow to lemon-yellow, and the surface is moist and shining. The growth upon potato is similar to that upon agar. Not pathogenic for rabbits or guinea- pigs. 221. PEDIOCOCCUS ALBUS (Lindner). Found in well water. Morphology. — Micrococci, solitary, in pairs, or in tetrads; frequently in pseudo-sarcina — accidental — groups. Biological Characters. — An aerobic, liquefying micrococcus. Grows in the usual culture media at the room temperature. Is destroyed by a tem- perature of CO0 C. in eight minutes. Upon gelatin plates forms spherical •colonies, around which liquefaction rapidly occurs, allowing the colonies to fall to the bottom of the liquefied gelatin, where they form irregular flocculi. In gelatin stick cultures, at the end of twenty-four hours, a deep channel of liquefaction has already formed, at the bottom of which a white, loose sedi- ment collects ; by the fourth day a pale-orange color is developed. Upon the surface of agar a broad, dry layer forms along the impfstrich ; old cul- tures have an orange color. Upon potato a dirty-white layer is developed. Produces an acid reaction in culture media. , NON-PATHOGENIC MICROCOCCI. 615 222. PEDIOCOCCUS ACIDI LACTICI (Lindner). Found by Lindner in mash from malt and in hay infusion. Morphology. — Micrococci, from 0.6 to 1 M in diameter, solitary, in pairs, or in tetrads — division in two directions. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying micrococcus. Grows very slowly in the usual culture media at the room temperature. Upon gelatin plates small colonies are developed, which are first colorless and later yellowish-brown. In agar stick cultures a moist, thin, colorless layer is slowly developed upon the surface. Upon potato a scanty, scarcely visible growth. Lactic acid is produced by the growth of this tetragenus in saccharine solutions. 223. PEDIOCOCCUS CEREVISIJE (Balcke). Found in beer and in the air of breweries. Morphology. — Micrococci, solitary, in pairs, or in tetrads. Biological Characters^ — An aerobic and facultative anaerobic, non- liquefying micrococcus. Grows slowly in the usual culture media at the room temperature. Is killed by a temperature of 60° C. maintained for eight minutes. Upon gelatin plates forms small, colorless colonies, which later are yellowish-brown. In gelatin stick cultures a white, leaf- like layer is formed on the surface and a grayish-white growth along the line of punc- ture. Upon agar a moist, grayish-white, iridescent layer with smooth mar- gins. Upon potato a scanty, scarcely visible growth. 224. MICROCOCCUS TETRAGENUS MOBILIS VENTRICULI (Mendoza). Found in the contents of the stomach. Morphology. — Micrococci, in tetrads, enclosed in a transparent capsule. Biological Characters. — An aerobic, non-liquefying, motile (?) tetrage- nus. Grows slowly in the usual culture media at the room temperature. Upon gelatin plates forms superficial colonies which are circular in outline, with well-defined margins, and dirty -white in color ; under a low power they are seen to be finely granular, both on the surface and in the interior. In gelatin stick cultures grows upon the surface as a dirty-white layer ; old cultures have a " sugar color " and give off an odor like that of skatol. The growth upon agar is similar to that upon gelatin. 225. MICROCOCCUS TETRAGENUS SUBPLAVUS (Von BeSSer). Found in nasal mucus. Morphology. — Spherical or oval cocci of medium size, usually associated in tetrads. Biological Characters. — An aerobic and facultative anaerobic tetra- genus. Does not grow in nutrient gelatin. Grows at the room temperature in nutrient agar. Upon agar plates forms flat, dirty -white colonies, 0.5 cubic centimetre in diameter, with shining surface and somewhat wrinkled margins ; under a low power these are seen to have a small brown nucleus surrounded by a grayish-brown, irregularly striped zone, around which is a wrinkled margin. The deep colonies are dark-green and punctiform, of very irregular form. Upon the surface of agar a broad, flat, grayish-white layer is formed, which in three or four days commences to turn brown at the mar- gins, and at the end of four weeks has the color of cultures of Staphylococcus Eyogenes aureus. Upon potato a pale-brown layer is developed along the ne of inoculation. 226. SARCINA AURANTIACA. Found in the air and in water. Morphology. — Micrococci, in pairs, with hemispherical elements, in tetrads,. c616 NON-PATHOGENIC MICROCOCCI. or in packets containing eight or more elements, as a result of division in three directions. The cells are smaller than those of Sarcina lutea. Biological Characters. — An aerobic, liquefying, chromogenic sarcina. Grows in the usual culture media at the room temperature. Upon gelatin plates forms small, spherical, granular colonies of an orange-yellow color. In gelatin stick cultures liquefaction occurs slowly along the line of inocula- tion, and in old cultures a yellow deposit is seen at the bottom of the trans- parent, liquefied gelatin. Upon the surface of agar an abundant, shining, golden-yellow layer is formed. Upon potato development is scanty and slow, being limited to the line of inoculation. 227. SARCINA LUTEA (Schroter). Found in the air. Morphology. — Micrococci, 1 // or more in diameter, associated in pairs, in groups of four, or in packets containing eight or more elements. Biological Characters. — An aero- bic, liquefying, chromogenic sarcina. Grows in the usual culture media at the room temperature. Upon gelatin plates small, spherical, yellow colonies are slowly developed. In gelatin stick cultures development occurs only on the surface in the form of a coarsely granular, yellow layer of moderate di- mensions; liquefaction of the gelatin occurs in old cultures and progresses slowly. Upon the surface of agar a rather thick, canary-yellow layer is quickly developed. Upon potato a sul- phur-yellow streak is slowly developed along the line of inoculation. FIG. 208.— Sarcina lutea, from an agar culture. X 1,000. From a photomicro- graph. • (Frankel and Pfeiffer.) 228. SARCINA FLAVA (De Bary). Found by Lindner in beer. Morphology. — Small micrococci in packets of sixteen to thirty-two ele- ments, which may be associated in irregular masses or united in larger packets of cubical form. Biological Characters. — An aerobic, liquefying, chromogenic sarcina. Grows in the usual culture media at the room temperature. Upon gelatin plates spherical, yellow colonies are developed, and liquefaction of the gel- atin around them commences in about four days. Upon the surface of agar forms a yellow layer. Upon potato the growth is scanty and limited to the impfstrich, yellow in color. 229. SARCINA ROSE A (Schroter). Found in the air. Morphology. — Large cocci in cubical packets with rounded corners, and about eight p thick. Biological Characters.— An. aerobic, liquefying, chromogenic sarcina. Grows in the usual culture media at the room temperature. In gelatin stick cultures liquefaction occurs rapidly, and old cultures acquire a red color, which by the addition of sulphuric acid is changed to bluish-green. Uponograr growth is slow, and small, cartilaginous-looking masses are de- veloped. Upon potato an abundant, intensely red layer is quickly de- veloped. In bouillon development is rapid and a red sediment is deposited. NON-PATHOGENIC MICROCOCCI. 617 230. SARCINA ALBA (Eisenberg). Found in the air and in water. Morphology. — Small cocci, associated in pairs or in tetrads. Biological Characters. — An aerobic, liquefying (very slightly) sarcina. Grows very slowly at the room temperature in the usual culture media. Upon gelatin plates forms small, round, white colonies. In gelatin stick cultures forms a white, button-like mass at point of puncture ; scanty growth along line of inoculation. Upon potato grows very slowly in the form of a yellowish- white layer limited to the line of inoculation. 231. SARCINA CANDIDA (Reinke). Found in the air about breweries. Morphology. — Micrococci of 1.5 to 1.7 n in diameter, solitary, in pairs, or in tetrads; under certain circumstances, as for example in hay infusion, multiplication occurs in three directions, forming sarcina-like packets. Biological Characters. — Anaerobic, liquefying sarcina. Grows slowly at the room temperature in the usual culture media. Upon gelatin plates forms shining white, spherical colonies, which later have a yellowish color and are surrounded by liquefied gelatin. In gelatin stick cultures causes rapid liquefaction along the line of puncture. Upon the surface of agar forms a white, moist, shining layer with smooth margins. 232. SARCINA PULMONUM (Hauser). Found in the sputum of patients with phthisis. Morphology. — Micrococci, from 1 to 1.5 /a in diameter; usually associated in tetrads, but may form cubical groups of eight cells. Biological Characters. — An aerobic, non-liquefying sarcina. Grows slowly in the usual culture media at the room temperature. Upon gelatin plates in three days white, puiictiform colonies are seen ; later these still re- main small; under a low power they are seen to be coarsely granular. In gelatin stick cultures the development is very scanty along the line of punc- ture ; upon the surface a round, sharply denned layer of a pearl -gray color is developed ; later this becomes tolerably thick, is grayish-brown in color, glistening, and has more or less folded and irregular margins. The growth upon potato is very scanty and is confined to the line of inoculation. This sarcina is said by Hauser to form endogenous spores which may be demonstrated by Neisser's method of staining, and which have great resistance to heat. When cultivated in urine it causes ammoniacal decomposition of the urea. 233. SARCINA VENTRICULI (Goodsir). Found in the contents of the stomach of man and animals. Morphology. — Spherical or slightly oval cells, about 2.5 jit in diameter; united in cubical groups with rounded corners, containing eight elements, and then associated in larger "packets." Biological Characters. — An aerobic, non-liquefying sarcina. Grows rapidly at the room temperature in suitable media. Upon gelatin plates, at the end of thirty-six to forty-eight hours, hemispherical, yellowish colonies are developed upon the surface ; these contain cocci in pairs and tetrads, but not in cubical packets. In hay infusion development occurs upon the surface in the form of small, brownish scales, while a brownish, flocculent deposit is seen at the bottom of the tube ; the sarcina form is well developed. Upon potato a dry, colorless strip develops along the impfstrich in twenty- four hours; later this acquires a chrome-yellow color. Upon the surface of blood serum flat, round, pale yellow colonies are formed along the line of inoculation. 618 NON-PATHOGENIC MICROCOCCI. 234. MICROCOCCUS AMYLOVORUS (Burrill). Synonym. — Micrococcus of pear blight. Found in the diseased branches of pear trees affected with ' ' blight " — "fire blight." Discovered by Burrill (1878) and carefully studied by Ar- thur (1886). Morphology.— Oval cells, from 1 to 1.25 n long and 0.5 to 0.75 n broad; usually solitary, often in pairs, and occasionally in a linear series of four. In fluid culture media form zooglcea, which are spherical or oblong and may be from thirty to forty /f long and twenty to thirty /j. broad ; these zoogloea masses may be solitary, or united in pairs or short chains ; they are found es- pecially upon the surface of the culture medium, and after a time present a wrinkled appearance, giving some resemblance to the external surface of the brain. Biological Characters. — An aerobic, non-liquefying, motile micrococcus (bacillus?). Grows in various media at the room temperature. According to Arthur, this micrococcus exhibits active "swarming movements" when undergoing rapid multiplication in a favorable medium : ' ' The bacteria move rapidly back and forth, in and out, among each other, but never in a straight line to any distance. " An infusion of potato constitutes a favorable cul- ture medium. In this medium, at a temperature of 25° to 30° C., turbidity commences in twenty-four hours and bubbles of carbon dioxide are given off ; in forty -eight hours the liquid is entirely turbid and a thin, whitish pel- licle has formed on the surface, at the same time a sediment commences to form at the bottom of the tube. Upon the surface of nutrient gelatin de- velopment is very scanty and scarcely perceptible. In the interior small, punctif orm colonies are developed ; these are spherical or oval, and may at- tain a diameter of 0.5 millimetre. Upon slices of unripe pear, in two or three days, fine milky drops like beads of dew are developed. Upon potato a thin, moist, somewhat yellowish layer is developed. The experiments of Bun-ill and o'f Arthur show that the disease known as pear blight is produced by applying cultures of this ' ' micrococcus " to healthy branches of the pear tree. 235. ASCOCOCCUS BILLROTHII. Found by Billroth in putrefying flesh infusion. Morphology. — Small cocci, united in peculiar colonies. Upon the sur- face of liquid media form a cream- like layer in which numerous small, spherical or oval masses are seen. Under the microscope these are seen to consist of a jelly-like, cartilaginous, extremely resistant envelope from ten to fifteen ju. thick ; in the interior of this one or more spherical or oval masses of cocci are enclosed, which are from twenty to seventy n or more in diameter; the cocci are closely crowded and united by an unusually firm and scanty intercellular sub- stance. Biological Characters. — Aerobic. Grows at the room temperature. Produces a strongly alkaline reaction in culture media, due to the develop- ment of ammonia. According to Cohn, a greenish- white, slimy mass is de- veloped upon slices of beet, and in the juice of the sugar beet it produces a slimy fermentation. FIG. 209.— ASCOCOCCUS Billrothii. NON-PATHOGENIC MICROCOCCI. 619 236. LETJCONOSTOC MESENTEROIDES (Cienkowski). Synonyms. — Froschlaichpilz ; Pilz der Dextrangarung. Found upon the beet juice and the molasses of sugar factoi'ies, where it develops as large, jelly-like masses resembling frog spawn ; also upon raw or cooked carrots or sugar beets. Morphology. — The reproductive elements are spherical or oval, and from 1.8to2ju in diameter; they consist of a firm, membranous envelope with shining contents. When these germinate — according to Van Tieghem — the external layer of the cell wall is ruptured in an irregular way and a middle layer is extruded to form a thick, jelly-like envelope, while the inner layer remains in contact with the protoplasmic contents of the cell ; the cell and its envelope then become elongated and binary division of the reproductive element occurs; this process is repeated in the segments, and as a result a chain of spherical elements enclosed in a sausage-shaped mass of jelly is de- veloped ; later the chains become curved and break up into longer or shorter fragments, which are enclosed in an irregular gelatinous mass ; these zoog- Icea crowded together form small masses having a parenchymatous struc- ture ; these adhere to each other when they come in contact, and when dis- tributed in a liquid may be brought together by shaking to form a large, Jelly-like mass which ha's apparently been developed by the process of shak- ing, as the smaller masses suspended in the fluid could not readily be seen. The zoogloea masses have a cartilage-like consistence and can be cut into sections with a razor. The jelly is hyaline, but in beet juice often has a gray- ish color from the presence of impurities attached to its surface. When treated with a solution of haematoxylon it acquires a brown color. After some time the jelly is dissolved, and the cocci are set free and in a suitable medium again develop zooglcea. Van Tieghem has demonstrated the forma- tion of spores. These are formed when conditions are no longer favorable for the development of the vegetative cells ; here and there a cell in a chain is seen to increase in dimensions, and in the interior of this mother cell a con- siderable number of refractive spores are developed. Biological Characters. —This coccus is aerobic. It is readily cultivated in liquid media containing glucose. According to Van Tieghem, it appro- priates grape sugar directly and cane sugar indirectly — i.e., after it has been changed into grape sugar by a ferment produced by the coccus. Development is very rapid under favorable conditions. Thus Durin reports that a wooden tub in which beet juice had been kept, and upon the walls of which a thin, slimy mass of this microorganism remained attached, was filled with a neu- tral solution of molasses containing ten per cent of sugar. At the end of twelve hours the entire contents of the tub had been changed into a compact, jelly-like mass by the development of Leuconostoc mesenteroides. The chemical formula for the jelly is CnHioOio. IX. NON-PATHOGENIC BACILLI. A. Chroinogenic, Non-Liquefying Bacilli. 237. BACTERIUM LUTEUM (List). Found in water. Morphology. — Elliptical cells from 1.1 to 1.3 // long. Biological Characters. — An aerobic, non-liquefying, non-motile, chro- mogenic bacillus. Produces an orange-yellow pigment soluble in water, alcohol, and ether; not changed by alkalies, but at once destroyed by acids. Grows at the room temperature in the usual culture media. Upon gelatin plates forms irregular colonies upon the surface, of slimy consistence and orange-yellow in color ; under a low power these are seen to consist of many club-shaped, coarsely granular, zpoglcea masses, each one of which is made up of several segments. In gelatin stick cultures development occurs slowly along the line of puncture and an orange-yellow layer forms on the surface. In milk, at 30° C., a layer forms upon the surface in from twenty-four to thirty hours, and the milk below has a pale-yellow color ; later coagulation of the casein occurs. 238. BACILLUS AURANTIACUS (Frankland). ' Found in well water. Morphology.— Short, thick bacilli which vary greatly in their dimensions ; sometimes associated in pairs ; often grow out into long filaments. Biological Characters. — An aerobic, non-liquefying, slightly motile, chromogenic bacillus. Forms an orange-colored pigment. Grows at the room temperature in the usual culture media. Upon gelatin plates the super- ficial colonies are homogeneous, opaque, elevated masses of a pale-orange color ; the deep colonies spherical and granular. In gelatin stick cultures scarcely any development occurs along the line of puncture ; upon the sur- face a shining, orange-colored layer is formed. Upon agar a similar devel- opment occurs. Upon potato the growth is limited to the line of inoculation and is of shining, oimtge-red color. 239. BACILLUS BRUNNEUS (Adametz). Found in water. Morphology. — Small, slender bacilli. Biological Characters.— An. aerobic and facultative anaerobic, non- liquefying, non-motile bacillus. Forms a brown pigment. Forms spores. Grows in the usual culture media at the room temperature. Upon gelatin plates the superficial colonies appear as little drops of slimy consistence, but not viscid ; under a low power they are at first white and opaque, with circu- lar contour ; later, in ten to fourteen days, they are gray, and a brown color is given off from the lower surface. In gelatin stick cultures development XON-PATHOGENIC BACILLI. 621 occurs chiefly along the line of puncture ; upon the surface a milk-white, slimy growth is seen about the point of puncture ; this becomes about one millimetre thick and changes to a gray color, while a brown pigment is given off from its lower surface and also along the line of inoculation. 240. BACILLUS AUREUS (Adametz). Found in water and also upon the surface of the body in cases of eczema seborrhceicum — by Unna and Tommasoli. Morphology. — Slender bacilli, straight or slightly curved, from 1.5 to 4 n long and 0.5 /f broad ; often arranged in groups in which the bacilli lie paral- lel to each other; frequently in pairs or in long filaments. Biological Characters. — An aerobic, non-liquefying, slightly motile, chromogenic bacillus. Forms a golden-yellow pigment. Spore production not observed. Grows slowly at the room temperature in the usual culture media. Upon gelatin plates forms small superficial colonies of irregular form, which at the end of eight days still appear as small white points ; these later become pale-yellow and finally chrome-yellow in color ; they are opaque, have well-defined outlines, and may attain a diameter of one to four milli- metres; they vary in form, being round or elliptical, and later sometimes sausage-shaped. In gelatin stick cultures development occurs slowly upon the surface in the form of small, hemispherical colonies crowded together to form an irregular, dull-shining, dark chrome-yellow layer ; very scanty growth occurs along the line of puncture. Upon potato broad, glistening, hemispherical colonies are formed, which gradually become confluent and form a dark chrome-yellow layer which in old cultures acquires a deep red- dish-brown color. 241. BACILLUS FLAVOCORIACEUS (Eisenberg). Synonym. — Sulphur-yellow bacillus (Adametz). Found in water. Morphology. — Very small bacilli. Biological Characters. — An aerobic, non-liquefying, non-motile, chro- mogenic bacillus. Produces a sulphur-yellow pigment. Spore formation not observed. Grows at the room temperature. Upon gelatin plates forms small, round, sulphur-yellow colonies, which under a low power are seen to be coarsely granular and have a brownish-yellow centre surrounded by a pale-yellow marginal zone ; older colonies are irregular in outline. In gela- tin stick cultures a scanty, granular, grape-like growth occurs on the sur- face and along the line of puncture. 242. BACILLUS BEROLINENSIS INDICUS (Classen). Found in water — of the Spree. Morphology. — Slender bacilli with round ends, resembling the typhoid bacillus in form and dimensions ; usually solitary, sometimes united in pairs or in chains of three. The rods are surrounded by a delicate, transparent envelope. Biological Characters. — An aerobic, non-liquefying, motile, chromo- genic bacillus. Forms an indigo-blue pigment which is insoluble in water, alcohol, or chloroform, soluble in cold concentrated hydrochloric acid ; is decolorized by ammonia. Spore formation not demonstrated. Grows rap- idly at the room temperature in the usual culture media. Upon gelatin plates, at the end of three days, grayish-white colonies the size of a pin's head are developed ; by the fourth day these commence to acquire an indigo- blue color, the deep colonies around the margin and the superficial colonies more in the centre ; the uncolored margins of the superficial colonies are ir- regularly bulged and have a pearly lustre. In gelatin stick cultures a small, indigo-blue mass develops at the point of puncture at the end of twenty- 52 622 NON-PATHOGENIC BACILLI. four hours • in the course of three or four days this becomes a regular wall, and then extends slowly over the surface of the gelatin, forming a layer with irregular outlines. Upon the surface of agar the growth is very charac- teristic ; at the room temperature an indigo-blue streak is seen at the end of twenty-four hours, and in four or five days this becomes a tolerably thick layer, which gradually thins out at the edges and which has a deep indigo- blue color ; the surface is covered up to within two or three millimetres of the margin with a thin, shining layer of pigment similar to that seen upon a saturated solution of gentian violet. Upon potato, at the end of three or four days, an abundant layer, thickest in the middle and with well-defined margins, is formed ; this has a deep indigo-blue color, and also has a thin film of pigment on the surface when the potato has an acid reaction; when alkaline a thin, dirty-green layer is formed without the film of pigment on the surface, 243. BACILLUS CONSTRICTUS (Zimmermann). Found in water. Morphology. — Bacilli with round ends, from 1.5 to 6.5 ju in length and 0.75 a thick; the rods are constricted at intervals so as to form two to six or more segments, which in stained preparations are separated by an unstained interval. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, motile, chromogenic bacillus. Forms a Naples-yellow pigment. Spore formation not observed. Grows slowly at the room temperature in the usual culture media — no development at 30° C. and above. Upon gelatin plates forms, in four to five days, small, shining drops of a Naples-yellow color; the deep colonies under a low power are spherical, granular, and yellowish-gray in color; they have well-defined outlines, but the margins are irregularly dentate. In gelatin stick cultures development occurs along the line of puncture, and upon the surface a layer is developed which con- sists of irregular masses closely crowded together and which have a Naples- yellow color. Upon the surface of agar a tolerably thick, shining, Naples- yellow streak is developed along the impfstrich. Upon potato after a time a thin layer of a cadmium-yellow color is developed. 244. BACILLUS FLUORESCENS AUREUS (Zimmermann). Found in the Chemnitz water supply. Morphology. — Short bacilli, about 1.9 n long and 0.74 /* broad, usually in pairs and associated in longer or shorter groups; have long terminal flagella. Biological Characters. — An aerobic, non-liquefying, actively motile, chromogenic bacillus. Forms an ochre-yellow pigment. Spore formation not observed. Grows in the usual culture media — best at the room tempera- ture. Upon gelatin plates the deep colonies are small, yellowish- white, and spherical ; the superficial colonies are glistening, yellowish-gray, and have ill-defined margins ; under the microscope they are seen to be thickest in the middle and have irregular outlines, while the deep colonies are sphei'ical, pale-yellow, and granular. In gelatin stick cultures a scanty development occurs along the line of puncture, which after a time acquires a brownish color; upon the surface a thin layer is developed, which later becomes thicker at the centre and extends to the walls of the tube ; it has a yellow color. Upon the surf ace of agar an abundant ochrous or golden-yellow layer is developed. Upon potato the growth is similar but more scanty. 245. BACILLUS FLUORESCENS LONGUS (Zimmermann). Found in the Chemnitz water supply. Morphology.— Straight or slightly curved bacilli, about 0.83 n broad, and varying greatly in length — from 1.45 to 14 u and more. NON-PATHOGENIC BACILLI. 623 Biological Characters. — An aerobic, non-liquefying, motile, chromo- genic bacillus. Forms a grayish-yellow pigment. Spore formation not positively determined, but unstained bodies resembling spores are seen in the rods. The short bacilli only are motile, the longer filaments not. Upon gelatin plates the deep colonies are small, spherical, and white with a green- ish lustre; under a low power they are seen to be yellowish, sharply de- fined, and the interior is marked by contorted stripes. The superficial colonies are at first quite thin, nearly circular in form, and with a pearly lustre ; they increase rapidly in size, and at the end of three days appear as yellowish-green patches which may be as much as nine millimetres in dia- meter ; the margins are very thin and appear to be permeated with yellow- ish-white threads ; under a low power the superficial colonies are seen to be striped by broad, convoluted bands resembling the intestine of a small ani- mal. In gelatin stick cultures a very thin layer first forms, which later be- comes thicker at the centre and soon reaches the walls of the test tube ; at first it has a blue and later bluish-green fluorescence. Upon the surface of agar a rather thin layer is developed and the agar acquires a greenish-yellow color. Upon potato a thin, gray layer with a yellowish reflection and moist, shining surface extends over the surface. 246. BACILLUS FLUORESCENS TENUIS (Zimmermann). Found in the Chemnitz water supply. . Morphology. — Short, thick bacilli with round ends, from 1 to 1.85 fit long and about 0.8 M thick; one or both ends are somewhat pointed ; the rods are associated in irregular groups, and in old cultures often in chains con- taining four to six elements. Biological Characters. — An aerobic, non-liquefying, motile, chromo- genic bacillus. Forms a greenish-yellow, fluorescent pigment. Spore for- mation not determined. Grows rather rapidly in the usual culture media at the room temperature. Upon gelatin plates thin, shining superficial colonies are developed, which are irregularly circular in outline and have a radiate-cleft margin ; the gelatin acquires a green color far beyond their boundary. In gelatin stick cultures a thin layer is formed upon the surface, which afterwards becomes thicker, of a grayish-white color, and extends to the walls of the test tube in about four days ; the gelatin acquires a beautiful yellow color near the surface ; by reflected light the surface growth is at first blue and later bluish-green; the line of puncture is marked by a scanty development which does not become any more pronounced. Upon the sur- face of agar a smooth, gray, shining, and rather scanty layer is developed; the agar gradually acquires a greenish color. Upon potato a thin, grayish- yellow, shining layer is developed along the impfstrich; later this acquires a reddish-brown color. 247. BACILLUS FLUORESCENS NON-LIQUEFACIENS. Found in water. Morphology. — Short, slender rods with round ends. Biological . Characters. — An aerobic, non-liquefying, non-motile, chro- mogenic bacillus. Forms a greenish-yellow, fluorescent pigment. Spore formation not observed. Grows in the usual culture media at the room temperature — not in the incubating oven. Upon gelatin plates forms fern- shaped superficial colonies, around which the gelatin has a pearly lustre. In gelatin stick cultures very scanty growth occurs along the line of punc- ture ; the superficial growth has a fluorescent shimmer. Upon the surface of agar a layer is formed having a greenish color. Upon potato a rapidly de- veloped, diffuse, brownish layer is formed, and the surface of the potato acquires a grayish-blue color. 624 NON-PATHOGENIC BACILLI. 248. BACILLUS FLUORESCENS PUTIDUS (Flugge). Found in water. Morphology.— Short bacilli with round ends. Biological Characters. — An aerobic, non-liquefying, actively motile, chromogenic bacillus. Produces a greenish, fluorescent pigment. Does not form spores. Grows in the usual culture media at the room temperature. Upon gelatin plates the superficial colonies are small, and under a low power they are seen to be finely granular discs with a dark centre surrounded by a yellow zone, and a pale-gray margin which is serpentine in outline; older colonies are dentate and have a greenish shimmer ; the gelatin around ac- quires a fluorescent green color ; an odor of trimethylamin is given off. In gelatin stick cultures development occurs upon the surface only, as a thin, dirty- white layer ; in the course of a few days the gelatin commences to acquire a greenish, fluorescent color, most intense near the surface and gradually fading out below. Upon potato a thin, slimy, gray or brownish layer is developed. 249. BACILLUS ERYTHROSPORUS (Eidam). Found in water, in putrefying flesh infusion, etc. Morphology. — Slender bacilli with round ends ; often grow out into short filaments. Biological Characters. — An aerobic, non-liquefying, motile, chromo- genic bacillus. Forms a greenish-yellow, fluorescent pigment. Forms oval spores — from two to eight in each filament. Grows at the room temperature in the usual culture media — not in the incubating oven. Upon gelatin plates forms whitish colonies, which spread out over the surface of the gelatin as a wrinkled and furrowed layer, around which the gelatin acquires a greenish- yellow, fluorescent color; under a low power the colonies are seen to have an opaque, brownish centre surrounded by a transparent, greenish-yellow mar- ginal zone, an irregular outline, and the surface is marked by indistinct, ra- diating lines. In gelatin stick cultures an abundant growth occurs along the line of puncture and upon the surface ; the gelatin throughout gradually acquires a fluorescent green color by transmitted, and a yellow color by re- flected light. Upon potato a layer of limited dimensions is formed, which is at first reddish in color and later nut-brown . 250. BACILLUS VIRIDIS PALLESCENS (Frick). Morphology. — Bacilli which are somewhat longer and more slender than the typhoid bacillus ; form long filaments. Biological Characters. — An aerobic, non-liquefying, actively motile, chromogenic bacillus. Produces, in presence of oxygen, a pale bluish-green pigment, which later becomes yellowish-brown with a green fluorescence. Grows best at the room temperature. Spore formation not observed. Upon gelatin plates the deep colonies are small and spherical; superficial colonies flat, with irregular, well-defined margins and a granular surface — similar to Bacillus virescens, but larger and of more rapid development ; the colonies are green in color at first, but fade out to a pale bluish-green ; they are fre- quently slightly iridescent. In gelatin stick cultures growth occurs upon the surface only, of a green color which quickly fades out to a bluish-green. Upon the surface of agar the growth is the same as on gelatin. Upon potato a nut-brown, moist layer is formed, and the potato around it acquires a dirty- violet color. 251. BACILLUS VIRESCENS (Frick). Found in green sputum. Morphology. — Bacilli of about the size of the typhoid bacillus, three to four times as long as broad ; frequently grow out iiito long filaments. NOX-PATHOGENIC BACILLI. 625 Biological Characters. — An aerobic, non-liquefying, actively motile, fhromogenic bacillus. Forms a green pigment, which after several weeks becomes yellowish-brown and then dark-green and fluorescent. Spore formation not observed. Grows best at the room temperature— rather slowly. Upon gelatin, plates the deep colonies are small and spherical ; the superficial colonies are flat, with irregular but well-defined margins and a finely gran- ular surface; at the end of two to three days the colony and the surrounding gelatin have an intensely green color ; later the colony becomes thicker, softer, and of a still deeper green color. In gelatin stick cultures growth occurs on the surface only ; the gelatin acquires a decided green color. Upon agar an abundant development occurs upon the surface. Upon potato a nut-brown, moist layer is formed, and the potato around it acquires a dirty-violet color. In bouillon forms a layer upon the surface, which is with difficulty made to sink to the bottom, and not until it is broken up by shaking ; the upper por- tion of the bouillon has a green color. 252 BACILLUS IRIS (Frick). Morphology. — Very small, slender bacilli. Biological Characters. — An aerobic, non-liquefying, non-motile, chromo- genic bacillus. Produces a fluorescent green color, which later becomes yellowish-brown, and finally dark-green and fluorescent. Spore formation not observed. Grows best at the room temperature. Upon gelatin plates forms prominent, whitish, round, sharply defined superficial colonies with a, smooth, shining surface ; a green color is slowly developed. In gelatin stick cultures a prominent mass develops about the point of puncture ; no growth along the line of puncture. Upon potato a dry, pale-brown layer is formed. In bouillon no layer is formed upon the surface, and the bouillon is not colored 253. BACILLUS FUSCUS (Zimmermann). Found in water. Morphology.— Straight or curved bacilli with round ends, an irregular contour, here and there slightly bulging ; about 0.63yu in diameter and of various lengths. Biological Characters. —An aerobic, non-liquefying, non-motile, chromo- genic bacillus. Forms a dark chrome-yellow pigment. Spore formation not observed. Grows slowly at the room temperatm-e in the usual culture media — best at 30° C. Upon gelatin plates the deep colonies are punctiform -and yellowish-brown in color ; later they project from the surface in button form, and often have an irregular, knobby form ; under the microscope such colonies are seen to be made up of smaller, spherical masses. The deep col- onies, under a low power, are spherical or irregular in form, granular, and grayish-yellow or brownish -yellow in color ; the superficial colonies have a brownish-yellow central portion surrounded by a highly refractive marginal zone. In gelatin stick cultures a button-like mass develops at the point of puncture; later the growth extends over the surface, forming a thick, wrinkled layer, at first pale-yellow and then chrome-yellow in color. The growth upon agar is similar to that upon gelatin. Upon potato a dark chrome-yellow, friable layer is developed. 254. BACILLUS RUBEFACIENS (Zimmermann). Found in the Chemnitz water supply. Morphology. — Bacilli with round ends, from 0.75 to 1.65 /* long and about 0.32 n thick; united in pairs, or in chains consisting of several elements. Biological Characters.— An aerobic, non-liquefying, actively motile, chromogenic bacillus. Produces a pale-pink pigment. Grows best at the room temperature. Upon gelatin plates the deep colonies are spherical or lenticular in form, and white with a shade of yellowish-red ; the superficial 620 NON-PATHOGENIC BACILLI. colonies are flat, gray with a reddish tint ; under the microscope the deep colonies are seen to be granular, spherical, and yellowish or brownish in color. In gelatin stick cultures development occurs along the line of punc- ture, and upon the surface a tolerably thick, grayish-white layer is formed, which later has a yellowish tint, while the gelatin around it has a bluish- white lustre. In old cultures the gelatin frequently acquires a pale wine color. Upon agar a smooth, rather thick, bluish-gray layer is formed. Upon potato an abundant, yellowish-gray layer is developed, which later has a reddish-brown color, while the potato around it after forty -eight hours acquires a pink color. 255. BACILLUS STRIATUS FLAVUS (Von Besser). Found in nasal mucus — rare. Morphology.— Small, thick rods, often curved, of about the dimensions of the diphtheria bacillus. In preparations stained with methylene blue, has a striated appearance. In old preparations various involution forms are seen. Biological Characters. — An aerobic, non-liquefying, chromogenic bacil- lus. Forms a sulphur- yellow pigment. Grows at the room temperature in the usual culture media. Spore formation not observed. Upon gelatin plates forms thick, dry, granular colonies of a yellowish color. Upon agar plates projecting milk-white colonies of about 0.5 centimetre in diameter are developed ; later these acquire a sulphur-yellow color. Upon the sur- face of agar a white, thick layer develops along the impfstrich ; after some days this acquires a sulphur-yellow color. Upon potato a narrow, yellow stripe is developed along the impfstrich. 256. BACILLUS SUBPLAVUS (Zimmermann). Found in the Chemnitz water supply. Morphology. — Bacilli with round ends, from 1.5 to 3;* long and about 0.77 n broad ; united in chains of several elements. Biological Characters. — An aerobic, non-liquefying, motile, chromo- genic bacillus. Forms a pale-yellow pigment. Grows best at the room tem- perature. Spore formation not observed. Upon gelatin plates the deep colo- nies are small, yellowish-white, and spherical; these break through the surface and form hemispherical, yellowish-white, shining masses, which later extend over the surface and have an irregular outline; under a low power the surface is seen to have a pearly lustre ; they gradually acquire a dirty-yellow color. In gelatin stick cultures a thin, yellowish-gray layer with finely dentate margins forms about the point of puncture. Upon the surface of agar a pale-yellow layer gradually extends over the entire sur- face; the color gradually becomes darker and is finally between a pale chrome-yellow and yellow ochre. Upon potato a scanty, dull, clay-yellow layer is formed. 257. BACILLUS CYANOGENUS (Hueppe). Synonyms. — Bacterium syncyanum; Bacillus lactis cyanogenus; Bacil- lus of blue milk. Found in milk. Morphology. — Bacilli with slightly rounded corners, from 1.4 to 4 n long and from 0.3 to 0.5 n broad. Biological Characters. — An aerobic, non-liquefying, actively motile, chromogenic bacillus. Produces a grayish-blue pigment. Forms spores, which are located at the extremities of the rods, giving them a club shape. Grows rapidly at the room temperature or in the indicating oven. Upon gelatin plates, at the end of two days, small, punctiform, grayish -white colonies are developed ; the superficial colonies appear as slimy drops which NON-PATHOGEXIC BACILLI. 627 attain a diameter of one to two millimetres ; they are finely granular, have a dirty- white color and circular, smooth outline ; the gelatin around them ac quires a steely grayish-blue shimmer. Under a low power the deep colonies are seen as round discs with a dark centre and a brownish, granular mar- ginal zone with a sharply defined dark contour ; the superficial colonies have a dark-brown centre surrounded by a grayish-brown, and outside of this a narrow, yellowish, finely granular marginal zone with a sharply denned contour. In gelatin stick •„ t , v cultures a white mass develops around the point -^f/^L \L ) '^ />\! *// of puncture, and around it the gelatin acquires a ^ wr /}, dark steel-blue color. Upon agar a grayish layer is developed and the culture medium acquires a dark-brown color near the surface. Upon potato FIG. 210.— Bacillus cyano- a slimy, yellowish layer is developed, limited to genus; at a the rods contain the vicinity of the impf strich ; around this the po- spores, x TOO. (Flugge.) tato acquires a diffused grayish-blue color. Upon blood serum development occurs without the formation of pigment. In milk a slightly alkaline reaction is produced by the growth of this bacillus ; the casein is not coagulated ; at the surface and gradually extending down- ward a slate-blue color is developed, and upon the addition of an acid this changes to an intense blue. In milk which has not been sterilized and which contains acid-forming1 bacteria a sky-blue color is produced without the ad- dition of an acid. The pigment is produced most abundantly at a tempera- ture of 15° to 18° C. ; at 25" G. it is less abundant, and at 37° C. is not formed at all. Note. — Jordan gives an account of Bacillus cyanogenus which differs materially from that above given by Fliigge, and which leads to the belief that two or more different bacilli have been described under this name. Jordan's description agrees tolerably well, however, with that of Heim. The bacillus described by him was quite frequently found in Lawrence sewers and is described as follows : "Morphology. — Small bacilli with rounded ends, often oval in form. Occur in chains in all media, isolated individuals being quite the exception. The chain is usually long and its members cohere quite firmly. On no me- dium has there been observed anything resembling spore formation. The individuals are about 1.3 /* long and 0.8 ju broad. Motility : There is a slight independent movement to be observed in hanging drops. We have certainly ncD found this species to be ' very motile.' Temperature: Does not grow as well at 37° as at 21° C. Need of oxygen : Grows very scantily under the mica plate. Plate cultures : The young colonies below the surface of the gelatin are usually slightly oval, with a coarsely granular interior and an even, regular edge. Often, however, the colonies have a frayed, irregular appearance to the naked eye, and with the microscope show fine branchings from the centre. On coming to surface the colonies always spread out into a dull, dry expansion with irregularly hacked edges. Sometimes the colo- nies are surrounded by a light blue-green haze, which soon changes to a faint brown ; and this becomes deeper and deeper till the whole plate is colored an intense dark -brown. More often, however, in our experience, the brown color comes without a previous development of the blue. In slightly acid gelatin the blue color comes more surely and constantly than in the ordinary alkaline medium. The gelatin is not liquefied. Tubes — gelatin : In about three days there is a thin surface growth, smooth and faintly lus- trous. The contour is at first quite regular, with the edges only slightly toothed. There is only a slight growth along the inoculation line. The gelatin near the surface soon takes on a brown tint, and eventually the whole tube of gelatin is colored dark-brown. The blue coloration is not observed as well in tubes as on plates. This species grows fairly well in acid gelatin, but not so well as in alkaline. In the former the brown color invariably comes more slowly. Tubes— agar : In. three days there is a good surface growth, white and lustrous. The agar is colored dark-brown. The growth itself G28 NON-PATHOGENIC BACILLI. also assumes a brownish cast. Potato culture .' A very rapid growth on po- tato. In twenty-four hours the potato is colored a deep-brown color over nearly its whole surface. We have in no case observed a previous blue color- ation. The growth is brownish, thin, spreading-, and dry. Milk: The milk very slowly becomes a light-chocolate color. The reaction is slightly alka- line. Bouillon: In three days the bouillon has become slightly turbid. Month-old cultures are a deep brown, with a tough, thick skin on the sur- face. Effect upon nitrates : Nitrates are slowly reduced." Jordan remarks : "This is undoubtedly the 'bacillus of blue milk' de- scribed originally by Hueppe." 258. BACILLUS FUSCUS LIMBATUS (Scheibenzuber). Obtained from rotten eggs. Morphology. — Short rods, occasionally united into filaments. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, actively motile, chromogenic bacillus. Produces a brown pig- ment. Spore formation not observed. Grows at the room temperature in the usual culture media. Upon gelatin plates colonies are developed in the form of small brown masses of circular outline and often surrounded by a paler marginal zone which is two to three times as broad as the brown in- terior. In gelatin stick cultures a branching growth is seen along the line of puncture, and the short branches are beset with small projections ; the gelatin in the vicinity of the line of growth acquires a brown color. Upon the surface a scanty development occurs. Upon the surface of agar a super- ficial layer, beneath which the medium acquires a dark-brown color, Upon potato a brown layer is developed. 259. BACILLUS LATERICEUS (Eisenberg). Synonym.— Ziegelroter bacillus (Adametz). Found in water. Morphology. — Bacilli, from three to five times as long as broad; grow out into short curved filaments. Biological Characters. — An aerobic, non-liquefying, non-motile, chro- mogenic bacillus. Forms a brick-red pigment. Grows very slowly at the room temperature. Spore formation not observed. Upon gelatin plates forms punctiform, brick- red colonies ; under a low power these are seen to be spherical, finely granular, and brownish-red in color ; the opaque centre is surrounded by a more transparent marginal zone. In gelatin stick cultures a tolerably thick, slimy, brick-red layer is developed ; very scanty growth along the line of puncture. Upon potato a brick-red layer, 260. BACILLUS SPINIFERUS (Unna). Found upon the surface of the body in cases of eczema seborrhceicum. Morphology. — Straight or slightly curved bacilli, 2 /* long and 0.8 to 1 f-t broad ; solitary or in pairs, frequently seen in irregular groups or lying parallel to each other in bundles. Biological Characters. — An aerobic, non-liquefying, chromogenic ba- cillus. Forms a grayish-yellow pigment. Spore formation not observed. Grows rather slowly at the room temperature. Upon gelatin plates, at the end of eight days, the superficial colonies are prominent, shining, skin- colored, and circular in outline, from one to two millimetres in diameter ; under a low power they are seen to be irregular in outline, covered with coarse projections ; later finely granular, with a margin surrounded by small, thorn-like projections, which after a time form a radiating aureole and give the colony a porcupine-like appearance. In gelatin stick cultures, at the end of six days, an irregular, wrinkled layer about two millimetres broad is formed upon the surface; this is grayish-yellow upon the folds and bluish NON-PATHOGENIC BACILLI. 029 in the furrows; as this extends the folds become more prominent and more decidedly yellow in color, the margins are irregular, dentate, and thin; along the line of inoculation very small, yellowish, punctiform colonies are developed. Upon the surface of agar the growth is similar to that upon gelatin. Upon potato development is very slow; first as a glistening, yel- lowish-white line along the impfstrich; at the end of three weeks here and there along1 this line small, grayish-yellow, glistening masses are seen. 261. BACILLUS KUBESCENS (Jordan). Found in sewage at Lawrence, Mass. Morphology. — Bacilli with round ends, about 4 jn long and 0.9 n broad, often slightly curved ; solitary, in pairs, or in short chains. Biological Characters. — An aerobic, non- liquefy ing, motile, chromoge- nic bacillus. Forms a pale-pink pigment. Spore formation not observed. Movements slow. Grows best at the room temperature. Upon gelatin plates the deep colonies are spherical or oval ; on coming to the surface they form a projecting, porcelain- white drop; they slowly increase in size, and after a time have a slight brownish color. In gelatin stick cultures a pro- minent porcelain-white mass is formed upon the surface at the point of in- oculation; very scanty growth along line of puncture; grows in slightly acid gelatin. Upon the surface of agar a white and lustrous layer is quickly de- veloped, which is at first smooth, but later becomes wrinkled ; in cultures about three weeks old a slight pinkish tinge is seen. Upon potato the growth is rapid and abundant; it is at first light-brown in color, and this gradually changes to pink; at the end of three weeks there is a projecting growth which has a delicate flesh-pink color ; the potato itself is not coloi'ed. Milk is not coagulated and has an alkaline reaction ; in old cultures a slight pinkish tinge is observed near the surface. Bouillon becomes slightly tur- bid and a heavy white precipitate is formed. At the end of several weeks a thick, tenacious film forms upon the surface. 262. BACILLUS ALLII (Griffiths). Synonym. — Bacterium allii. Found upon the surface of putrefying onions. Morphology. — Bacilli with round ends, from 5 to 7 /« long and about 2. 5 p broad ; solitary or in pairs ; form zooglcea. Biological Characters. — An aerobic, chromogenic bacillus. Produces a green pigment which is soluble in alcohol. Grows tolerably well on nu- trient agar, and produces a bright green pellicle on the surface of this me- dium. Sulphuretted hydrogen is given off by the cultures in small quan- tities. B. Chromogenic, Liquefying Bacilli. 263. BACILLUS FULVUS (Zimmermann). Found in the Chemnitz water supply. Morphology. — Short bacilli with round ends, from 0.88 to 1.3 ju in length and 0.77 // broad ; solitary, in pairs, or in chains containing several elements. Biological Characters. — An aerobic, liquefying, non-motile, chromoge- nic bacillus. Grows slowly at the room temperature — best at 30° C. Spore formation not observed. Forms a gamboge-yellow-colored pigment. Upon gelatin plates the deep colonies are irregularly spherical, oval, or elliptical, granular, and yellowish -gray in color; they are usually surrounded by pale- yellow patches. The superficial colonies are reddish-yellow, drop-like, and at the end of eight days measure about one millimetre in diameter. In gela- tin stick cultures a prominent, arched mass of irregularly circular contour and of the color of gamboge is formed about the point of puncture ; a scanty 53 630 NON-PATHOGENIC BACILLI. growth occurs along the line of inoculation, which has a slightly yellowish color; liquefaction commences after some weeks. Upon the surface of agar an abundant, glistening layer of a gamboge-yellow color. Upon potato development occurs slowly along the impfstrich; the growth has at first an india-yellow color, which later becomes ochre yellow. 264. BACILLUS HELVOLUS (Zimmermann). Found in the Chemnitz water supply. Morphology. — Bacilli from 1.5 to 4.5 // in length and about 0.5 n broad; usually in pairs, but also in chains of four or more elements and in long filaments. Biological Characters.— An aerobic, liquefying, motile (rotary motion only), chromogenic bacillus. Forms a Naples-yellow pigment. Spore for- mation not observed. Grows rather rapidly at the room temperature. Upon gelatin plates the deep colonies are small, spherical, and of a pale-yellow color ; when they come to the surface they form a prominent pale-yellow drop, which lies in a shallow cavity. The superficial colonies for some time have a sharply defined contour and an irregular outline ; by transmitted light they have a dark-brown color. In gelatin stick cultures a button-like mass of a Naples-yellow color forms about the point of puncture ; this gradu- ally extends until it nearly reaches the walls of the test tube ; in the n.ean- time the gelatin below is slowly liquefied and a saucer-shaped depression is formed ; liquefaction progresses very slowly. Upon the surface of agar an abundant layer of a Naples-yellow color is developed. Upon potato a tolerably thick and abundant, shining, yellow layer, which frequently has a shade of green. 265. BACILLUS OCHRACEUS (Zimmermann). Found in the Chemnitz water supply. Morphology. — Bacilli with round ends, from 1.25 to 4.5 /* long and from 0.65 to 0.75 /f broad; usually in pairs, or in chains containing several ele- ments, also in long jointed filaments. In stained preparations a capsule-like envelope may often be seen. Biological Characters. — An aerobic, liquefying, motile, chromogenic bacillus. Forms an ochre- yellow pigment. Spore formation not observed. Motions slow and serpentine. Grows rather slowly — best at the room tem- perature. Upon gelatin palates forms small, pale-yellow, spherical colonies, which gradually increase in size and cause the gelatin above them to become liquefied, so that they lie at the bottom of a saucer-shaped cavity ; here the color is more intense and becomes a golden ochrous yellow ; later the yel- low mass extends in diameter, and is of a paler yellow color at the mar- gin than at the centre. Under a low power the colonies are seen at first as granular, yellowish-brown discs ; later they are covered with wart-like pro- jections. ID gelatin stick cultures liquefaction occurs at first in funnel form, and when it extends to the walls of the tube it gradually progresses downward ; a pale-yellow deposit is seen at the bottom of the liquefied gela- tin, which later acquires an ochrous-yellow tint. Upon the surface of agar an ochrous-yellow layer is formed, which at the end of four to six weeks has a breadth of about six millimetres at the lower portion of the impfstrich. Upon potato a thin, ochrous-yellow layer is developed. 266. BACILLUS PLICATUS (Zimmermann). Found in the Chemnitz water supply. Morphology.— Small, thin bacilli with rounded ends, about 0.48/* (?) in length and 0.45 /* broad; usually in pairs, but also in chains of four or more elements. Biological Characters. — An aerobic, liquefying, non-motile, chromo- genic bacillus. Forms a grayish-yellow pigment. Spore formation on NON-PATHOGENIC BACILLI. 631 observed. Grows best at the room temperature. Upon gelatin plates small, yellowish-white colonies of irregular form are developed beneath the surface, which under a low power are seen to present hemispherical projections ; when they break through the surface they have a mulberry -like appearance and yellowish-white color. In gelatin stick cultures a wrinkled, yellowish- white layer with irregular contour is developed; at the end of one or two weeks it is depressed and the gelatin gradually undergoes liquefaction : an abundant development of small, granular, yellowish- white colonies occurs along the line of puncture. Upon potato a thin layer is formed, which soon becomes dry and friable and has a grayish-yellow color. 267. BACILLUS JANTHINUS (Zopf). Synonym. — Yiolet bacillus. Found in water and in sewage (at Lawrence, Mass.). Morphology. — Very small, slender bacilli, often associated in short fila- ments; about 2 n long and 0.5 to 0.6 ft broad. Biological Characters. — An aerobic, liquefying, motile, chromogenic bacillus. Forms a bluish-violet pigment. Spore formation not observed. Grows best at the room temperature. Upon gelatinplates the deep colonies are spherical or oval, with an even contour ; upon coming to the surface they spread out as a broad, irregular expansion with deeply notched edges ; these superficial colonies are at first thin, but later increase in thickness ; a deep-violet color soon appears, sometimes near the centre of the colony, sometimes around its edges; the gelatin is very slowly liquefied. In gelatin stick cultures a scanty growth, without color, is developed along the line of puncture, and a thin, violet-colored layer upon the surface. The gelatin is slowly liquefied, and an abundant violet-colored precipitate is seen at the bottom of the liquefied gelatin in old cultures. Upon the surface of agar a rather tough and coherent layer is developed, which has a dark-violet color. Upon potato the development is rapid and covers the entire surface ; the growth frequently has a beaded appearance, and the membranous growth is with difficulty detached from the surface; it has a black-violet color. In milk an abundant development occurs without producing coagulation, and causing it to acquire a violet color. Reduces nitrate to nitrite very rapidly and completely. (Above characters given by Jordan.) 268. BACILLUS VIOLACEUS LAURENTIUS (Jordan). " Found in large numbers in the effluent of Tank 1 " at Lawrence, Mass. Morphology. — Bacilli with round ends, from 3 to 3. 6 /* long and 0.7 ft broad; often in pairs, and sometimes in chains of four or five elements. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, actively motile bacillus. Produces a dark- violet pigment. Spore for- mation not observed. Grows best at the room temperature. Upon gelatin plates the deep colonies, at the end of two days, are small, spherical, and coarsely granular ; they usually have a radiating margin, and a dark centre ; upon coming to the surface a thin, irregular expansion occurs and the gela- tin is quickly liquefied in the vicinity of the colony ; at the centre a violet- colored spot is seen, and around this a zone of slightly clouded liquid gelatin ; liquefaction progresses rapidly, but the central violet mass does not mate- rially increase in size. In gelatin stick cultures liquefaction occurs rapidly along the line of inoculation; the liquefied gelatin is clouded and of a violet color, and an abundant dark- violet precipitate accumulates at the bottom of the tube. Upon agar an abundant development occurs, which at first has a dark-violet color and later becomes jet-black. Upon potato an abundant growth of a dark- violet color spreads over the entire surface; this soon be- comes jet-black, except near the edge. In milk the bacillus develops rapidly and abundantly, causing rapid coagulation of the casein and an acid reac- tion; the milk acquires a deep blue- violet color. In ordinary bouillon the C32 NOX-PATHOGENIC BACILLI. violet color is not developed, but when nitrates are added the growth is more luxuriant and a rich violet color is produced. Nitrates are reduced to- ni- trites by this bacillus, rather slowly. 269. BACILLUS TREMELLOIDES (Schottelius). Found in the Freiburg water supply by Tils. Morphology. — Bacilli with round ends, from 0.75 to 1 n long and 0.25 n broad, associated in friable masses. Biological Characters. — An aerobic, liquefying, chromoqenic bacillus. Forms a golden -yellow pigment. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates the deep colonies appear as yellow points ; at the end of forty-eight nours the superficial colonies appear as prominent yellow masses and the plate looks as if it were covered with coarse sand ; at the end of a few days the colonies measure several millimetres in diameter and do not subsequently increase in size ; under a low power these are seen £o be made up of cloud-like masses and have a yellow or yellowish-brown color, the contour is smooth or irre- gularly bulging; at the end of eight days the margin is surrounded by a golden-yellow, slimy layer and the colony commences slowly to sink into the gelatin. In gelatin stick cultures isolated, punctiform, yellow colonies are developed along the line of inoculation, and upon the surface a colony which resembles those upon gelatin plates ; at the end often to fourteen days the superficial layer is slowly depressed as a result of liquefaction of the underlying gelatin. Upon the surface of agar a layer is developed which is at first dry and granular, later slimy and of a golden-yellow color. Upon potato a yellow layer is developed which may attain a thickness of several millimetres; after a long time the growth is surrounded by a golden-yellow, slimy zone, and no further extension occurs upon the surface of the potato. In milk, at the end of thirty-six hours, a strongly acid reaction is produced and the fluid becomes viscid. 270. BACILLUS CUTICULARIS (Tils). Found in water. Morphology. — Bacilli from 2 to 3 n long and 0.3 to 0.5/f broad; may grow out into short filaments. Biological Characters. — An aerobic, liquefying, motile, chromogenic bacillus. Spore formation not observed. Forms a yellow pigment. The shorter rods are slightly motile, the longer filaments not. Grows in the usual culture media at the room temperature. Upon gelatin plates the deep colonies appear under the microscope as brownish discs with an irregular but smooth contour. The superficial colonies are yellowish-brown in color, with well-defined contour; after several days the centre commences to be depressed and the gelatin is quickly liquefied ; the colony then consists of a membranous film surrounded by a well-defined zone of liquefied gelatin; finally the gelatin of the plate is entirely liquefied and these membranous colonies float upon the surface. In gelatin stick cultures, at the end of two days, liquefaction commences near the surface and progresses rapidly; a membranous layer is formed on the surface. Upon potato development is slow, in the form of a pale-yellow layer, which later becomes slimy and dark-yellow. In milk a pale-yellow, membranous layer is formed upon the surface in from twenty-four to thirty-six hours, and an odor of sulphuretted hydrogen is developed. 271. FLESH-COLORED BACILLUS (Tils). Found in water. Morphology.— Bacilli of 2 j* in length and 0.5 t* broad ; in hanging-drop cultures always seen solitary and in active motion. NON-PATHOGENIC BACILLI. 633 FIG. 211.— Bacillus arbores- cens, from a gelatin culture. X 1,000. (Frankland.) Biological Characters.— An aerobic, liquefying, motile, chromogenic bacillus. Forms a dark flesh-colored pigment. Spore formation not ob- served. Grows at the room temperature in the usual culture media. Upon gelatin plates, at the end of two days, circular cavities with well-defined margins and filled with liquefied gelatin are seen; under a low power the centre of the colonies is seen to be more opaque and is surrounded by con- centric rings, alternately of lighter and darker color, while the marginal zone is colorless and appears finely granular. In gelatin stick cultures liquefaction occurs rapidly along the line of puncture, in funnel shape; at the lower part of the funnel a deposit of a pale-pink color accumulates and the liquefaction ceases to extend. Upon the surface of agar it grows rapidly and forms a thick, slimy, pale-pink layer. Upon potato a layer is formed which is first pale and later dark flesh-colored. 272. BACILLUS ARBORESCENS (Frankland). Found in the London water supply. Morphology.— Bacilli with round ends, about 2.5 ^ long and 0.5 /^ broad; often united in pairs, or in chains of three or four ^^_ elements ; also form long, flexible filaments. /^^^ -^ Biological Characters. — An aerobic, liquefy- — -S. ing, chromogenic bacillus. Spore formation not observed. Oscillating movements only. Grows in the usual culture media at the room tempera- ture. Upon gelatin plates the colonies, at the end of twenty-four hours, consist of a thin axial trunk with root-like offshoots at both ends; later the body becomes thicker and the branching extremities are so strongly developed that the whole has the ap- pearance of a sheaf of wheat ; the naked-eye appearance, in this stage, is also peculiar, and the colony is seen as an iridescent bundle, constricted in the middle and with the ends striped in- a radial direction; later the gelatin is liquefied slowly and the central part of the colony acquires a yellow color, while the periphery is beautifully iridescent. In* ge- latin stick cultures, by the second day, an iridescent layer is seen on the surface; in the middle of this the gelatin is slightly depressed and filled with a semi-fluid, yel- lowish mass; along the line of puncture a transparent, grayish cloudiness is seen ; liquefaction progresses slowly at the sur- face, and a funnel is formed, at the bottom of which the yellow deposit gradually in- creases; along the line of puncture no further changes occur. Upon the surface of agar a, rather thin, dirty orange colored layer is formed, the margins of which are slightly iridescent and striped in a radial direction. Upon potato a thick, glistening stripe of a deep orange-red color is formed along the line of in- oculation ; the surface of this is covered with irregular protuberances. 273. BACILLUS CITREUS CAD AVERTS (Strassmann). Found in a cadaver fifty hours after death from accidental shooting — in blood from a vein. Morphology. — Oval bacilli, 0.9 ju. long and 0.6 /^ broad, usually united in chains. Biological Characters. — An aerobic, liquefying, non-motile, chromogenic bacillus. Forms a yellow pigment. Spore formation not observed. Grows slowly at the room temperature. Upon gelatin plates forms small, pale- FIG. 212.— Colony of Bacillus arbo- escens. X 100. (Frankland.) 634 NON-PATHOGENIC BACILLI. yellow colonies, around which the gelatin is liquefied in circular form. In gelatin stick cultures liquefaction occurs at the surface, with formation of an air bubble at the surface, a scanty, yellow deposit at the bottom of this, clear liquefied gelatin below, and a yellow deposit on the concave floor of the liquefied gelatin; below this, along the line of puncture, small colonies are seen. The cultures give off an odor of sulphuretted hydrogen. Upon the surface of agar a narrow, yellow layer forms along the impfstrich. Upon potato a dry, lemon-yellow, slightly granular layer is developed. 274. BACILLUS MEMBRANACEUS AMETHYSTINUS (Eisenberg). Found in a specimen of well water from Spalato. Morphology. — Short bacilli with round ends, from 1 to 1.4 ju long and 0.5 to 0.8/* broad; in irregular groups; some of the rods in stained preparations show end staining. Biological Characters . — An aerobic, liquefying, non-motile, chrpmogenic bacillus. Produces a dark-violet pigment which has a metallic lustre. Spore formation not determined. Grows at the room temperature in the usual culture media — no development at 37. 5° C. Upon gelatin plates, at the end of two to three days, colonies are developed the size of a poppy-seed, which have a homogeneous, dark- violet color ; later a ring of liquefied gela- tin forms around each colony, which gradually extends, and at the end of one to two weeks the entire gelatin is liquefied and the colonies are seen floating upon its surface, not much larger than when liquefaction com- menced. When the colonies are not so closely crowded superficial colonies are seen at the end of three to four days, which resemble those of the typhoid bacillus ; they have a yellowish- white color and a dentate margin ; at the end of ten to fourteen days the gelatin around these colonies commences to soften and a dark- violet color extends from the centre to the periphery ; at the end of three to four weeks the colonies are seen floating upon the surface of the scarcely liquefied gelatin, as large violet layers. In gelatin stick cul- tures a yellowish-white layer with dentate borders develops upon the surface, which at the end of ten to fourteen days begins to acquire a violet color at the centre, which gradually extends to the periphery; liquefaction occurs slowly, so that at the end of about four weeks a thick, violet-colored layer rests upon the softened and depressed surface of the culture medium; at the end of three to four months the gelatin in the tube is completely liquefied. Upon the surface of agar a yellowish- white, milky, thick layer is formed, which commences to acquire a violet color at the end of eight to ten days, and at the end of three to four weeks is seen as a wrinkled, dark- violet layer with a metallic lustre, which is easily lifted entire from the sur- face of the culture medium. Upon potato a dirty-yellow or olive-green layer is slowly formed and gradually extends from the line of inoculation. In bouillon development is very slow ; at the end of several weeks the dark- brown bouillon is seen to have a violet film upon the surface and a deposit of the same color at the bottom of the tube. 275. ASCOBACILLUS CITREUS (Unna and Tommasoli). Found upon the surface of the body of individuals with eczema sebor- rhoeicum. Morphology. — Straight or curved bacilli, 1.3 jtt long and 0.3 1* broad, soli- tary, in pairs, or in irregular groups. Biological Characters. — An aerobic, liquefying, motile, chromogenic bacillus. Produces a lemon-yellow pigment. Spore formation not observed. Grows in the usual media at the room temperature — slowly in gelatin, rapidly upon agar and potato. Upon gelatin plates, at the end of two weeks, promi- nent, opaque, yellow, punctiform colonies are developed upon the surface, while the deep colonies are scarcely visible with the naked eye ; under a low power they are seen to be grayish-yellow, opaque, sometimes like little NOX-PATHOGEXIC BACILLI. 635 drops of oil and sometimes a conglomeration of minute spherical masses. Later the deep colonies are oval, dark, sharply defined, and as large as a pea ; those nearer the surface are conglomerate ; those upon the surface are partly homogeneous, pale-yellow, and round; some show an opaque, con- glomerate mass in the centre, with a more transparent, yellowish-green marginal zone ; some have a form resembling that of Saturn with its rings. In gelatin stick cultures a slimy, thick, lemon-yellow layer develops upon the surface ; this is gradually depressed in the middle, while the margins re- main elevated and granular ; along the line of puncture small colonies are developed which form a funnel above ; at the end of five to six weeks the gelatin in the funnel, when shaken, appears pap-like, and the layer floats upon its surface, while some greenish flocculi are seen below. Upon the surface ofagar development is rapid and the entire surface is covered within a few days; below, the layer consists of an abundant jelly-like growth, covered with protuberances resembling drops of honey ; above, it has an orange color and creamy consistence, and is covered with numerous small, spherical or oval masses. Upon potato a slimy, lemon-yellow layer is quickly developed and extends over the entire surface ; at the margins this is more transparent and albuminous in appearance ; at the end of two weeks the central portion has a greenish-yellow color, distributed like the veins of a grape leaf, with smaller, pale-yellow veins. 276. BACILLUS CCERULEUS (Smith). Found in water of the Schuylkill River. Morphology. — Bacilli from 2 to 2.5 p long and 0.5 u broad, frequently as- sociated in chains. Biological Characters. — An aerobic, liquefying, chromogenic bacillus. Produces a beautiful blue pigment. Spore formation not observed. Grows slowly at the room temperature. Upon gelatin plates forms superficial colo- nies having a blue color, around which the gelatin is liquefied. In gelatin stick cultures cup-shaped liquefaction occurs at the surface and a blue color is developed, while below a scanty, colorless growth occurs along the line of puncture. Upon the surface ofagar a bluish layer is formed. Upon po- tato at first a beautiful dark-blue layer, which later acquires an intense blue-black color. 277. BACILLUS FLUORESCENS LIQUEFACIENS. Found in water and in various putrefying infusions — very common. Morphology. — Short bacilli, in pairs with a constriction at the point of junction. Biological Characters. — An aerobic, liquefying, motile, chromogenic bacillus. Forms a greenish-yellow, fluorescent pigment. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates whitish colonies are developed upon the surface, which may attain a diameter of three millimetres; a ring of liquefied gelatin forms around each ; under a low power the colonies are seen to have a sharp con- tour and irregularly circular outline, a dark-brown, finely granular centre surrounded by a finely granular marginal zone of a yellow color, which be- comes more transparent and grayish- white toward the edge; the gelatin gradually acquires a greenish tint. In gelatin stick cultures a whitish growth occurs along the line of puncture ; a small funnel of liquefied gelatin is first seen near the surface, and this has an air bubble above; gradually the liquefaction extends to the walls of the test tube and also in a downward direction, forming a superficial layer of liquefied gelatin, upon the floor of which a thick white deposit is formed; the gelatin below this has a green- ish-yellow fluorescence and the liquefied gelatin also, although less pro- nounced. Upon potato an abundant brownish layer is developed along the line of inoculation. 630 NON-PATHOGENIC BACILLI. 278. BACILLUS FLUORESCEXS LIQUEFACIENS MIXUTISSIMUS (Unna and Tommasoli). Found upon the surface of the body in cases of eczema seborrhoeicuin. Possibly identical with the previously described species. Morphology. — Bacilli with round ends, usually constricted in the middle, from 1.5 to 2 u long and 0.3 u broad ; often united to form filaments. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile, chromogenic bacillus. Forms a greenish-yellow, slightly fluo- rescent pigment. Forms spherical spores. Grows rapidly in the usual cul- ture media at the room temperature. Upon gelatin plates, at the end of three to four days, the colonies consist of an outer yellowish- white zone of transparent, liquefied gelatin and a thick, pale-brown centre which is made up of grayish granular material. In gelatin stick cultures, at the end of three days, a broad funnel of liquefied gelatin is formed above, which is about one centimetre deep and five millimetres in diameter at the surface; the liquefied gelatin is clouded, greenish-yellow, and contains some whitish nocculi, while a thick whitish deposit accumulates at the bottom ; at the end of eight days the gelatin is entirely liquefied and a thick, opaque, gray- ish-yellow, fluorescent layer floats upon the surface. Upon the surface of agar a slimy, moist, smooth, pale-brown layer is developed. Upon potato a broad, compact, flat layer is quickly developed; this has a pale-brown color and elevated, sharply defined margins; the potato acquires a dark color. 279. BACILLUS FLUORESCENS NIVALIS (Sclimolck). Found in ice water and snow from Norwegian glaciers. (Probably iden- tical with No. 277.) Morphology. — Short bacilli, often united in chains. Biological Characters. — An aerobic liquefying, motile, chromogenic bacillus. Forms a bluish-green, fluorescent pigment. Spore formation not observed. Upon gelatin plates whitish, punctiform colonies are formed, which spread out upon the surface as round discs and cause liquefaction of the surrounding gelatin ; the non-liquefied gelatin in the vicinity acquires a bluish-green fluorescence. In gelatin stick cultures liquefaction occurs in funnel form, and the liquefied gelatin acquires a greenish fluorescence like that of Bacillus liquefaciens fluorescens. Upon the surface of agar a whitish layer is formed and the culture medium acquires a fluorescent color. Upon potato a brownish layer is developed. 280. BACILLUS LACTIS ERYTHROGENES (Hueppe). Synonym. — Bacillus of red milk. Found in red milk, and by Baginsky in the faeces of a child. Morphology. — Short bacilli with round ends, from 1 to 1.4^ long and from 0.3 to 0.5 /f thick; in bouillon cultures may grow out into short fila- ments. Biological Characters. — An aerobic, liquefying, non-motile, chromogenic bacillus. Produces a yellow pigment which is destroyed by acids and is developed either in the presence or absence of light; and a red pigment which is absorbed by the culture medium and is produced most abundantly in an alkaline or neutral medium in the absence of light. Does not form spores. The cultures give off an intense and disagreeable odor. Grows at the room temperature in the usual culture media — more rapidly at 28° to 35° C. \Jpongelatinplates small, spherical colonies are developed, Avhich are at first grayish- white and later yellow in color; after a time the sur- rounding gelatin is liquefied in saucer shape and acquires a pale-pink color. In gelatin stick cultures a rather thin, round layer is developed upon the surface, which is at first whitish and later yellow in color; the gelatin XOX-PATHOGENIC BACILLI. 637 around it acquires a pale-pink color, and after a time liquefaction occurs ; along- the line of puncture the development is scanty ; at the end of ten to twelve days a slightly clouded, pink liquid is seen at the upper part of the test tube, ia which well-defined yellow colonies are suspended, while the unliquefied gelatin below has a pink color. Upon the surface of agar a glistening, yellowish layer is slowly developed. Upon potato development is more rapid and an extended layer is formed, which is first grayish-white and later yellow in color; the potato acquires a dark color which later becomes yellowish -red ; at 37° C. , at the end of six to eight days, an intense golden-yellow color is developed. In bouillon development is rapid and yellowish cloudiness of the culture medium is seen. In milk the casein is slowly precipitated and later is peptonized, with a neutral or alkaline re- action of the medium ; a stratum of blood-red serum is seen above the pre- cipitated casein, and above this a yellowish-white layer of cream. 281. BACILLUS GLAUCUS (Maschek). Found in water. Morphology. — Slender bacilli of various lengths. Biological Characters. — An aerobic, liquefying, non-motile, chromogemc bacillus. Produces a gray pigment. Spore formation not observed. Grows rapidly in the usual culture media at the room temperature. Upon gelatin plates forms round, gray colonies wTith sharply defined outlines; on the fourth day the centre becomes intensely gi*ay, th'e margin brown and folded in a radial direction ; on the eighth day liquefaction has occurred and the colony sinks beneath the surface. In gelatin stick cultures development is rapid both upon the surface and along the line of puncture ; the entire gela- tin is quickly liquefied and a gray deposit is seen at the bottom of the tube. Upon the surface of agar a gray layer is quickly developed. Upon potato the growth is at first of a dirty- white; later a slimy, dark-gray layer is formed. 282. BACILLUS LIVIDUS (Plagge and Proskauer). i Found in the Berlin water supply. Morphology. — Slender bacilli of medium size. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile, chromogenic bacillus. Produces an intense blue-black pigment. Spore formation not observed. Grows slowly in the usual culture media at the room temperature. Upon gelatin plates the colonies at first resemble drops of ink ; the gelatin around them is slowly liquefied and a bluish- violet deposit is seen at the bottom of the liquefied gelatin. In gelatin stick cultures a colorless growth is seen along the line of puncture and a violet layer upon the surface ; liquefaction occurs very slowly. Upon the surface of agar a beautiful blue-black layer is developed. Upon potato an abundant layer of a violet color is formed along the line of inoculation. 283. BACILLUS INDICUS (Koch). Found in the contents of the intestine of a monkey, by Koch, while pur- suing his cholera investigations in India. Morphology. — A short and slender bacillus with round ends. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile, chromogenic bacillus. Spore formation not observed. Produces a brick-red pigment. Grows rapidly in the usual culture media — best in the incubating oven. Upon gelatin plates the deep colonies are white and punctiform ; under a low power they are seen to be granular, irregular in form, and of a greenish-brown color. The superficial colonies quickly cause liquefaction of the gelatin and form circular depressions with a well-defined outline and grayish contents, which under the microscope appear as dense, finely granular, grayish-yellow masses, the edges of which appear to be 54 038 NON-PATHOGENIC BACILLI. fringed with short fibres ; later the gelatin acquires a pale-red color, which gradually becomes more intense. In gelatin stick cultures liquefaction is rapid along the entire line of inoculation ; a wrinkled, red film forms upon the surface and grayish- white flocculi accumulate at the bottom of the lique- fied medium. Upon the surface of agar an abundant layer, covering the entire surface, is quickly developed in the incubator ; this usually acquires a brick -red color, but the margins of the layer, or even the entire growth, may remain colorless, especially in cultures kept in the incubating oven. Upon potato development occurs very rapidly along the line of inoculation; this soon acquires the characteristic color. Blood serum is liquefied by this bacillus. Large quantities (twenty cubic centimeti'es) of a pure culture in- jected into a vein or into the peritoneal cavity of a rabbit or guinea-pig cause the death of the animal in from three to twenty hours; at the autopsy an intense gastro-enteritis is, found. 284. BACILLUS PRODIGIOSUS. Synonyms.-' ^Micrococcus prodigiosus; Monas prodigiosa. This bacillus has long been known, having attracted attention because of the blood-red stains which it causes upon farinaceous substances, such as boiled potatoes, moist bread, etc. It was described by Ehrenberg under the name of Monas prodigiosa. At times, in certain parts of Europe, it has been exceptionally abundant, and the bloody-looking patches produced by its rapid development upon favorable media have been regarded with ap- prehension, by the superstitious. Morphology. — Short bacilli with rounded ends, which are sometimes so short as to be scarcely distinguishable from micrococci, but also occur as rod-shaped cells and short filaments ; frequently in pairs and occasionally in chains containing ten or more elements — especially in acid media. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, usually non-motile, chromogenic bacillus. Although usually described as non-motile, this bacillus is said under certain circumstances to be capable of spontaneous movements. According to Schottelius, these are best seen when it is cultivated in strongly diluted liquid media and under urifavoiv able conditions of growth. Forms a red pigment which is soluble in alcohol and ether, but not in water; this is only formed in presence of oxygen; it is changed to a pale-red color by the action of acids, and the deep- red color is restored by ammonia and other strong alkalies. The pigment is not seen in the interior of the bacterial cells, but the chromogenic substance formed by them develops the color outside of the cells, where it is seen in the form of granules. The formation of pigment is influenced not only by the presence of oxygen, which is essential to its production, but also by conditions re- lating to temperature, constitution of the culture medium, etc. By continu- ous cultivation in the incubating oven a non-chromogenic variety may be obtained, and the same result is obtained by continuous cultivation in acid bouillon. But under favorable conditions color production again returns after a few successive transplantations upon potato or nutrient agar, if the cul- tures are kept at the room temperature and freely exposed to the air. Spore formation has not been observed, but this bacillus retains its vitality for a long time in a desiccated condition. Cultures give off a strong odor of tri- methylamm; and in culture media containing sugar, fermentation occurs with production of alcohol and carbon dioxide. This bacillus grows rapidly in the ordinary culture media — best at the room temperature or a little above (25° C.). Upon gelatin plates small, white, punctiform colonies are developed below the surface, and upon the surface round, granular colonies which quickly cause liquefaction of the gelatin; saucer-like depressions are Eroduced, at the bottom of which the colony is seen as a whitish mass which iter acquires a deep-red color, first appearing at the centre. In gelatin stick cultures liquefaction quickly occurs along the entire line of inoculation and rapidly extends until the medium is completely liquefied ; pigment for- NON-PATHOGENIC BACILLI. 639 mation occurs at the surface only, but after a time the entire medium is colored red by the deposition of granular, colored masses from the surface growth. Upon the surface of agar an abundant purplish-red layer is formed, but the color is not absorbed by the culture medium. Upon potato very rapid and abundant development occurs at the room temperature, forming a thick, purplish-red layer which after some days has the color of undissolved fuchsiii and a metallic lustre. Blood serum is liquefied by this bacillus. In milk the development of Bacillus prodigiosus causes a precipi- tation of the casein and a deep-red color of the medium. When cul- tivated for some time in acid media the peptonizing (liquefying) power of the bacillus is greatly reduced, as well as its chromogenic power. It has been showji by Roger that animals which are not susceptible to the disease known as malignant oedema, become infected and die when inoculated with the malignant-oedema bacillus and at the same time with one or two cubic centimetres of a culture of Bacillus prodigiosus. But this bacillus alone has no decided pathogenic power. An interesting discovery made by Pawlowsky is the fact that when rabbits are inoculated simultaneously with a virulent culture of the anthrax bacillus and with a culture of Bacillus prodigiosus they recover from the inoculation, the chemical products of one bacillus having apparently the power to neutralize the toxic substances to which the other owes its pathogenic potency. 285. BACILLUS MESENTERICUS RUBER. Synonym. — Rothen Kartoffelbacillus (G-lobig). Found upon potatoes. Morphology. — Slender bacilli with round ends, united in pairs, in chains of four, or in long filaments. Biological Characters. — An aerobic, liquefying, motile, chromogenic bacillus. Produces a reddish-yellow or pink pigment. Forms oval spores which have a great resistance against high temperatures and germicidal agents. Grows rapidly, especially at a temperature of 45° C. ; also at the room temperature in the usual culture media. Upon gelatin plates, at 15° to 20° C., at the end of two days deep colonies are formed wnich are spheri- cal and of a yellow color; when these come to the surface they spread out as a fine network, around the margins of which projecting points are seen ; liquefaction commences on the fom-th day and this network vanishes, leav- ing a grayish-brown, friable mass at the bottom of the liquefied medium. In gelatin stick cultures, at the end of three or four days, a cloudy white growth is developed along the line of puncture, and liquefaction occurs in funnel shape near the surface ; this soon exends to the walls of the tube and downward, and a thin film is formed on the surface. Upon potato, at 15° C., at the end of three days the surface is covered by a thin, viscid, slimy, yellowish, finely wrinkled layer; at 37° C. the entire surface is covered in twenty-four hours with a reddish-yellow or pink layer; in forty- eight hours this extends over the lower surface of the potato also, except where it is in contact with the receptacle in which it is placed. 286. BACILLUS PYOCYANUS ft (Ernst). Found in pus from bandages colored green. Morphology. — Slender bacilli from 2 to 4 u long — occasionally 5 to 6 // — and from 0.5 to 0.75 n broad; sometimes united in pairs, or chains of three elements. Biological Characters. — An aerobic, liquefying, actively motile, chromo- genic bacillus. Produces a yellowish-green pigment; when old cultures 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 for- mation has not been demonstrated. Grows in the usual culture media at the 640 NON-PATHOGENIC BACILLI. room temperature — more rapidly at 35° C. Upon gelatin plates colonies are formed resembling those of the well-known Bacillus pyocyanus, but lique- faction 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 pro- gresses more rapidly than is the case with Bacillus pyocyanus under the same circumstances ; on the fifth day a bluish-green color is developed ; 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, green- ish-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 de- veloped; the growth on potato has a more or less wrinkled appearance; 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 intense 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 pyocyanus. In milk a green color is developed at the surface, the casein is precipitated and sub- sequently peptonized. 287. BACILLUS MYCOIDES ROSEUS (S(^holl). Found in the soil. Morphology . — Resembles the anthrax bacillus. Biological Characters. — An aerobic, liquefying, chromogenic bacillus. Produces a red pigment when cultivated in the absence of light. Spore formation not reported. Grows rapidly at the room temperature. Upon gelatin plates forms colonies of interlaced filaments which cause liquefaction of the surrounding gelatin. In gelatin stick cultures liquefaction rapidly occurs; a red layer is formed upon the surface, and a sediment of the same color is seen at the bottom of the liquefied medium, but the gelatin itself is not colored. Upon the surface of agar, in the dark, a pink layer is de- veloped, while in the light it is white. 288. BACILLUS ROSACEUM METALLOIDES (Dowdeswell). Morphology. — Bacilli from 0.6 to 0.8 n broad and about twice as long. Biological Characters. — An aerobic, liquefying, motile (usually not mo- tile), chromogenic bacillus. Forms a magenta-red pigment which has a metallic lustre. Spore formation not observed. Grows best at 15° C. ; no development at 35C C. ; is destroyed in five minutes by a temperature of 55° C. Upon gelatin plates, at 15 C , superficial colonies are developed, which in the course of a few days are elevated, colorless discs about two milli- metres in diameter ; under a low power the centre appears dark, the mar- gin transparent and granular; later the colony acquires a red color and liquefaction of the surrounding gelatin occurs. In gelatin stick cultures a red layer is developed upon the surface, and later a broad funnel of lique- fied gelatin is slowly developed. Upon the surface of agar a pale-red layer is formed. Upon potato, at 15° C., a thick layer quickly covers the entire surface ; this has a beautiful red color, especially near the margins. 289. BACILLUS viscosus (Frankland). Resembles very closely, and is perhaps identical with, Bacillus fluores- cens liquefaciens. NON-PATHOGENIC BACILLI. 641 Found in uu filtered river water. Morphology. — Bacilli with round ends, 1.5 to 2 /< long1 and about three or four times as long- as broad ; usually united in pairs. Biological Characters. — An aerobic, liquefying, motile, chromogenic ba- cillus. Produces a fluorescent green pigment. Spore formation not ob- served. Grows rapidly at the room temperature in the usual culture media. Upon gelatin plates the deep colonies appear under a low power as finely granular discs with a smooth contour ; when they come to the surface the margin is fringed and hair-like offshoots extend into the gelatin ; at the same time liquefaction occurs around the colonies and rapidly extends ; each liquefying1 colony is surrounded by a fluorescent green zone, and the lique- fied gelatin has the same color. In gelatin stick cultures liquefaction in funnel shape is already seen on the second day, and the liquefied gelatin is filled with whitish flocculi, while a slight green, fluorescence is seen near the surface ; liquefaction progresses rapidly, and a viscid layer of a greenish- white color forms upon the surface, while the liquefied gelatin below is more or less clouded and has a fluorescent green color ; an abundant flocculent deposit is seen at the bottom of the tube. Upon the surface of agar a smooth, glistening, greenish- white layer is formed along the line of inocula- tion and the agar quickly acquires a green color. In bouillon, at the end of two days, the liquid is clouded, and later a thin, greenish-white layer forms upon the surface, while the bouillon acquires a green color. Upon potato a chocolate-colored, moist-shining layer quickly extends over the entire surface. 290. BACILLUS VIOLACEUS. First found in the water of the Spree at Berlin, and since by Frankland in the water of the Thames and of the Lea. Morphology. — Bacilli with round ends, about 1.7 /t longand 0.8 /* broad; usually in pairs; may grow out into long filaments. Biological Characters. — An aerobic and facultative anaerobic, liqiiefy- ing, motile, chromogenic bacillus. Produces a dark -violet pigment. Forms oval spores, which are located in the centre of the rods. Upon gelatin plates, at the end of two days, the colonies are seen to be irregular in out- line and granular; on the fourth day a funnel of liquefied gelatin is formed by each colony, and under a low power an opaque mass surrounded by con- voluted, filamentous offshoots is seen at the bottom of this ; later the funnel of liquefied gelatin increases in dimensions and the colony acquires a deep- violet color. In gelatin stick cultures liquefaction occurs rapidly along the line of inoculation as a funnel-shaped sac ; the liquefied gelatin is clouded, and a violet-colored deposit is seen at the bottom of the tube. Upon the sur- face of agar a smooth, shining layer of a deep-violet color is quickly de- veloped. Upon potato growth is limited to the line of inoculation and a dark- violet stripe is slowly developed. Blood serum is liquefied by this1 bacillus. 291. BACILLUS SULFUREUM (Holschewnikoff). Morphology.— Bacilli with round ends, from 1.6 to 2 A p long and 0.5 n broad. Biological Characters.— An aerobic and facultative anaerobic, liquefy- ing, motile, chromogenic'bacillus. Forms, in the absence of oxygen, a red- dish-brown or red pigment. Produces sulphuretted hydrogen in sterilized urine and certain other media when cultivated in the absence of oxygen. Spore formation not observed. Grows in the usual culture media at the room tem- perature— also- in the incubating oven. Upon gelatin plates small, puncti- form colonies are developed within forty -eight hours, which, when they reach the surface, cause a funnel-shaped liquefaction ; as the liquefaction progresses very slowly the liquefied gelatin is dried and the funnel- shaped cavities are filled with air. lu gelatin stick cultures small colonies are de- veloped along the line of puncture, and liquefaction in funnel shape occurs 042 NON-PATHOGENIC BACILLI. very siowiy ; in contact with the air the colonies are white : in the absence of oxygen liquefaction does not occur and the colonies have a reddish- brown or red color. Upon the surface of agar, in the incubating oven, a slimy, gray layer is quickly developed. L pon potato no growth occurs in the presence of oxygen ; when it is excluded a reddish-brown layer is de- veloped. In milk, at the end of ten days, solution of the casein commences without previous coagulation. 292. BACILLUS RUBIOUS (Eisenberg). Found in water. Morphology. — Bacilli of medium size, with blunt ends, often united in long filaments. Biological Characters. — An aerobic, liquefying, motile, chromogenic. bacillus. Forms a shining, brownish-red pigment. Spore formation not observed. Grows very slowly in the usual culture media at the room tem- perature — not in the incubator at 37° C. Upon gelatin plates form spheri- cal, finely granular colonies, which are of a reddish color at the centre and around which the gelatin is slowly liquefied. In gelatin stick cultures liquefaction occurs slowly and a brownish-red pigment is formed. Upon the surface of agar a brownish-red layer is developed, which quickly ex- tends over the surface. A similar development occurs iipon potato — not limited to line of inoculation. Blood serum is liquefied by this bacillus. ' 9 * FIG. 213. FIG. 214. FIG. 213.— Bacterium terrno of Vlgnal, from a bouillon culture. X 1,300. (Vignal.) FIG. 214.— The same from a culture fifteen days old. X 1500. (Vignal.) 293. BACTERIUM TERMO OF VIGNAL. Found in the salivary secretions of healthy persons by Vignal, and de- scribed under the name of Bacterium termo, which was formerly given to various motile bacilli encountered in putrefying infusions, many of which have been differentiated by modern methods and are described under differ- ent names. Morphology. — Bacilli from 1.5 to 2 n long, constricted in the middle, and 0.5 to 0.7 /abroad; never u*nited in chains or filaments; possess terminal flagella. Biological Characters. — An aerobic, liquefying, motile, chromogenic bacillus. Forms in gelatin cultures a fluorescent yellowish-gray pigment. Spore formation not determined. Grows at the room temperature in the usual culture media — better at a higher temperature. Gelatin cultures give off a putrefactive odor. Upon gelatin plates, at the end of twenty -four hours, small, white colonies are seen ; at the end of forty-eight hours these have a diameter of two to five millimetres; the centre of the colony is white and opaque, and it is surrounded by a zone of liquefied gelatin which is. NON-PATHOGEXIC BACILLI. 643 clouded and more or less granular in appearance ; the area of liquefaction increases and the opaque central colony disappears, while the margins of the liquefied gelatin are clouded and whitish. In gelatin stick cultures, at the end of two days, liquefaction has occurred all along the line of puncture and a broad funnel is formed above, while below a mass of white flocculi fills the narrow tube; at the end of three days the gelatin is completely liquefied, it is opalescent, fluorescent, and greenish; on the fifth or sixth day it has a yellowish-green color and a strong odor of putrefaction. Upon the surface of agar, at a temperature of 36° C., it forms circular, grayish- white, almost transparent colonies ; these rapidly coalesce to form a layer of uniform thickness, which is easily broken up. In bouillon the liquid is at first clouded throughout, and at the end of a week has a green color, while the bacilli are seen at the bottom as a pulverulent white deposit. Blood serum is slowly liquefied and gives off a strong putrefactive odor. Upon potato, at the end of forty-eight hours in the incubating oven, a glairy,, grayish layer is formed the size of a five-franc piece ; later this acquires a pale-yellow color. 294. BACILLUS BUCCALIS MINUTUS. Synonym. — Bacillus g, Vignal. Found by Vignal in the salivary secretions of healthy persons. Morphology. — A very short bacillus, with round ends, almost as broad as long; in cultures upon agar the length is from 0.5 to 1 /< — usually about 0.7 n ; in neutral bouillon it is from 1 to 1.7 /« long; in old cultures involu- tion forms are common ; in stained preparations the two ends are more deeply stained than the central portion. Biological Characters. — An aerobic, liquefying, chromogenic bacillus. Produces a yellow pigment. Spore formation not observed. Motility not mentioned. Grows slowly at the room temperature. Upon gelatin plates, at the end of forty-eight hours, the colonies are round, with refractive con- tour and of a mastic-yellow color; they are but slightly elevated and the gelatin commences to liquefy around them. In gelatin stick cultures, at the end of forty-eight hours, a yellowish-white growth is seen along the line of puncture, and upon the surface a layer having the same color and sev- eral millimetres in diameter has developed ; by the fourth day the surface growth has increased to twice the size and is yellow at the centre, while the periphery is white; the growth along the line of puncture is abundant and consists of small, closely crowded colonies; below the surface growth a cup- shaped cavity filled with clouded liquefied gelatin is seen ; by the sixth day a small funnel of liquefaction has formed, the liquefied gelatin is clear and contains some white flocculi in suspension ; by the twelfth day the gelatin in the tube is completely liquefied, an abundant yellow deposit is seen at the bottom and the liquefied gelatin has the same color. Upon the surface of agar golden-yellow plaques are developed, which are easily removed with the platinum needle. In bouillon a thin, iridescent pellicle is formed upon the surface and the fluid below is clouded, while an abundant yellow deposit accumulates at the bottom. Does not grow well in acid bouillon. Upon potato, at the end of forty-eight hours, a thin and extended layer is formed of a yellow color, which later has a brownish tint. 295. BACILLUS OF CANESTRINI. Found in larvae and bees from infected hives in Italy (1891). Morphology. — Bacilli with rounded ends, from 4 to 6 ^ long and about 2 n broad; the isolated elements ai*e somewhat longer than those in chains; solitary, in pairs, or in chains whicli may contain numerous segments. Biological Characters. — An aerobic, liquefying, motile, chromogenic bacillus. Forms a pink pigment. The movements are slow and oscillating. Forms oval spores 3 /< long and 1.5 /i broad. Grows at the room tempera- 044: XOX-PATHOGENIC BACILLI. ture— better at 37° C. Stains with the aniline colors and by Gram's method. In gelatin stick cultures causes liquefaction of the gelatin; after some days the upper portion of the liquefied gelatin has a pink color, while at the bot- tom an abundant white sediment collects. Upon the surface of agar forms a white layer which contains numerous spores. Liquefies blood serum ; in this medium the bacilli are surrounded by a capsule, and are frequently seen in long chains containing fifteen to twenty elements, all enclosed in a sin- gle capsular envelope. Upon potato development is rapid at 37° C. ; at the end of twenty -four hours a wine-colored layer is formed. Not pathogenic for mice or guinea-pigs. Experiments made with pure cultures show that it is pathogenic for bees and their Iarva3, and that it is the cause of an infec- tious malady which is destructive to these insects in certain localities (in. Italy). C. Nou-chromogenic, Non-liquefying Bacilli. 296. BACILLUS UBIQUITUS (Jordan). Found in sewage at Lawrence, Mass. ; also in water and in the air — "ap- parentlv abundant everywhere " ( ordan). Morphology. — Bacilli from 1.1 to 2 /* long and about 1 ft broad— reseinble micrococci ; quite variable in f orm ; in bouillon short filaments are some- times formed. Biological Characters. — Aja.aSrobio andfacultativeanaerobic, non-lique- fying, non-motile bacillus. Spore formation not observed. Grows at the room temperature in the usual culture media— also at 37° C. Upon gelatin plates forms small, sphe- rical or oval colonies, which have a yellowish tins-e; at the end of two days the superficial colonies are prominent, white, and glistening, resembling a drop of milk ; they gradually in- crease in diameter, become somewhat irregular in outline, and acquire a dull brownish tint. Under a low power the young colonies are seen to be finely granular and to have a smooth con- tour. In gelatin stick cultures development occurs upon the surface and along the line of puncture, producing a '' nail-shaped " growth at the end of a week; the color is at first a lustrous porcelain- white, which later changes FIG. 215. - Bacillus ubiquitus. to a j^} brownish-gray ; grows well in slightly x 1,000. From a photomicrograph. acid gelatin _ Upon the surface of agar a whit- ish-gray layer is developed which has a slightly metallic lustre. Upon potato a shining, white growth of limited extent. In milk coagulation occur-s quickly at 37° C., and the milk acquires a strongly acid reaction. Reduces nitrates vigorously. " This species apparently resembles quite closely the Bacillus candicans de- scribed by the Franklands" (Zeit.fiir Hyg., Bd. vi., page 397). " It differs from that, however, among other respects, in its capacity for reducing ni- trates and in its mode of growth upon agar and potato " (Jordan). 297. BACILLUS CAXDICANS (Fraiiklaiid). Found in the soil. Morphology. — Short, thick bacilli, resembling micrococci ; often form short filaments. Biological Characters. — An aerobic, non-liquefying, non-motile bacillus. Does not form spores. Grows slowly at the room temperature in the usual culture media. Upon gelatin plates the superficial colonies resemble drops of milk ; the deep colonies under a low power are seen to be spherical, slightly NON-PATHOGENIC BACILLI. ' 645 granular at the margins, and have a smooth contour. In gelatin stick cultures the superficial growth is like a drop of milk in appearance; very scanty growth along the line of puncture at first, later a row of spherical colonies is seen ; the surface growth in old cultures has a slightly reddish tint. Upon the surface of agar a thin, transparent, grayish- white layer with smooth margins. Upon potato an abundant layer is developed. 298. BACILLUS ALBUS (Eisenberg). Found in water. Morphology. — Short bacilli with blunt ends, often united in short chains. Biological Characters. — An aerobic, non-liquefying, motile bacillus. Spore formation not observed. Grows slowly at the room temperature — not in the incubator at 37° C. Upon gelatin plates forms round, white colonies the size of a pin's head. In gelatin stick cultures grows slowly, forming a white line along the puncture and a small, button-like, white mass at the point of entrance. Upon the surface of agar forms a milk- white layer. Upon potato a dirty yellowish-white growth, limited to the line of inoculation. 299. BACILLUS ACIDI LACTICI (Hueppe). Found in sour milk. Morphology. — Bacilli from 1 to 1.7 V long and from 0.3 to 0.4/* broad; usually in pairs, sometimes in chains of four. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, non-motile bacillus. Forms spherical spores, which are located at the ends of the rods. Grows slowly at the room temperature in the usual culture media. Upon gelatin plates forms small, white, punctiform colonies, which later develop into shining, porcelain-colored discs with a transparent margin; under a low power they have a yellowish tint in the centre and thin, irregular margins. In gelatin stick cultures small colonies are de- veloped along the line of puncture, and later a dry, glistening, soft, grayish- white, and tolerably thick layer is developed upon the surface. Upon potato an extended, yellowish-brown layer is formed. In. milk lactic acid is pro- duced, the casein is precipitated, and carbon dioxide is given off. 300. BACILLUS LIMBATUS ACIDI LACTICI (Marpmann). Found in fresh cow's milk. Morphology. — Short, thick bacilli, usually in pairs; every rod is sur- rounded by a capsule which is not stained by the aniline colors. Biological Characters. — An aerobic, non-liquefying, non-motile bacil- lus. Does riot form spores. Grows slowly in the usual culture media at the room temperature — also in the incubator. Upon gelatin plates, at the end of twenty-four hours, forms milk-white, punctiform colonies. In gela- tin stick cultures scanty development occurs along the line of puncture, and upon the surface a flat, irregular layer of a white, pus-like color is formed. In milk, at the end of twelve hours, a slightly reddish color is seen; at the end of twenty -four hours coagulation of the casein and a strongly acid reaction — lactic acid ; does not produce gas. 301. BACILLUS LACTIS PITUITOSI. Synonym. — Bacillus der schleimigen Milch (Loffler). Found in milk. Morphology. — Tolerably thick, slightly curved bacilli, which very quick- ly break up into small segments resembling micrococci. Biological Characters. — An aerobic, non-liquefying bacillus. Grows 55 646 NON-PATHOGENIC BACILLI. rather rapidly at the room temperature. Spore formation not determined. Upon gelatin plates forms white colonies with well-defined contour, from one-quarter to one-half millimetre in diameter; by transmitted light these are grayish-brown in color and present a slightly radial striped appearance. Upon the surface of agar a dirty- white layer is developed. \Jponpotato a grayish-white, tolerably dry layer. In milk a slightly acid reaction is pro- uced, and a very viscid substance having a peculiar odor is formed, espe- cially in the lower portion of the liquid ; this can be drawn out into long threads. 302. BACILLUS AEROGEXES (Miller). Found in the alimentary tract of healthy persons. Morphology. — Small bacilli of various lengths. Biological Characters. — An aerobic, non-liquefying, motile bacillus. Spore formation not observed. Grows at the room temperature. Upon gelatin plates forms spherical, homogeneous, transparent, white or slightly yellowish colonies ; older colonies sometimes appear to be formed of con- centric rings. In gelatin stick cultures development occurs along the line of puncture and the growth has a yellowish color; upon the surface a thin, pearl-gray layer with dentate margins is formed ; in old cultures the line of inoculation acquires a dark-brown color and is surrounded by a pale-brown halo. Upon potato a dry layer of a dirty bluish-yellow color, and with irregular outlines, is slowly developed. 303. BACTERIUM AEROGEXES (Miller). Found in the alimentary tract of healthy individuals. Morphology. — Short rods, solitary or in pairs. Biological Characters.^ An aerobic and facultative anaerobic, non- liquefying, motile bacillus. Spore formation not observed. Grows at the room temperature in the usual culture media. Upon gelatin plates forms sharply denned, yellowish colonies, which are marked by dark lines radi- ating from the centre toward the margin. In gelatin stick cultures devel- opment occurs along the entire line of puncture and the growth has a brown- ish-yellow color ; upon the sui'face a soft, flat, grayish- white mass is formed about the point of puncture. Upon the surface of agar a soft, grayish- white layer. Upon potato a soft layer with irregular margins and of a slightly yellowish-white color. 304. HELICOBACTERIUM AEROGEXES (Miller). Found in the alimentary tract of healthy persons. Morphology. — Slender bacilli, solitary or in chains ; grow out into long, undulating or spiral threads. Biological Characters. — An aerobic, non-liquefying, motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates forms transparent, white or slightly yellowish colonies of various forms — spherical, oval, snail-shaped, spindle- shaped, spiral, etc. In gelatin stick cultures grows upon the surface as a thin, bluish, scarcely visible, dry layer, which covers the entire surface at the end of forty-eight hours; along the line of puncture a uniform, light- yellowish growth. Upon agar the growth is not characteristic. Upon po- tato a layer is formed which has a dry surface, indented margins, and a yellowish-brown color. 305. BACILLUS AQUATILIS SULCATUS xo. I. ( Weichselbaum). Found in the Vienna water supply. Morphology. — Bacilli resembling" the bacillus of typhoid fe\erinform and dimensions. NON-PATHOGENIC BACILLI. 647 Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, motile bacillus. Spore formation not observed. Gi*ows rapidly at the room temperature — not so well in the incubating oven. Growth occurs at a temperature as low as 5° to 7° C. Upon gelatin plates, at the end of two days, the superficial colonies are seen as flat discs with a thicker and whitish centre and very thin, bluish, notched margins ; under a low power the surface is seen to becovered with fine lines or furrows which cross each other at various angles; the color is white, with a yellowish tint at the centre ; later very numerous lines are seen crossing each other in all direc- tions at the centre, while the periphery is still white and is marked by more delicate lines. The deep colonies are round and yellowish. In gelatin stick cultures a flat, white layer with notched edges is seen at the end of twenty- four hours ; this becomes thicker and is of less diameter than the surface growth of the typhoid bacillus under similar conditions. Upon the surface of agar, at 37. 5° C., a tolerably thick, white layer is developed; this has an odor like that of milk. Upon potato, at 37.5° C., the growth is invisible, the line of inoculation having only a moist appearance ; at the room tem- perature this is also the case at first, but later a very thin, moist-shining, of ten cream-colored layer with raised edges is seen, and the potato around this acquires a bluish-gray color, which again fades out. 306. BACILLUS AQUATILIS SULCATUS NO. ii. (Weichselbaum). Found in the Vienna water supply. Morphology. — Short bacilli with round ends, of the form and dimensions of the shorter typhoid bacilli. Biological Characters. — An aerobic an d facultative anaerobic, non-lique- fying, motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature — not so well in the incubating oven. Grows at a lower temperature than the typhoid bacillus — 5° to 7° C. Upon gelatin plates, at the end of two days, the superficial colonies are similar to those of the typhoid bacillus and of the preceding species, but somewhat thicker and not visibly notched ; under the microscope they are seen to be in- distinctly notched and marked by lines, although not so distinctly as are the colonies of Bacillus aquatilis sulcatus No. I. ; the centre of the disc-shaped colonies is yellowish, the periphery white; after three days they become thicker and* the notching of the margins and lines upon the surface are no longer seen, while the entire colony, with the exception of the outer margin, has a yellowish color. In gelatin stick cultures a whitish, rather thick layer of limited dimensions is formed upon the surface. Upon the surface of agar, in the incubator, a grayish- white layer is developed in twenty -four hours. Upon potato, at the room temperatui'e, a bluish-gray color is first seen along the line of inoculation, and a yellowish- gray or yellowish-brown layer is subsequently developed ; this may become tolerably abundant, while the original color disappears ; potato cultures give off a slight uriiious odor. 307. BACILLUS AQUATILIS SULCATUS NO. in. (Weichselbaum). Found in the Vienna water supply. Morphology. — Very short bacilli, frequently resembling micrococci. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, motile bacillus. Spore formation not observed. Grows at the room temperature and at 5° to 73 C. — not so well in the incubating oven. Old cultures give off a disagreeable odor. Upon gelatin plates, at the end of two or three days, the superficial colonies are disc-shaped, with a thicker, white centre and a very thin, bluish periphery; the margin is notched; under the microscope the surface is seen to be marked with lines; later the colonies increase in thickness and diameter and lose the bluish color ; the system of fine lines also disappears, and the surface is covered with numerous short lines and furrows; the yellow color extends from the centre nearer to 648 NON-PATHOGENIC BACILLI. the periphery. In gelatin stick cultures, at the end of twenty-four hours, a very thin, white layer with notched edges is developed ; this extends rapidly to the margins of the tube. Upon the surface of agar an abundant grayish- white layer is quickly developed in the culture oven ; this has an odor re- sembling that of milk. Upon potato, at the room temperature, a discolora- tion of the line of inoculation is seen at the end of twenty -four hours ; later an abundant pale-yellow layer with raised margins is developed which has an odor of herring brine; at the end of nine days the potato around the growth has a bluish-green color. 308. BACILLUS AQUATILIS SULCATUS NO. iv. (Weichselbaum). Found in the Vienna water supply. Morphology. — Bacilli of various lengths; often grow out into filaments. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, tnotile (the short rods only) bacillus. Spore formation not demonstrated. Grows slowly at the room temperature — still more so in the incubating oven. Upon gelatin plates the colonies are first visible on the fourth day; the superficial colonies are thin, bluish discs with notched mar- gins and a somewhat thicker, whitish centre; under a low power the surface is seen to be covered with fine lines, and the larger colonies have a yellowish color in the centre ; later they increase in thickness and diameter and acquire a yellow color throughout, while the system of lines is replaced by more numerous and shorter lines and furrows. In gelatin stick cultures develop- ment is very slow, but at the end of several days a grayish- white layer with notched margins is developed, which gradually extends to the walls of the test tube. Upon the surface of agar, at 37° C., the growth is very scanty at the end of six days ; at the room temperature a grayish- white layer of mode- rate thickness is formed within two days. Upon potato no growth occurs either in the incubator or at the room temperature. 309. BACILLUS AQUATILIS SULCATUS NO. V. (Weichselbaum). Found in the Vienna water supply. Morphology. — Bacilli with round or pointed ends, of various lengths; somewhat thicker than the typhoid bacillus. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, motile bacillus. Spore formation not observed. Grows at the room temperature in the usual culture media — not in the incubating oven. Upon gelatin plates forms colonies resembling those of Bacillus aquatilis sulcatus No. I. , which become visible in two or three days. In gelatin stick cultures a layer of moderate thickness is formed, which gradually extends over the surface ; this is grayish- white at first and later has a yellow color like that of the yolk of an egg. Upon the surface of agar no growth occurs in the incubator, but at the room temperature an abundant, viscid, yellow- ish layer is developed. Upon potato, at the room temperature, a bluish-yel- low layer is formed and the potato around it acquires a dark-gray color, which disappears later, while the vegetation after a time has a honey -yellow color. 310. BACILLUS MULTIPEDICULUS (Flugge). Found in the air and in water. Morphology. — Long: slender bacilli. Biological Characters. — An aerobic, non-liquefying, non-motile bacil- lus. Spore formation not observed. Grows at the room temperature in the usual culture media. Upon gelatin plates forms spherical or oval opaque colonies, which under a low power are seen to give off at certain points of the periphery broad, segmented outgrowths consisting of round zoogloea NON-PATHOGENIC BACILLI. 649 masses; at the end of two or three days the oval, white, superficial colonies are seen with the naked eve to be surrounded with these outgrowths, whicli resemble the feet and antennae of certain insects. In gelatin stick cultures a white layer is developed upon the surface which gives off short, isolated outgrowths. Upon potato a smooth, dirty-yellow layer of limited extent is developed. 311. BACILLUS CYSTIFORMIS (Clado). Found in the urine of a patient with cystitis. Morphology. — Very short and slender bacilli. Biological Characters. — An aerobic, non-liquefying, motile bacillus. Grows slowly at the room temperature. Upon gelatin plates forms trans- parent, yellowish colonies, first round and later oval in form; from the fourth to the seventh day a granular elevation appears at the centre, around which a finely granular, yellowish zone is seen, and outside of this a broad, transparent zone with double contour. In gelatin stick cultures a scanty development occurs along the line of puncture, and on the surface a whitish layer is developed. Upon agar a yellowish- white layer. 312. BACILLUS HEPATICUS FORTUITUS (Sternberg). Obtained, by inoculation in a guinea-pig, from the liver of a yellow-fever cadaver. Morphology. — Resembles Bacillus coli communis in its morphology, but differs from this bacillus in being strictly aerobic. Biological Characters. — An aerobic, non-liquefying, non-motile bacillus. Does not form spores. Grows at the room temperature in the usual culture media. Upon gelatin plates the deep colonies are spherical, homogeneous or finely granular, and light-brown in color; at the end of four days they are more or less lobate. The superficial colonies are sbaped like a mamma, with striatious radiating from the centre, and are of a dark -brown color under the microscope. In gelatin stick cultures no growth occurs along the line of puncture, except to a slight extent near tbe surface; on the surface a white, button-like mass is formed about the point of puncture. Upon the surface of glycerin-agar the development is quite rapid at 35° C., the entire surface being nearly covered with a soft, milk-white growth within twenty- four hours. Upon potato, at the end of forty-eight hours, a rather dry and thick, cream-white growth forms along the line of inoculation ; the potato has a bluish discoloration, which subsequently disappears ; at the end of two weeks a rather thin, semi-fluid, light-brown layer covers the entire surface. Not pathogenic for rabbits — single experiment. 313. BACILLUS INTESTIXUS MOTILIS (Sternberg). Obtained from the contents of the intestine of yellow-fever cadavers. ^Morphology. — Resembles Bacillus coli communis in its morphology, but differs from this bacillus in being very actively motile, in its colonies upon gelatin plates, etc. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, actively motile bacillus. 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 deep colonies are spherical, homogene- ous, and of a pale-straw color; the superficial colonies resemble little drops of water and are of a pale-brown color by transmitted light. In gelatin stick cultures pale straw-colored colonies are developed all along the line of puncture, and a rather thin, translucent, whitish layer forms upon the surface; sometimes a nebulous outgrowth occurs from the line of puncture, and tufted outlying colonies are formed throughout the gelatin; at other times, in old cultures, a few feathery tufts sprout out from the line of punc- 650 . NON-PATHOGENIC BACILLI. ture. Upon potato the growth is rather thin and of a pale-yellow color, not extending far from the line of inoculation. Does not form gas in a sac- charine solution — agua coco. 314. BACILLUS CAVI^E FORTUITUS (Sternberg). Obtained, by inoculation in a guinea-pig, from the liver of a yellow- fever cadaver, preserved for forty-eight hours in an antiseptic wrapping. Morphology. — Bacilli with round ends, from 1 to 4/« long and 0.5 to 0.8 // broad; often in pairs. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, actively motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates, at the end of three days, the colonies are small, spherical, and under the microscope light-brown in color ; later opaque, or sometimes with an opaque centre surrounded by a transparent zone. In gelatin stick cultures there is. a scanty growth about the point of puncture; growth occurs all along the line of puncture in the form of spherical, translucent, straw colored colo- nies, which have a pearly lustre by reflected light. Upon potato, at the end of a week, development has occurred in the form of small, dirty-yellow masses. Does not form gas in a saccharine liquid — agua coco. 315. BACILLUS COLI siMiLis (Sternberg). Obtained from a piece of liver — of man — kept in an antiseptic wrapping for forty-eight hours. Morphology. — A bacillus with round ends, from 1 to 3 >" long and from. 0.4 to 0.5 p thick; solitary or in pairs. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, non-motile bacillus. Spore formation not observed. Grows at the room temperature in the usual culture media — better at 37° C. Upon gelatin plates, at the end of two days, the deep colonies are spherical and pale-brown in color; later they become opaque. The superficial colonies are at first translucent, homogeneous, drop-like elevations ; later .they are quite thin and have a pale-brown color. In gelatin stick cultures a translucent growth with irregular margins is developed upon the surface, and a rather scanty line of growth is seen along the track of the inoculating needle. On potato, at 22° C., a thick, dirty -white or pale-brown layer is developed along the impfstrich. Not pathogenic for rabbits or guinea-pigs. 316. BACILLUS FILIFORMIS HAVANIENSIS (Sternberg). Obtained in anaerobic cultures from the liver of a yellow-fever cadaver. Morphology. — Long and slender bacilli, about 0.3>" in diameter, and forming long, homogeneous filaments in aerobic cultures, while the bacilli are shorter and thicker in anaerobic cultures in glycerin-agar. Biological Characters. — An anaerobic and facultative aerobic, non- liquefying, non-motile bacillus. Does not form spores. In gelatin stick cultures a scanty growth occurs along the line of puncture ; no growth on the surface. In anaerobic, glycerin-agar roll tubes colonies are developed which are spherical or irregular in outline, of a pale-brown or straw color by trans- mitted light, white and opaque by reflected light ; the superficial colonies are thin and translucent, and have a bluish lustre by reflected light ; later they appear as opaque, cream-like masses with irregular contour. In nutrient agar a scanty, milk-white growth occurs upon the surface and an opaque, branching growth along the line of puncture. No growth upon potato. Grows in neutral bouillon, causing a slight opalescence, and later a scanty white deposit at the bottom of the tuoe. Not pathogenic for rabbits or guinea-pigs. NON-PATHOGENIC BACILLI. 651 317. BACILLUS MARTINEZ (Sternberg). Obtained from the liver of a yellow-fever cadaver, kept for forty-eight hours in an antiseptic wrapping. Morphology. — A short, oval bacillus from 1 to 1.2/f long and from 0.5 to 0.8 n broad. 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 the deep colo- nies are spherical and translucent ; superficial colonies shaped like a mam- ma, with a central nipple-like projection, the surface covered with mosaic markings. In gelatin stick cultures a thin, translucent, scanty growth upon the surface, and large, spherical, translucent colonies along the line of puncture. In glycerin-agar stick cultures growth to the bottom of the line of puncture, and scanty development on the surface. 318. BACILLUS EPIDERMIDIS (Bizzozero). Synonym. — Leptothrix epidermidis. Found attached to scales of epidermis from between the toes. Morphology. — Bacilli from 2.8 to 3 // long and 0.3 /* broad. Biological Characters. — An aerobic, non liquefying bacillus. Forms long, oval spores— at 25° C. in three days. Grows best at a temperature of 15° to 20° C. Very scanty growth in gelatin. Grows upon the surface of agar. Upon potato, at 15° to 20° C., the development at first is in the form of vis- cid, transparent, drop-like colonies, which gradually coalesce and form a rather thick layer. 319. BACILLUS NODOSUS PARVus (Lustgarten). Found in the healthy urethra of man. Morphology. — Bacilli from 1.2 to 2.4 n long and 0.4 /* broad; one ex- tremity often presents an irregular club shape ; usually united in pairs, in which the elements lie parallel or are united at an acute angle. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, non-motile bacillus. Grows best in the incubating oven. Spore formation not observed. Very slow and scanty growth in nutrient gelatin. Upon the surface of agar, at 37° C., at the end of twenty-four hours a white line of growth is seen along the line of inoculation; at the end of two to three days this has a breadth of five to six millimetres, and the central por- tion of the layer is white, chalky in appearance, porous, and lustreless; around this is a smooth, flat, glistening, grayish- white marginal zone one to two millimetres broad. In agar stick cultures, at the end of five to eight days, growth is seen along the line of puncture as a white stripe made up of confluent spherical colonies, while at the point of puncture a small, stearin- like drop is seen. 320. BACILLUS HYACINTHI SEPTICUS (Heinz). Found in diseased hyacinths. Morphology. —Bacilli with round ends, 4 to 6 n long and about 1 n broad ; always solitary. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, motile bacillus. Spore formation not observed. Grows at the room temperature. Old cultures have a sti'Oiig putrefactive odor. Upon gelatin plates the superficial colonies are flat, shining, bluish-white in color with a somewhat darker centre, transparent, and about two millimetres in diameter; the deep colonies are oval with rather sharp poles, yellowish- white, and lustreless. In gelatin stick cultures growth occurs all along the G52 NON-PATHOGENIC BACILLI. Hue of puncture, and on the surface as a shining layer of moderate extent. Upon agar the growth is similar to that upon gelatin. Upon potato, at the end of thirty-six hours, a dirty-yellow, slimy, granular layer. 321. BACTERIUM GLISCROGENUM (Malerba). Found in urine which was viscid and acid in reaction. Morphology.— Oval bacilli, from 0.57 to 1.14 u long and 0.41 jit broad. Biological Characters. — An aerobic, non-liquefying, motile bacillus. Spore formation not observed. Grows at the room temperature in the usual culture media — better at 30° to 37° C. Upon gelatin plates, at the end of two days, puuctiform colonies are seen, which gradually increase in size, are per- fectly round, granular, and lenticular in form ; later there is a wavy depres- sion of the surface of the gelatin and gas bubbles are formed in the interior. In gelatin stick cultures growth occurs along the line of puncture and upon the surface in nail shape ; the growth along the line of inoculation consists of disc-shaped, isolated colonies closely piled one upon another. Upon the surface of agar, at the room temperature, a granular, opalescent stripe is developed in from three to five days ; at 37° C. an abundant development occurs in twenty-four hours; a white, viscid film forms upon the surface of the condensation water. Upon potato a yellowish or yellowish-brown layer is developed, and in the course of a few days numerous gas bubbles are formed, which become confluent ; later the growth becomes viscid and ex- tends over the entire surface. In bouillon diffuse cloudiness is seen at the end of twenty-four hours, and the fluid becomes viscid ; at the end of four to five days a whitish layer forms upon the surface. 322. BACILLUS OVATUS MINUTISSIMUS (Unna). Found upon the skin in cases of eczema seborrhoeicum. Morphology. — Short oval bacilli with pointed ends, 0.6 to 0.8/* long and 0.4 // broad; associated in irregular groups. Biological Characters. — An. aerobic- and facultative anaerobic, non- liquefying bacillus. Spore formation not observed. Grows at the room temperature in the usual culture media. Gelatin and potato cultures give off a strong and disagreeable odor. Updn gelatin plates, at the end of eight days, the superficial colonies are the size of a mustard seed, prominent, spherical, grayish-white, shining, and resemble a drop of a solution of gum tragacanth; the deep colonies are punctiform and grayish- white in color; later the superficial colonies become flat, or occasionally preserve the form of a small pearl ; these are almost one centimetre in diameter, round, finely granular, yellowish-gray, with a darker centre and a more transparent peri- pheral zone; the margins are notched. The deep colonies, under a low power, are seen to be spherical or oval, opaque, finely granular, dark-yel- lowish in the centre and paler at the periphery; they may attain the size of a pea. In gelatin stick cultures growth is rather rapid upon the surface in form of an abundant, slimy, grayish-white layer with irregular outlines; this later becomes dry and opaque, and presents small, spherical protu- berances which are more transparent; along the line of puncture numerous grayish- white, closely crowded, punctiform colonies. Upon a^ar the growth is similar to that upon gelatin. Upon potato an abundant, grayish-white, dull-glistening layer. 323. CAPSULE BACILLI OP SMITH. Theobald Smith has described three species or vai-ieties ( 0 of capsule ba- cilli, resembling Friedlander's bacillus, obtained by him from the intestine of swine. These he designates by the letters a, b, and c. Morphology. — Slight morphological differences were detected by culti- vating all under the same circumstances in peptone-bouillon . XOX-PATHOGEXIC BACILLI. 653 a. 1.2 /* long and 0.8 to 0.9 H broad ; capsule not visible in hanging1 drop. b. 1.6 to 1.8 Jt long and 0.8 to 0.9 ju thick; capsule clearly visible in hanging drop ; usually in pairs ; both ends somewhat thickened as in a. c. Bacilli somewhat thicker than b ; capsule not visible. Friedlander's bacillus, 1 to 2 n long and 1 M thick. A second examina- tion of all four, made at the end of forty-eight hours, showed no apparent difference in a, b, and Friedlander's bacillus, while c remained shorter than the others. None of the species retained their color when treated by Gram's method. Biological Characters. — Aerobic and facultative anaerobic, non-liquefy- ing, non-motile bacilli. Upon gelatin plates — a forms colonies resembling those of Bacillus coli communis; they spread out in a thin layer, tbe margins of which are very thin and bluish by trans- mitted light ; the contour is irregular ; later the colonies become thicker, white, and opaque, and flap-like processes may be given off from the mar- gins which double the original diameter of the colony — of four to five mil- limetres; in the centre a small, button-like elevation is seen. b. The colonies are at first thicker and more opaque than those of a ; they reach a diameter of four millimeti'es, and are thinner toward the mar- gin, which is irregular in outlhie; a small central prominence is usually observed. c. The colonies are thicker, circular in outline, with smooth margins, and attain a diameter of five millimetres ; the central projection is strikingly large. In peptone-bouillon, at 36° C., at the end of five hours a, b, and c all cause the culture medium to be decidedly clouded, while Friedlander's ba- cillus only causes a slight cloudiness at the end of twenty-four hours. At the end of a week the cultures of a show a dense clouding and an abun- dant deposit at the bottom of the tube, but 110 layer on the surface ; the cul- tures of b a similar cloudiness and deposit, and also a thick, gelatinous layer on the surface ; the cultures of c a densely clouded medium with a thin film upon the surface. Friedlander's bacillus, cultivated in the same medium, showed a slight cloudiness and a few fragments of a mycoderma floating upon the surface. In the cultures of b the whole fluid becomes very viscid and can be drawn out into long threads ; the same character is developed later and to a less extent in cultures of a ; and in c the superficial film is somewhat viscid — cultures of Friedlander's bacillus do not exhibit this character. All of the cultures have an alkaline reaction, and those of a, b, and c after a time have a disagreeable odor. In milk, at 37° C., at the end of a week coagulation is not complete, although the fluid is very thick; b causes milk to be quite thick in two days, and to be completely coagulated in four days; c, at the end of a few days, causes the lower half of the milk to coagulate, and at the end of a week the whole is firmly coagulated ; Friedlander's bacillus in milk did not produce any apparent change. The milk cultures of a, b, and c had an acid reac- tion ; at the end of two weeks some viscid serum was seen above the coagu- lum in a, and the culture smelled like sour dough ; at the same time the whole coagulum was viscid in the culture of b and a similar odor was per- ceived ; the culture of c showed a superficial layer of serum which was not viscid, and the odor was that of cheese. Upon potato a and b developed a thin, shining, grayish, transparent layer ; the growth of c upon potato was thick and cream- white, resembling that of Friedlander's bacillus. In cultures containing glucose, and in mashed potato, c produced considerable quantities of gas— equal parts of COa and of H. Upon agar the growth of all is similar in appearance, but that of a and b is very viscid, that of c less so, while Friedlander's bacillus was destitute of this character. Smith concludes his description of these bacilli by the remark that they may be identical with Bacillus lactis aerogeiies of Escherich. 054 NON-PATHOGENIC BACILLI. 324. BACILLUS PUTRIFICUS COLI (Bienstock). Found in human faeces. Morphology. — Slender bacilli, about 3 ju long, often shorter, frequently- united in long filaments. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, actively motile bacillus. Forms large spherical end spores, which give the bacilli the form of a drumstick; the motile, spore bearing rods always advance with the spore in front. Grows at the room tempera- ture. Upon nutrient gelatin a layer having a pearly lustre is developed ; later this has a yellowish color and is homogeneous in appearance. This bacillus was supposed by Bienstock to be constantly present in faeces, and to be especially concerned in the decomposition of albuminous substances. Its characters of growth have not been determined with precision. 325. BACILLUS SUBTILIS SIMULANS NO I. (Bienstock). Found in human fasces. Morphology. — Bacilli with round ends, resembling Bacillus subtilis ; grows out into long filaments, which become segmented and form short chains of two to five elements; or, more commonly, separate into single rods. Biological Characters. — An aerobic, non-liquefying, non-motile bacillus. Grows best at 37° to 39° C. Forms oval spores, in which germination oc- curs at the two poles simultaneously, leaving the central portion of the new- formed bacillus bulging from the presence of the spore membrane. Upon nutrient agar this bacillus grows out in the form of a mesentery, yellowish- white ' ' veins " running in all directions, which are united with each other smaller anastomosing branches. 326. BACILLUS SUBTILIS SIMULANS NO. II. (Bienstock). Found in human faeces. Morphology. — The same as the preceding species. Biological Character s. — An aerobic, non-liquefying bacillus. Grows very rapidly — best at 37° to 39° C. Upon the surface of agar forms a glistening, white layer, which is at first smooth and later has a somewhat uneven sur- face, while the margins are surrounded by grape-like outgrowths. Imper- fectly described. 327. BACILLUS STRIATUS ALBUS (Von Besser). Found in nasal mucus from healthy individuals. Morphology. — Small, thick bacilli, of about the size of the diphtheria bacil- lus ; often more or less curved ; in preparations stained with methylene blue the bacilli have a striped appearance; club-shaped involution forms may be seen. Biological Characters. — An aerobic, non-liquefying bacillus. Spore formation not observed. Grows at the room temperature in the usual cul- ture media. Upon gelatin plates forms small, dry, superficial colonies. Upon agar plates forms prominent, milk-white colonies, one-half centime- tre in diameter, which under the microscope have a brown nucleus sur- rounded by a paler brown marginal zone. Upon the surface of agar forms a flat, shining, grayish-white layer. Upon potato a scanty, transparent,, jelly-like layer. 328. BACILLUS STOLON ATUS (Adametz). Found in water. Morphology. — Bacilli two and one half times as long as thick. NON-PATHOGENIC BACILLI. Biological Characters. — An aerobic, non-liquefying, actively motile bacillus. Spore formation not observed. Grows rather quickly in the usual culture media at the room temperature. Upon gelatin plates the deep colo- nies are small, spherical or oval, finely granular, whitish or yellowish- brown; the superficial colonies are whitish or brownish, elevated, hemi- spherical, with sharply defined outlines, and about one /* in diameter. In gelatin stick cultures a granular growth is slowly developed along the line of puncture and a white layer about the point of inoculation ; at the end of two to three weeks the upper part of the line of puncture forms a saucer or flask-shaped cavity and the walls are covered with a white layer; there is, however, no liquefaction of the gelatin. Upon agar plates the growth is very characteristic : branches are given off from a central point ; these are variously bent and give off numerous smaller, wavy branches ; these colo- nies extend only upon the surface and may have a diameter of two to three centimetres ; under a low power they are seen as very thin, finely granular, yellowish layers with club-shaped branches. Upon potato forms a dirty- white layer. FIG. 216.— Bacterium Zopfli ; a, long filament with commencement of "ball formation"; b shows the breaking up into short rods; c, further breaking up into spherical elements ; e,f, spirilla- like filaments, x 740. (Kurth.) 329. BACILLUS VENTRICULI (Raczynssky). Found in the stomach of dogs fed exclusively on meat. Morphology — Bacilli from 1.5 to 3 /* long and 1 n thick; united in pairs or in chains of four. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, motile bacillus. Does not form spores. Grows slowly at the room temperature. Upon gelatin plates forms round colonies, which are opaque at the centre and become more transparent toward the margin, which is surrounded by a dark contour. In gelatin stick cultures small, white, punctiform colonies are developed along the line of puncture. Upon agar a whitish layer is formed. This bacillus is said to cause the peptonization of albuminous substances when the reaction is acid or neutral. 656 NON-PATHOGENIC BACILLI. 330. BACTERIUM ZOPFH (Kurth). Found in the intestine of chickens. Morphology. — Bacilli from 2 to 5 fj. long and 0.75 to 1 ju broad; form long filaments, which in gelatin cultures are folded and coiled in a peculiar man- ner. In liquid media straight filaments only are seen. The coiled filaments in gelatin cultures form tangled balls, in which they subsequently break up into short rods and finally into spherical bodies which appear to be repro- ductive elements. Biological Characters. — An aerobic, non-liquefying, actively motile bacillus. Forms spherical spores (?), which are not highly refractive and stain deeply with the aniline colors. Grows best at the room temperature ; at 30° to 37° C. the bacillus is no longer motile ; at 37° to 40° C. involution forms are developed. Upon gelatin plates, at the end of twenty-eight hours, white, punctiform colonies are developed, from which as a centre a mass of fine, radiating filaments is given off; among these numerous small, white points aue distributed, which under the microscope are seen to be spherical zoogloea masses of a brownish-yellow color ; the centre of the colo- nies consists of bundles of interlaced or parallel filaments. In gelatin stick cultures, at the end of twenty-four hours, a thick, white layer is developed along the upper portion of the line of puncture, and later radiating lines of growth are given off from this; these cross each other in various directions and resemble the mycelium of a fungus. No development occurs on blood serum. 331. BACTERIUM ZURNIANUM (List). Found in water. Morphology. —Short rods with slightly pointed ends, from 1.2 to 1.5 n long and 0.6 to 0.8 /* broad; in stained preparations the ends are most deeply stained, giving the rods the appearance of diplococci. Biological Characters. — An aerobic, non-liquefying, non-motile bacil- lus. Does not form spores. Grows in the usual culture media at the room temperature — better at 25° to 30° C. Upon gelatin plates forms spherical, dirty-white or gray, extremely viscid colonies, which develop into slimy, grape-like masses. In gelatin stick cultures a grape-like mass upon the surface and scanty growth along the line of puncture. Upon potato, at 25 J to 30° C., a slimy, translucent, gray or yellowish-white layer extends over the surface within forty-eight hours. 332. BACILLUS OF COLOMIATTI. Obtained from xerotic masses from the eye of a child and in certain forms of conjunctivitis. Morphology. — Bacilli which correspond with the bacillus of mouse septi- caemia in length, associated in irregular masses. Biological Characters. — An aerobic, non-liquefying, non-motile bacillus. Does not grow at the room tempei^ature. Forms spherical spores, which lie at the ends of the rods. Does not grow in nutrient gelatin or on potato. Upon the surface of agar a thin film is developed at 34° to 39° C. , which gives to the surface a fatty lustre. Upon blood serum, at 35° to 39° C., de- velopment occurs along the line of inoculation as a dull-gray layer with a fatty lustre, which has a breadth of two to three millimetres and a rosette- like form. 333. BACILLUS SCISSUS (Frankland). Found in the soil. Morphology. — A short, thick bacillus of variable dimensions; usually about 1 /LI broad and 1 to 2 it long ; resembles Bacillus prodigiosus, and, like NON-PATHOGENIC BACILLI. 657 this, may easily be mistaken for a micrococcus ; sometimes in pairs or in short chains. Biological Characters.— An aerobic, non-liquefying, non-motile bacillus. Spore formation not observed. Grows slowly in the usual culture media at the room temperature. Upon gelatin plates the deep colonies are yellowish and punctiform ; under a low power they are seen to be opaque in the mid- dle and have dentate margins; when they break through to the surface they form small, prominent masses, and from these superficial colonies are de- veloped which have a pale-greenish tint, a deeply dentate margin, and are seen under the microscope to be finely granular In gelatin stick cultures a very thin, smooth, shining layer is developed upon the surface; this has an irregular and deeply dentate margin; the gelatin acquires a greenish tint; no development occurs along the line of puncture. Upon the surface of agar a smooth, shining layer with wrinkled margins is formed ; the agar acquires a green tint. Upon potato a shining, flesh-colored layer extends over a considerable portion of the surface. jrf?Sv,® , ^ %% j^i FIG. 217. FIG. 218. FIG. 217.— Bacillus scissus. X 1,000. (Frankland.) FIG. 218.— Bacillus scissus; superficial colony on gelatin plate, x 100. (Frankland.) 334. BACILLUS NO. I. OF FULLES. Found in the soil. Morphology. — Bacilli from 1 to 1.2 u long and 0.6 ja broad. Biological Characters. — An aerobic, non-liquefying, motile bacillus. Spore formation not observed. Upon gelatin plates forms spherical, finely granular colonies ; under the microscope these have a yellowish-brown cen- tre surrounded by a yellowish marginal zone ; later these differences in color are not seen, and to the naked eye the colonies are bluish-white. In gelatin stick cultures a thin layer forms upon the surface which is not characteris- tic. In bouillon, at the room temperature, the liquid becomes densely clouded and white flocculi are deposited at the bottom of the tube. Upon potato a moist, dirty -yellow layer is developed. Not pathogenic for mice or for guinea-pigs. 335. BACILLUS NO. II. OF FULLES. Found in the soil. Morphology. — Small bacilli with round ends; resemble cocci. Biological Characters. — An aerobic, non-liquefying bacillus. Spore for- mation not observed. Presents oscillatory movements only. Upon gelatin plates somewhat elevated, round, grayish-white colonies are developed upon the surface ; the deep colonies are small, yellowish in color, and under a low power are seen to be finely granular, brownish-yellow, and with sharp con- tours. The superficial colonies attain a diameter of two millimetres and have a brownish-yellow color. In gelatin stick cultures the growth is at 658 NON-PATHOGENIC BACILLI. first nail-shaped ; later it spreads out upon the surface. Upon agar a thin, white layer with irregular outlines is developed. In bouillon, at the room temperature, a dense cloudiness is developed and a thick, slimy deposit ac- •cumulates at the bottom of the tube. Upon potato a moist, granular, yel- lowish-white layer is developed. In milk an acid reaction is not produced. 336. BACILLUS PHOSPHORESCENS GELIDUS (Forster). Found in phosphorescent sea fish. Morphology. — In recent cultures the bacilli appear as small rods with slightly rounded ends and about three times as long as broad ; in cultures more than twenty-four hours old they are thicker and nearly oval in form. Biological Characters. — An aerobic, non-liquefying bacillus. Spore formation not positively determined. Grows slowly at the room tempera- ture, and even as low as 0' C. ; is killed in a few hours by exposure to a temperature of 35° to 37° C. Cultures freely exposed to the air are phos- phorescent in the dark when kept at a temperature between 0° and 20° C. — phosphorescence ceases at 32° C. Upon gelatin plates, at the end of forty- eight hours, small, punctiform colonies are developed ; under the microscope these are seen to be spherical, grayish-white in color with a greenish shim- mer ; later granular, yellowish, and with somewhat irregular outlines. In gelatin stick cultures a white layer is developed upon the surface; very scanty growth along the line of puncture. Upon agar the growth is simi- lar to that 011 gelatin. Upon potato a broad, white layer is developed. 337. BACILLUS SMARAGDINO-PHOSPHORESCENS (Katz). Obtained from a herring from the fish market at Sydney, New South Wales. Resembles Photobacterium phosphorescens (Cohn) described by Beyer- inck, and Photobacterium Pfliigeri (Ludw.). Morphology. — Bacilli with somewhat pointed ends, about 2 n long and 1 n broad ; solitary or in pairs ; in recent cultures closely resemble cocci. 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 eighteen hours, the deep colonies are seen as grayish points, and the superficial as thin, grayish- white drops ; under a low power these are pale-gray with a yellow- ish tint, finely granular, and transparent near the margins, which are finely dentate ; diameter from 0.3 to 0.45 millimetre. -The deep colonies, under the microscope, are seen to be oval or lemon-shaped, with a smooth, well-defined contour, and about 0.15 millimetre in diameter; they consist of a broad cen- tral portion surrounded by a narrow central and still narrower marginal zone ; zone formation not observed in colonies in eight-per-cent gelatin. At the end of twenty days the superficial colonies attain a diameter of two milli- metres ; they are flat, have irregular outlines, and are composed of a rela- tively small central portion of a yellowish color, surrounded by a slate-col- ored marginal zone ; the deep colonies at this time (twenty days) are about 0.6 millimetre in diameter and yellowish- white in color — under the micro- scope straw-yellow. In gelatin stick cultures (six per-cent gelatin) a thin, white line of growth is seen along the line of puncture, and a flat, round, grayish-white layer, with a stearin lustre, is developed upon the surface; this acquires a diameter of about five millimetres. Cultures made by Katz for a year in six-per-cent gelatin gave no evidence of liquefaction, but subse- quently the same bacillus, cultivated in six-per-cent gelatin containing 2.7 per cent of sodium chloride, caused liquefaction of the gelatin beneath the surface growth, which gradually extended downward. Growth occurs upon the surface of agar, but this is not a very favorable medium. In neutral bouillon development occurs, and a diffuse cloudiness is seen, but no growth occurs in simple flesh infusion; when, however, 2.5 per cent of sodium NON-PATHOGENIC BACILLI. 659 chloride is added to this it constitutes a favorable medium. Upon the sur- face of sterilized milk a tolerably abundant, sticky, glistening- layer of a cream-white color is developed. Upon cooked egg a thin, grayish-white, phosphorescent layer is formed. No growth on potato which has an acid reaction, but when the acidity is neutralized with a solution of sodium phos- phate a thin, brownish-yellow layer is developed. Small quantities of a pure culture added to sea water cause it to exhibit a very decided phospho- rescence, and the addition of sodium chloride to culture media favors the growth of the bacillus and its phosphorescent power. Free access of oxygen is essential for the growth and phosphorescence of this species. The color of the light given off by fresh cultures is emerald-green ; it is less intense in ao-ar cultures than in cultures in nutrient gelatin, bouillon, or upon cooked fish. 338. BACILLUS ARGENTEO-PHOSPHORESCENS NO. I. (Katz). Obtained from sea water at Elisabeth Bay, Sydney, New South Wales. Morphology. — Slender, slightly curved bacilli with pointed ends, about 2.5 jit long and one-third as thick as they are long; solitary or in pairs; oc- casionally in long, wavy filaments. Biological CJiaracters. — An aerobic, non-liquefying, motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature — not in the incubator at 35° C. Upon gelatin plates (six-per-ceiit gelatin), at the end of twenty hours, the superficial colonies ap- pear as flat, shining, transparent drops from 0.4 to 0.6 millimetre in dia- meter; under the microscope they are seen to be homogeneous and usually circular in outline, with a dentate margin. The deep colonies are spherical or short oval; they have a smooth, well-defined contour and pale-yellow color. At the end of forty- eight hours the superficial colonies are granular, pale-yellow, with an undulating contour, and about 1.25 millimetres in dia- meter; at the same time the deep colonies are pea-yellow and uniformly granular ; later the deep colonies present three well-defined zones ; the super- ficial colonies also, at the end of twenty days, present two or three distinct zones; they attain a diameter of about three millimetres. In eight-percent gelatin the deep colonies, at the end of two days, are oval, and under the microscope show three well-defined zones; the superficial colonies present the same appearance in from four to seven days, at which time they have a diameter of three millimetres — later as much as seven millimetres. In gela- tin stick cultures (six per cent) development occurs upon the surface as a flat, shining, usually circular layer, about one cubic centimetre in diameter, and of a greenish-yellow or wax-like color. No growth occurs in acid gela- tin. In old gelatin cultures containing 2.7 per cent of sodium chloride liquefaction sometimes occurs at a temperature approaching that at which the gelatin would become liquid. In bouillon a diffuse cloudiness is pro- duced and a film is formed upon the surface ; no growth occurs in flesh in- fusion without the addition of peptone and salt, but the addition of 2.5 per cent of sodium chloride to neutral flesh infusion makes it a favorable me- dium. Upon the surface of sterilized fish a pale-yellow, glistening, sticky layer is developed. Upon cooked egg a thin, grayish- white layer. No growth on potato. Phosphorescence depends upon the presence of certain salts — especially sodium chloride— in the culture medium, and upon the free access of oxygen. In bouillon cultures, when a mycoderma has formed upon the surface this shows phosphorescence, while the liquid below does not. The light given off is of a silver-white color, and a recent culture upon the sur- face of gelatin gives sufficient light to enable one to determine the time from a watch in a dark room. 339. BACILLUS ARGENTEO-PHOSPHORESCENS NO. II. (Katz). Obtained from phosphorescent pieces of fish found in the market at Syd- ney, New South Wales. 000 NON-PATHOGENIC BACILLI. Morphology. — Bacilli with round ends, about 2. 7 n long and 0. 67 n broad; occasionally form short filaments. Biological Characters. — An aerobic, non-liquefying, non-motile bacil- lus. Spore formation not observed. Grows in the usual culture media at the room temperature — not so well at 32° to 34° C. Upon gelatin plates (six percent), at the end of twenty-four hours at 18° to 20° C., the superficial colonies have a diameter of 0.5 millimetre and resemble little drops of stea- rin; they have a circular and sharply defined contour, are homogeneous, and of a pale yellowish-gray color ; at the end of forty-eight hours they are grayish-yellow, finely granular, with irregular contour, and about one milli- metre in diameter. The deep colonies are much smaller, have a greenish- yellow color, granular contents, and a smooth, well-defined contour. The deep colonies which come to the surface spread out to form a bluish-gray, shining disc, which may attain a diameter of six millimetres. In gelatin stick cultures scanty development along the line of puncture, and a grayish- white, glistening layer of limited extent upon the surface. Upon the sur- face of ten-per-cent gelatin a bluish-gray band with cloudy margins along the impfstrich. In. bouillon cultures the liquid is diffusely clouded, but no mycodermais formed upon the surface. Upon sterilized fish a shining, sticky layer of a yellowish color — here and there lemon-yellow; the cultures have a silver-white phosphorescence in the dark, and a small quantity added to a considerable quantity of sea water causes this to be phosphorescent. The presence of certain salts, and especially of sodium chloride, and free contact with oxygen, is favorable to the growth of this bacillus and to the development of phosphorescence. 340. BACILLUS ARGENTEO-PHOSPHORESCENS NO. III. (Katz). Obtained from a fragment of cuttlefish which was phosphorescent, at Sydney, New South Wales. Morphology. — Resemble Bacillus argenteo-phosphorescens No. II., but the rods are a little thinner and are motile; frequently united in pairs and occasionally form short filaments. Biological Characters. — An aerobic, non-liquefying, motile bacillus. Spore formation not observed. Grows at the room temperature in the usual culture media— not so well at 32° to 34° C. Upon gelatin plates, at the end of twenty-four hours at 18° to 20° C., the superficial colonies appear as white scales with irregular outlines, sometimes wrinkled, and marked with fine lines or furrows, about 0.45 millimetre in diameter. The deep colonies are spherical, oval, or lemon-shaped, homogeneous, and greenish-yellow in color; at the end of forty-eight hours th^y appear finely granular and divided into two zones. At the end of a week the superficial colonies attain a diameter of about three millimetres ; they are bluish-gray and cloudy in appearance, quite thin, and have a notched or dentate margin. Upon the surface of gelatin stick cultures a layer is developed which extends nearly to the walls of the tube, and which becomes very thin at the margins. Development occurs upon the surf ace of agar, but this is not a very favorable medium. In bouil- lon a diffuse cloudiness is produced and a mycoderma forms upon the surface. The addition of 2.5 per cent of sodium chloride to bouillon or other media is favorable to the growth and phosphorescence of this bacillus, as of those previously described. Upon sterilized fish a viscid, glistening, yellowish layer is developed. No growth upon acid potato. The cultures give off a silver- white, phosphorescent light. Bouillon cultures commence to give off light from the surface at the end of four or five days, and at the end of eight days the floating mycoderma may give a light by which the time can be dis- cerned,^in a dark room, from a watch ; the light is a " bluish-greenish white.'* NON-PATHOGENIC BACILLI. 661 D. Non-chromogenic, Liquefying Bacilli. 341. BACILLUS CYANEO-PHOSPHORESCENS (Katz). Obtained from sea water at Little Bay, near Sydney, New South Wales. Very nearly related to Bacillus phosphoresceiis of Fischer (Katz). Morphology. — Bacilli with round ends, about 2.6 ju long and 1 ft thick ; solitary oriii pairs; occasionally grow out into long filaments. Stains by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile bacillus. Spore formation not observed. The cultures give off a bluish phosphorescence which has a faint greenish tint. Grows in the usual culture media at the room temperature — best at 26° C. ; very scanty growth upon nutrient agar at 32° to 34° C. Upon gelatin plates, at the end of eighteen hours, colonies are already visible ; those upon the surface and those in the interior of the gelatin are of about the same dimensions — 0.25 to 0.4 millimetre; under the microscope they are seen to be finely granular, have a sharply defined, smooth contour and a dark-gray color; the superfi- cial colonies are finely granular and pale yellowish-gray in color. At the end of forty-eight hours the superficial colonies are surrounded by a broad girdle of liquefied gelatin, at the bottom of which they lie in contact with the glass plate ; they are of a dirty brownish-yellow color and irregular in outline; the liquefied gelatin is pale-gray or yellowish-gray by transmitted light, and contains here and there granular masses scattered through the more finely granular structure. At this time the deep colonies are yet well defined, have irregular outlines, and are of a dirty yellowish-brown color ; they have a diameter of about 0.3 to 0.5 millimetre, and are surrounded by a zone of liquefied gelatin about 0.05 to 0.1 millimetre in diameter ; this is finely granular, light-brown or light-gray in color, and marked by delicate radial striations. When the colonies are crowded upon a plate liquefaction may be complete at the end of eighteen hours ; the liquefied gelatin gives off a peculiar odor. In gelatin stick cultures (six-per-cent gelatin) liquefaction commences beneath the surface growth, and at the end of forty-eight hours a shallow cavity of Avatch-glass form is seen which has a diameter of about five millimetres — temperature of 20° to 22° C. ; at the bottom of this a gray- ish-white layer is seen, and below this a line of development along the track of the inoculating needle; this is surrounded by a narrow zone of liquefac- tion. At the end of three or four days liquefaction at the surface reaches the walls of the test tube ; when liquefaction is complete a yellowish, viscid mass is seen at the bottom of the tube and a mycoderma upon the surface; the gelatin is at first diffusely clouded and later becomes transparent; it has a yellowish color, which gradually changes to reddish-brown. In six-per- cent gelatin containing 2.7 per cent of sodium chloride development is espe- cially abundant and rapid, and short, radiating processes are given off into the gelatin in advance of liquefaction. In bouillon diffuse cloudiness occurs and a mycoderma forms upon the surface. No development occurs in simple flesh infusion, but the addition of 0.5 per cent of sodium chloride to this constitutes a medium in which growth occurs — still better 2.5 per cent. Upon sterilized fish a glistening, sticky layer of a yellowish or yellowish- brown color, in the thicker places, is developed. Phosphorescence depends upon the free access of oxygen and the presence of certain salts, especially sodium chloride. A very minute quantity of a culture added to sea water causes it to exhibit phosphorescence. Cultures upon the surface of agar give sufficient light to enable one to distinguish printed letters in a dark room. No growth upon potato. 342. BACILLUS ARGENTEO-PHOSPHORESCENS LIQUEFACIENS (Katz). Obtained from sea water at Bondi Bay, near Sydney, New South Wales. Resembles Photobacterium luminosum of Beyerinck. 56 662 NON-PATHOGENIC BACILLI. Morphology. — Straight or slightly curved bacilli with round ends; about two /* long and one-third as broad; grow out into filaments of various lengths. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile bacillus. Spore formation not observed. Cultures give off a silvery phosphorescence, which is less intense than with the previously de- scribed species. Grows at the room temperature in the usual culture media — best at 25° C. ; does not grow in the incubating oven at 34° C. Upon gelatin plates, at the end of twenty-four hours at the room temperature, small, hyaline discs are developed, which under the microscope are seen to be finely granular and light-brown in color; they are irregularly circular in outline and about 0.7 millimetre in diameter; the deep colonies are con- siderably smaller, mulberry-like in structure, and straw-yellow in color. At the end of forty-eight hours shallow liquefaction has occurred beneath the superficial colonies, in watch-glass form, and about two millimetres in diameter ; under the microscope a central mass of a straw- yellow color is seen, around this a narrow, light-brown zone with granular contents, and outside of this a broader peripheral zone, from which fine, radiating outgrowths are given off into the non-liquefied gelatin. At the same time (forty-eight hours) the deep colonies have a diameter of 0.3 to 0.45 millimetre and a more or less polygonal contour; they are straw-yellow in color and consist of a finely granular central mass, surrounded by a slender, marginal zone which is marked by radial striations. After complete liquefaction of the gelatin the colonies, which remain attached to the glass plate, have a lemon-yellow color. In gelatin stick cultures (six per cent) liquefaction occurs beneath the super- ficial layer which is developed, in form of a shallow watch glass, and gradually extends in diameter and depth; growth also occurs along the line of puncture, and the cultures resemble those of Bacillus cyaneo-phosphores- cens, but with less rapid development and liquefaction of the gelatin ; also without the formation of hair-like outgrowths into the non-liquefied gelatin. The addition of 2.7 per cent of sodium chloride is favorable for the development of this as for the previously described species of phosphorescent bacilli; on the other hand, the addition of two per cent of glucose exercises a restraining influence upon the growth of all the species studied by Katz. In bouillon a diffuse cloudiness is produced by the growth of this bacillus, and a mycoderma is formed upon the surface. No growth occurs in simple meat infusion, but an abundant development when 2.5 per cent of sodium chloride is added to this. Upon sterilized fish a shining, sticky, yellowish- gray layer is developed. No growth upon potato. 343. BACILLUS PHOSPHORESCENS INDICUS (Fischer). Found in sea water from the Gulf of Mexico. Morphology. — Bacilli with rounded and pointed ends, from two to three times as long as broad; length from one-sixth to one-quarter the diameter of a red blood corpuscle ; solitary or in pairs ; also in short filaments. Stains readily with the aniline colors, but unstained places are often seen in the interior of the rods. Biological Characters. — An aerobic, liquefying, motile bacillus. Spore formation not observed. Cultures, especially upon animal substances and in presence of certain soda salts, exhibit a decided phosphorescence in the dark ; this depends upon free access of air, and is most marked at a tem- perature of 25° to 30° C. It is no longer manifested at a temperature of 0° C., and is neutralized by putrefaction. Grows in the usual culture media at the room temperature — not so well in the incubating oven. Upon gelatin plates, at the end of thirty-six hours, small, round, grayish- white, punctiform colonies are developed ; under a low power these are seen to be spherical, with well-defined outlines, and have a sea-green color with a pink shimmer; later they become granular, have a wavy outline and a dirty-yellow color. In gelatin stick cultures, at the end of four days, a grayish-white line of NON-PATHOGENIC BACILLI. 063 growth is seen along1 the track of the inoculating needle, and at the surface a cup-shaped depression, the size of a hempseed, which contains air; in older cultures the gelatin is liquefied near the surface and a thin, dirty-yellow film swims upon it. Upon the surface of agar a grayish-white layer is devel- oped. Upon potato, at 15° to 20° C., a thin and broad white layer. Upon blood serum a narrow, grayish-white stripe, which extends to a tolerably deep channel, with irregular margins, from 0.5 to 1 centimetre wide; this is lined with a slimy, grayish-white growth. Cooked fish or flesh constitutes a favorable medium for the growth of this bacillus. By means of the phos- phorescent light given off by cultures of this bacillus, Fischer has succeeded in making photographs not only of the cultures, but of a watch dial placed between two cultures — an exposure of twenty-four hours' duration and a very sensitive dry plate were required to accomplish this. 344. BACILLUS PHOSPHORESCENS INDIGENUS (Fischer). Found in sea water from the harbor at Kiel and upon phosphorescent herring. Morphology. — Bacilli with round and slightly pointed ends; somewhat shorter than Bacillus phosphorescent Indicus, but of the same thickness — from 1.3 to 2.1 u long and 0.4 to 0.7 /* broad; solitary or in pairs; may grow out into filaments. Biological Characters. — An aerobic, liquefying, motile bacillus. Cul- tures give off a bluish- white, phosphorescent light — not so intense as that from Bacillus phosphorescens Indicus ; phosphorescence depends upon free access of oxygen. Sea water to which a small amount of a culture is added is phosphorescent in the dark Spore formation not observed. Grows at the room temperature in the usual culture media— more slowly than Bacillus phosphorescens Indicus. Grows at 5° to 10° C., and even below; at a tem- perature of 32° C. development still occurs, but the cultures do not exhibit phosphorescence. Upon gelatin plates the gelatin is depressed about the small spherical colonies, and at the end of a week cylindrical cavities filled with air, and not more than one millimetre in diameter, are formed in the gelatin ; at the bottom of these, on the surface of the plate, the colonies are seen ; these are the size of a pin's head, thin, disc-formed, and dirty yellow in color ; under a low power very young colonies are seen to be circular, with well-defined margins, and of a pale sea-green color; here and there reddish- shimmering granules are seen in the otherwise homogeneous contents ; the older colonies are made up of irregular, dirty yellowish-gray masses. In gelatin stick cultures, at the end of a week, a conical cavity forms near the surface, which is filled with air and is lined with a thin, friable growth; at the surface the mouth of the cone measures about two millimetres in diame- ter; this cavity increases in dimensions without containing any liquefied gelatin; in old cultures it may be three to five millimetres in diameter and two to three centimetres deep, the walls being covered with a thin layer of bacilli, and a mass of the same accumulating at the bottom. No growth occurs upon potato or upon blood serum. 345. BACILLUS CIRCULANS (Jordan). Found occasionally in. water from the Merrimac Eiver. Morphology. — Bacilli with round ends, from 2 to 5 n long and about 1 H broad ; usually solitary, but sometimes in loosely connected chains of three or four elements. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile bacillus. Forms oval spores, which are located at the ends of the rods and are of about the same diameter as these. Grows in the usual culture media at the room temperature — better at 37° C. Upon gelatin plates, at the end of two days, round, brownish colonies become visible ; under a low power the liquid contents of these colonies are seen to be in mo- 664 NON-PATHOGENIC BACILLI. tioii, owing to the active movements of the individual bacteria; the motion may be compared to the circulation of protoplasm in a cell ; this is seen after forty-eight hours of growth, but ceases, as a rule, on the third day, at which time the contents are seen to be coarsely granular ; a round, deep, even de- pression is formed in the gelatin, which at the end of several weeks may have a diameter of a centimetre; the contour of the colonies is usually smooth, but may be somewhat lobate and irregular. In gelatin stick cul- tures development is slow along the upper part of the line of puncture, and a conical-shaped cavity is formed, at the bot- tom of which a precipitate accumulates, while above the conical cavity is empty owing to the slowness of growth and drying-up of the liq- uefied gelatin. The upper part of the cone often has a somewhat ringed appearance. This bacillus grows well in a slightly acid medium. Upon the surface of agar development occurs along the line of puncture, and a very thin, translucent layer is formed upon the surface. Upon potato a slow and scanty growth having about the same color as the cut surface of the potato. In milk a slightly acid reaction oc- curs and the casein is slowly precipitated; FIG. 219. — Bacillus circulans, after cultivation for several months the bacil- from an agar culture five days old. lus no longer caused coagulation of milk. In x 1,000. (Jordan.) bouillon a cloudiness is seen at the end of three or four days, and a considerable slimy preci- pitate is formed ; no film forms upon the surface. Nitrates are slowly re- duced to nitrites by this bacillus. 346. BACILLUS SUPERFICIALIS (Jordan). Found frequently in sewage at Lawrence, Mass. Morphology. — Bacilli with round ends, about 2.2 ft long and 1 n broad ; solitary or in pairs. Biological Characters. — Anaerobic, liquefying, motile bacillus. Spore formation not observed. Grows at the room temperature in the usual cul- ture media— better at 37° C. Upon gelatin plates colonies become visible at the end of forty-eight hours; under a low power they are seen to be divided by irregular lines into angular lumps, giving a cracked appearance to the whole colony, which is irregularly spherical in form. Upon coming to the surface the colonies spread out to form a round, finely granular disc, which to the naked eye looks like a projecting, translucent drop; this slowly in- creases in dimensions, and the surrounding gelatin is slowly liquefied ; after some days the colony has an opaque, yellowish-brown centre and a translu- cent edge. In gelatin stick cultures there is a very scanty growth along the line of puncture and more abundant development upon the surface; lique- faction proceeds slowly, and by the end of ten days may reach the walls of the test tube. This bacillus grows well in acid gelatin. Upon the surface of agar a moist, lustrous, gray, translucent layer is developed; at the end of several weeks this growth is still smooth and glistening, and has a light- brown tint. Does not grow upon potato. Does not cause coagulation of milk, which, however, acquires a slightly acid reaction. In bouillon a dif- fuse cloudiness is slowly developed, and after some time a scanty white sediment is seen ; no film forms upon the surface. 347. BACILLUS RETICULARIS (Jordan). Found in water at Lawrence, Mass. Morphology.— Bacilli with slightly rounded ends, about 5 n long and 1 /u broad ; often united in chains of eight to ten elements. NON-PATHOGENIC BACILLI. 665 Biological Characters. — An aerobic, liquefying, motile bacillus. Spore formation not observed. Motion slow and sinuous. Grows at the room temperature in the usual culture media — much better at 37° C. Upon gela- tin plates the deep colonies send out long, spiral filaments, which give a hazy appearance to the colony ; under a low power the colony, surrounded by ra- diating filaments, resembles a jelly-fish with streaming tentacles; the sur- rounding gelatin is slowly liquefied. Oncoming to the surface the colony spreads out as an irregular expansion, under which the gelatin is slowly liquefied, and at the same time dried out so as to form shallow, cup-shaped depressions.; the surface of these cavities is mottled in appearance, as if cov- ered with a fine, irregular reticulation. In gelatin stick cultures, at the end of two days, the growth at the surface resembles a cup with flaring edges ; liquefaction occurs slowly, and the liquefied gelatin is dried by evaporation, so that the cup-shaped cavity gradually increases in dimensions ; it is lined with a reticulated growth similar to that seen in the colonies upon gelatin plates ; at the end of three days fine filaments begin to grow out from the line of puncture, but these do not reach any considerable length. This bacillus exhibits a scanty development under a mica plate. Upon the surface of agar ,a prominent, dull, dry layer is slowly developed. Upon potato a white, •dull, dry layer is developed at the end of two days, and at the end of five days this has a characteristic woolly appearance. Milk acquires an acid re- .action and is slowly -coagulated — fifteen to twenty days at the room tempe- rature. Bouillon slowly becomes turbid and a slight viscid sediment is formed. Nitrates are rapidly reduced to nitrites by this bacillus. 348. BACILLUS HYALINUS (Jordan). Found in large numbers in the sand of Tank 13 at Lawrence, Mass., "at ;a time when the tank was nitrifying well.'' Morphology. — Bacilli with round ends, from 3. 6 to 4 /* long and 1.5 JJ. broad ; usually united in short chains. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, actively motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature — better at 37° C. Upon gelatin plates the colonies are visible at the end of twenty-four hours; they are seen to consist of a dark central nucleus surrounded by a broad, translucent zone, which gives the colonies a hazy appearance ; under a low power the interior is seen to present a coarsely fibrillar appearance, with short fibrils radiating from the edge. In two days the colonies attain a diameter of about one and one-half centimetres; they are round in contour, with a distinct, opaque, yellowish margin, from which radiating fibrils are given off ; the interior is slightly translucent. In gelatin stick cultures, at the end of two days, a long and narrow, funnel-shaped growth is seen; the gelatin is rapidly lique- fied and is at first -clouded, with a white deposit at the bottom of the funnel; later a lustrous and tenacious white layer is seen upon the surface and a slight flocculent deposit at the bottom, while the liquefied gelatin between is entirely transparent. This bacillus grows well in acid gelatin. Upon the surface of agar a dry., grayish, spreading growth is rapidly developed ; when four or five days old small, warty projections are seen ; growth also occurs along the line of puncture in agar stick cultures. Upon potato, at the end of two days, the growth is just visible; at the end of four days a dry, whitish-gray, spreading layer is developed; later small protuberances .are seen upon the surface of this. In milk a strongly acid reaction with co- agulation of the casein is produced in seven days. In bouillon a diffuse cloudiness is quickly produced ; a viscid sediment is formed and a myco- derma forms upon the surface. This bacillus reduces nitrates vigorously and rapidly. 349. BACILLUS CLOAC.E (Jordan). Isolated from sewage at Lawrence, Mass. — "one of the most common ibacteria in sewage." 666 NON-PATHOGENIC BACILLI. Morphology. — Short oval bacilli with round ends, from 0.8 to 1.9 /* long1 and from 0.7 to 1 /* broad; frequently united in pairs. Biological Characters. — An aerobic and facultative anaerobic, lique- fying, actively motile bacillus. Spore formation not observed. Grows in- the usual culture media at the room temperature — better at 37° C. Upon gelatin plates, at the end of one or two days, spherical, yellowish colonies are seen ; upon coming to the surface these form a slight bluish expansion with irregularly notched edges, and liquefaction of the gelatin quickly oc- curs; under the microscope the colonies are seen to have an opaque centre surrounded by a translucent zone and a darker margin ; the interior is finely granular ; liquefaction of the entire plate occurs within three or four days. In gelatin stick cultures development occurs along the line of puncture, and liquefaction rapidly occurs; later an iridescent film is seen upon the surface of the gelatin and an abundant flocculent, whitish sediment at the bottom of the tube. This bacillus grows well in slightly acid gelatin. Upon the sur- face of agar a moist, slimy, porcelain-white layer is developed ; an abun- dant growth also occurs along the line of puncture in agar stick cultures. Upon potato a prominent, yellowish-white layer is quickly formed. In milk coagulation occurs in about four days, with a strongly acid reaction . In bouillon a diffuse cloudiness is seen at the end of two days, and at the end of ten to fourteen days an abundant whitish sediment is seen ; a slight film forms upon the surface; this falls to the bottom when the tube is disturbed; the bouillon is still clouded at the end of two or three weeks. This bacillus reduces nitrates, in bouillon, vigorously. 350. BACILLUS DELICATULUS (Jordan). Found in the water supply at Lawrence, Mass. Morphology.— Bacilli about 2 jo. long and 1 ju. broad; often united in pairs or in short chains. Biological Characters. — An aerobic, liquefying, actively motile bacil- lus. Spore formation not observed. Grows in the usual culture media at the room temperature — somewhat better at 37° C. Upon gelatin plates the young colonies are seen as whitish, homogeneous spheres with a regular, ra- diating margin ; at the end of two days liquefaction of the surrounding gelatin occurs In gelatin stick cultures liquefaction progresses rapidly along the line of puncture and is complete in about seven days ; a thick, whitish layer is then seen upon the surface, and an abundant flocculent, brownish deposit at the bottom of the tube ; the liquefied gelatin remains clouded. Grows well in slightly acid gelatin. Upon the surface of agar a wrinkled, grayish layer is developed, which later becomes porcelain-white and glistening ; development also occurs along the line of puncture in agar stick cultures. Upon potato a thin, spreading, gray layer is developed. Milk acquires a strongly acid reaction and is coagulated. Bouillon quickly becomes clouded ; a white film forms upon the surface and a white deposit is seen at the bottom of the tube. Nitrates are quickly and completely re- duced to nitrites by this bacillus. Does not grow at a temperature below 15° C. , and quickly dies out in artificial culture media— in a few weeks. 351. BACILLUS AQUATILIS (Frankland). Found in water — often almost the only microorganism in water from a deep well in the chalk formation at Kent. Morphology. — Bacilli about 2. 5 y« in length; grow out into filaments of 17 /* or more in length — resembles Bacillus arborescens (Frankland). Biological Characters. — An aerobic, liquefying, motile bacillus. Ex- hibits oscillatory movements only. Spore formation not observed. Grows very slowly in the usual culture media at the room temperature. Upon gelatin plates the deep colonies are at first smooth in outline, later the mar- NON-PATHOGENIC BACILLI. 667 gin is irregular; when the colony reaches the surface a very slow liquefac- tion of the gelatin commences and the appearance of the colonies becomes very characteristic. From the yellowish- brown centre twisted bundles of filaments are given off which are at first also of a yellowish-brown color, but gradually become colorless toward the periphery. In gelatin stick cul- tures development is extremely slow ; upon the surface a small, yellowish mass is formed at the point of puncture, while the line of inoculation is scarcely visible ; liquefaction occurs later and the liquefied gelatin is clouded ; when liquefaction has once commenced it progresses more rapidly. Upon the surface of agar a glistening, yellowish layer is developed which extends but little beyond the impfstrich. In bouillon a diffuse cloudiness is pro- duced and a whitish sediment is seen at the bottom of the tube; no film is formed upon the surface. Upon potato scarcely any development occurs — a faint yellowish stripe along the impfstrich only. Reduces nitrates with formation of ammonia. 352. BACILLUS DIPFUSUS (Frankland). broad; solitary or in Found in the soil. Morphology. — Bacilli about 1.7 j^ long and 0.5 pairs; also grow out into long, flexible filaments. Biological Characters. — An aerobic, liquefying bacillus. Exhibits ro- tatory and oscillatory movements only. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates the superficial colonies after a time have a very characteristic ap- pearance; they extend from the original centre as a broad, thin, bluish-green layer. Under a low power the deep colonies re- semble colonies of the cholera spirillum; they are nearly sphei'ical, coarsely gran- ular, and have somewhat jagged mar- gins ; later the margins are still more ir- regular and finely dentate, while the surface near the margin appears coarsely granular; when the colonies come to the surface the centre is no longer well de- fined, but remains granular, while about it a very characteristic surface growth occurs. In gelatin stick cultures the development is almost limited to the sur- face, upon which a smooth, thin, shin- ing, somewhat greenish-yellow layer is formed; liquefaction of the gelatin be- neath this progresses very slowly. Upon the surface of agar a very thin, shining, smooth layer of a feebly yellow or cream color is developed. In bouillon a diffuse cloudiness is developed and a greenish-yellow sediment is formed, while some flocculi may float upon the surface, which, however, do not constitute a film. Upon potato a thin, smooth, shining, faintly greenish-yellow layer is formed. FIG. 220. FIG. 221. FIG. 220.— Bacillus diffusus, from a gela- tin culture. X 1,000. (Frankland.) FIG. 221.— Bacillm diffusus; superficial colonies in nutrient gelatin. X 100. (Frankland.) 353. BACILLUS LIQUIDUS (Frankland). Found in river water from the Thames ; very common. Morphology.— Short and thick bacilli with round ends; usually in pairs, of which the length varies from 1.5 to 3.5 M ; differ greatly in dimensions. Biological Characters. — An aerobic, liquefying, motile bacillus. Spore formation not observed. Grows at the room temperature in the usual cul- ture media. Upon gelatin plates the deep colonies, under a low power, are seen to be spherical, with smooth outlines ; when liquefaction commences 668 NON-PATHOGENIC BACILLI. the margin becomes somewhat granular and jagged, while the centre re- mains opaque; development and liquefaction of the surrounding gelatin progress rapidly, so that the colonies soon coalesce. In gelatin stick cul- tures development occurs along the entire line of puncture, and a broad funnel of liquefied gelatin is formed in the course of a few days; this is clouded and contains numerous flocculi ; later the surface is covered with a a thin film, which sinks to the bottom when the test tube is shaken. Upon the surface of agar development also occurs rapidly, forming a smooth, shining layer on both sides of the impfstrich. In bouillon a diffuse cloudi- ness is developed ; an abundant deposit collects at the bottom of the tube, and a film is formed upon the surface. Upon potato a thick, flesh-colored layer covered with protuberances and having a dull, moist surface. This bacillus reduces nitrates with the production of nitric acid 354. BACILLUS VERMICULARIS (Frankland). Found in water from the river Lea. Morphology. — Bacilli with round ends, from 2 to 3 jn long and 1 u broad; may grow out into ' ' worm-shaped " filaments. Upon potato oval spores are formed; these are about 1.5 u long and 1 ju broad; they are formed in the centre of the rods, and remain attached to each other in chains of consider- able length. Biological Characters. — An aerobic, liquefying bacillus. Exhibits os- cillatory movements only. Forms oval spores. Grows slowly in the usual culture media at the room temperature. Upon gelatin plates the deep col- onies have a somewhat irregular contour ; upon the surface flat colo- |^| £r nies are developed, which have ir- J Q. ^K regular margins composed of wavy ^ bundles of bacilli closely crowded together; the centre of the super- ficial colonies is rough and wrinkled ; later liquefaction slowly occurs and Fio. 222.— Bacillus vermicuiaris; a, from a ge- the colonies sink beneath the surface latin culture; b, spore-bearing filaments from of the gelatin. In gelatin stick cul- a potato culture, x i,ooo. (Frankland..) tares a moist, shining, gray layer with dentate margins is developed upon the surface ; this does not extend far from the point of puncture ; a scanty growth is seen along the line of puncture; after some time the gela- tin commences to liquefy beneath the surface growth. Upon the surface of agar a smooth, shining layer of a gray color is slowly developed. In bouillon the liquid remains clear, and a white, flocculent growth is seen at the bottom of the tub. Upon potato a thick, flesh-colored layer with irregular outlines. Eeduces nitrates to nitrites. 355. BACILLUS NUBILUS (Frankland). Found in filtered London water. Morphology. — Bacilli about 3 JLI long and 0.3 /n broad; solitary or in short chains; grow out into long filaments in bouillon cultures — these maybe twisted in spiral form; upon potato the short bacilli are often curved, and the long filaments may have a spiral form. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing bacillus. Exhibits rotatory movement's without change of location. Spore formation not observed. Grows slowly at the room temperature in the usual culture media. Upon gelatin plates the colonies are very charac- teristic; at the end of forty -eight hours small clouded spots are seen, which are not sharply defined ; under the microscope these are seen with difficulty by transmitted light ; on the third day, when the-gelatin, commences to be NON-PATHOGENIC BACILLI. GG9 softened, these colonies are seen to consist of a network of interlaced fila- ments ; at the centre a thicker portion is often seen in the cloud-like colonies of interlaced threads; liquefaction progresses rapidly, the colonies become confluent, and soon the plate is destroyed. In gelatin stick cultures the gelatin is liquefied upon the surface, but liquefaction does not progress rap- idly ; the liquefied gelatin is clouded, and a yellowish deposit accumulates at the bottom of the liquefied gelatin ; along the line of puncture, almost to the surface, no growth is seen, but around it a row of flat, horizontal rings are developed, the diameter of which increases from above downward; the lower end of the line of puncture is uniformly clouded ; the rings described are made up of delicate, cloud-like masses ; the culture somewhat resembles that of the bacillus of mouse septicaemia; liquefaction progresses slowly, but finally the entire amount of gelatin is liquefied. Upon the surface of agar a thin, opalescent, bluish-white layer is formed, the fringed margins of which show a violet fluorescence. In bouillon a diffuse cloudiness is developed, and a dirty- white deposit collects at the bottom of the tube, while a very thin film collects upon the surface ; this falls to the bottom, when the tube is shaken. Upon potato the growth is almost invisible and of a very faint yel- low color; it extends, however, over a great portion of the surface. 356. BACILLUS PESTIPER (Frankland). Found in the air. Morphology. — Bacilli with round ends, 2.3// long and 1 /* broad; often grow out into filaments. Biological Characters. — An aerobic, liquefying, motile bacillus. Spore formation not observed. Grows slowly at the room temperature. Upon gelatin plates forms colonies resembling those of Bacillus vermicularis, but the deep colonies are more regular and the superficial colonies have a smooth centre. Upon the surface of agar a shining, transparent layer with dentate margins. Upon potato a thick, irregular, flesh-colored layer. 357. BACILLUS FILIFORMIS (Tils). Found in water. Morphology. — Bacilli with round ends, 4 jn long and 1 u broad ; usually united in chains, which may consist of as many as ten segments which are indistinctly separated from each other. Biological Characters. — An aerobic, liquefying bacillus. Exhibits os- cillatory movements only. Forms, in potato cultures, large oval spores. Upon gelatin plates grayish-white colonies are developed which have a marbled structure; under a low power the deep colonies are seen to be finely granular and irregular in outline ; the superficial colonies have a dentate margin, which is composed of many bundles of bacilli closely crowded to- gether; the centre is somewhat rough, elevated, and granular; the colonies are colorless at the margin, but have a yellowish color at the (^nti-e; at the end of eight to ten days liquefaction commences and the colonies sink slowly beneath the surface. In gelatin stick cultures a moist layer with deeply dentate margins forms around the point of puncture; the gelatin is slowly liquefied and thick, flocculent masses of bacilli accumulate at the bottom. In bouillon growth occurs chiefly upon the surface in the form of a firm my- coderma. Upon the surface of agar a white layer is developed like that upon gelatin. Upon potato a thick, slimy, dirty- white layer, which later becomes dry and acquires a gray or brownish color. In milk coagulation occurs at the end of tliirty-six hours, and an odor of putrefaction is perceived. 358. BACILLUS DEVORANS (Zimmermann). Found in well water. Morphology. — Bacilli with round ends, from 0.09 to 1.2 /* long and about 0.74 // thick; solitarv, in pairs, or occasionally in short chains. 57 670 NON-PATHOGENIC BACILLI. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, actively motile bacillus. Spore formation not observed. Grows at the room temperature in the usual culture media. Upon gelatin plates the dee}) colonies appear as small, white spheres ; the superficial colonies are seen as round, white, not homogeneous masses at the bottom of a funnel of lique- fied gelatin; under a low power they have a granular-thready structure and yellowish-gray color, and the margins are surrounded by thread-like processes of various lengths. In gelatin stick cultures growth is visible along the line of puncture at the end of twenty-four hours; on the second or third day an air bubble is seen at the upper portion and a whitish growth below; the funnel-shaped cavity gradually extends in dimensions without a trace of liquid being seen, and the growth is distributed upon the walls and at the bottom of this cavity ; often liquefaction occurs. Upon the surface of agar a thin, uniform, gray layer is developed, which covers the entire sur- face at the end of two or three days. No growth upon potato. 359. BACILLUS GRACILIS (Zimmermann). Found in the Chemnitz water supply. Morphology.— Long bacilli with round ends, usually more or less curved, from 2.4 to 3.6 fj, long and about 0.77 ju broad; grow out into long filaments, which are bent at an angle or present several wave-like curves. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing bacillus. Forms oval spores 1.83 // long and 1.3 fi broad. Exhibits ro- tatory and oscillatory movements only. Grows slowly at the room tempe- rature— not in the incubating oven. Upon gelatin plates the deep colonies appear as small, grayish-white spheres, which at first are well defined, but later have very indistinct outlines; under a low power they are first seen as sharply defined, pale-yellow discs, which are gradually surrounded by nu- merous thread-like outgrowths, which after a time form a thick network. Upon the surface there is no development or small, yellowish-gray, drop-like colonies are seen ; at the end of five days these attain a diameter of four to six millimetres and are circular in outline ; the centre is round, nebulous, and bluish-gray, and outside of this one or two concentric, nebulous rings are seen. In gelatin stick cultures there is a scanty growth upon the sur- face, often appearing as an opalescent film, or the superficial layer of the gelatin is penetrated by radiating lines given off from centres of growth which do not extend above the surface. Along the line of puncture a row of whitish discs is developed which are largest above ; at the end of three to five weeks the upper portion of the gelatin is liquefied to some extent. Upon the surface of agar a thin, irregular layer of a bluish- white color is developed ; an abundant growth occurs along the line of puncture in agar stick cultures. Upon potato the development is very scanty. *360. BACILLUS GUTTATUS (Zimmermann). Found in the Chemnitz water supply. Morphology. — Bacilli with round ends, from 1 to 1.13 n long and 0.93 n broad ; at first solitary or in pairs, later united in chains containing several elements. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, actively mottle bacillus. Appears to form spherical spores (?). Grows best at the room temperature. Upon gelatin plates the deep colonies are small, grayish- white spheres; under a low power these are seen to be finely granular and have a gray or bluish-brown color. The superficial colonies appear as bluish-gray drops ; under the microscope a brownish shimmer is observed at the centre, while the sharply defined margins are colorless and are distinguished from the surrounding gelatin only by being less transpa- rent; later the outline sometimes becomes irregular. In gelatin stick cul- tures an irregular, bluish-white, shining layer is developed, the surface of NON-PATHOGENIC BACILLI. 071 which is frequently opalescent ; an abundant development occurs along the line of puncture, consisting1 of a series of spherical colonies : liquefaction oc- curs after a considerable time — four weeks. Upon the surface of agar a thin, grayish-white layer is developed along the iinpfstrich. Upon potato a tolerably abundant slimy, yellowish-green layer. 361. BACILLUS IMPLEXUS (Zimmermann). Found in the Chemnitz water supply. Morphology. — Bacilli with blunt ends, about 2.5 ju. long and 1.15 // broad; form long, jointed filaments. Biological Characters. — An aerobic, liquefying, non-motile bacillus. Forms oval spores about 1.6 n long and 0.95 /u thick. Grows at the room temperature — more quickly at 30° C. Upon gelatin stick cultures, at the end of twenty-four to thirty-six hours, whitish, punctiform colonies become visible; under a low power these are seen to be granular, opaque, and ir- regular in outline — more transparent toward the margin ; under a higher power (1 : 175) filaments are seen which are interwoven ; these grow out from the margins and again become contorted and interlaced ; at the end of three days the round colony sinks into the liquefied gelatin and appears then as an interlaced mass of fine white filaments. In gelatin stick cultures growth is visible along the line of puncture at the end of twenty -four hours, and at the end of seventy-two hours bundles of short threads radiate into the gelatin in all directions; liquefaction soon occurs, which quickly reaches the walls of the test tube at the surface; a thick, white layer forms upon the surface of the liquefied gelatin, and it is filled below with white flocculi. Upon the surface of agar a tolerably thick, white layer is formed which has a dull, shagreen-like surface and soon becomes wrinkled. Upon potato a yellowish or greenish- white, felt-like layer is formed. 362. BACILLUS PUNCTATUS (Zimmermann). Common in the Chemnitz water supply. Morphology. — Bacilli from 1 to 1.60 /i long — when in rapid multiplica- tion— and 0.77 f* broad; solitary, in pairs, or in chains containing several elements. Do not stain readily with the usual aniline colors. Biological Characters. — An aerobic, liquefying, actively motile bacillus. Spore formation not observed. Grows rapidly at the room temperature — still better at 30° C. Upon gelatin plates the colonies, on the third day, al- ready have a diameter of twelve millimetres and have caused a saucer- shaped liquefaction of the gelatin ; in the grayish-blue liquid whitish, punc- tiform collections of bacteria are seen, which are often united with each other by whitish strings. In gelatin stick cultures liquefaction occurs in . stocking shape; the liquefied gelatin is uniformly clouded, and an abundant white deposit accumulates at the bottom of the tube. Upon the ' surface of agar a delicate, gray, glistening layer with a perfectly smooth surface is developed. Upon potato an abundant brownish or flesh-colored layer soon extends over the entire surface; gradually this acquires a darker hue. 363. BACILLUS RADIATUS AQUATILIS (Zimmermann). Found in the Chemnitz water supply. Morphology. — Bacilli about 0.65 n broad and from 1 to 0.5 fj. long. Biological Characters. — An aerobic, liquefying bacillus. The shortest rods only exhibit slight movements. Spore formation not observed. Grows best at the room temperature Upon gelatin plates, at the end of two days, irregular, bluish-white colonies are seen, which often have a white point in the middle; under the microscope the colonies have a root-like appearance, being surrounded by simple and branching mycelial-like offshoots; at the G73 NON-PATHOGENIC BACILLI. end of three days these colonies are nearly spherical, and under the microscope resemble 1 ittle balls of wool, from the margins of which fine filaments are given otf ; finally they break through the gelatin and appear upon the surface as one or more small, transparent droplets. At the end of four days the gelatin is liquefied in saucer shape; in the middle is seen a yellowish- white or cream- colored mass ; around this a ring of a still deeper color, and from this deli- cate offshoots radiate toward the periphery of the saucer-shaped cavity. In gelatin stick cultures a thin, round layer appears upon the surface, which is often delicately wrinkled in a radial direction ; below development occurs along the line of puncture in the form of a slender funnel, and 011 the third day liquefaction commences ; a yellowish deposit collects at the bottom of the liquefied gelatin, which is clouded throughout and contains numerous flocculi of various dimensions. Upon the surface of agar a smooth, glis- tening layer is developed, which is yellowish-brown by transmitted light and pale bluish-green by reflected light. Upon potato an ochrous-yellow layer is formed, which may have a reddish-brown tint. 364. BACILLUS VERMICULOSUS (Zimmermann). Found in water. Morphology. — Bacilli with round ends, 1.5 ju long and about 0 85 /j. broad ; united in pairs or chains of three, or in long, worm-like filament in which segmentation is very indistinct; the bacilli are surrounded by a slimy envelope. Biological Characters. — An aerobic, liquefying bacillus. The smaller rods exhibit a rotatory or oscillating motion. Spore formation not observed. Grows slowly at the room temperature— better at 25° to 30° C. Upon gela- tin plates the deep colonies appear as small, white spheres ; under a low power they are seen to be nearly round, well defined, gray, and granular. The superficial colonies are flat, gray, drop-like discs; under a low power they are seen to be irregular in outline, with a wavy or bulging contour; the interior is marked with paler lines, which cross each other in various di- rections, dividing the colony into mesh-like fields; later this appearance is only seen at the margin. In gelatin stick cultures a pale-gray, viscid layer with finely notched margins is developed upon the surface ; after the fourth day, when this has attained a diameter of about seven millimetres, liquefac- tion commences and the superficial growth becomes depressed ; liquefaction extends slowly toward the walls of the tube, and very slowly in a down- ward direction, until about one-third of the gelatin is liquefied ; at the bot- tom of this clouded liquid an abundant reddish-gray sediment is formed. Upon the surface of agar a flat, smooth, glistening layer is developed ; later the surface of this is opalescent. Upon potato an abundant yellowish-gray, shining layer is formed. 365. BACILLUS AEROPHILUS (Liborius). Found as an accidental contamination, probably from the air. Morphology. — Slender rods of various lengths, about two-thirds as thick as Bacillus subtilis; frequently united in jointed filaments. Biological Characters. — A strictly aerobic, liquefying, non-motile ba- cillus. Forms oval spores. Grows in the usual culture media at the room temperature. Upon gelatin plates small, punctiform colonies are developed at the end of forty hours; under alow power these are seen to be oval or pear-shaped, well defined, and of a yellowish-gray color; liquefaction quick- ly occurs and the colonies do not increase materially in size. In gelatin stick cultures a broad, sac-like channel of liquefaction is formed, the upper part of the liquefied gelatin is opaque and yellowish-gray, while the lower portion is clearer simply contains suspended flocculi. Upon potato a yellowish layer is formed; this has a dull, smooth surface and a paraffin-like lustre; NON-PATHOGENIC BACILLI. (j^o later it becomes drier at the periphery, slightly granular, and has a striped appearance. 36G. BACILLUS MYCOIDES (Fltigge). Found in the soil and in water — common. Morphology. — Bacilli from 1.6 to 2.4 /* in length and about 0. 9 /* thick; usually in long filaments, which when stained are seen to be made up of separate elements ; as a rule, the long filaments are united in tangled bun- dles. Biological Characters. — An aerobic, liquefying, motile bacillus. Forms elliptical spores from 1.3 to 1.48 n long and from 0.74 to 0 9 u broad. Grows very rapidly — best at the room temperature. Upon gelatin plates the colo- nies first appear as cloudy, white spots, in which fine, white, interlaced threads are soon developed; very soon a mycelial-like branching occurs, giving the colony the appearance of the commencing growth of a microsco- pic fungus ; as long as the bundles of filaments remain beneath the surface of the gelatin they are delicate and slender, but when they reach the surface they spread out, lose their sharply defined outlines, and liquefy the gelatin. In gelatin stick cultures, at the end of eighteen hours, a superficial layer of about four millimetres in diameter has formed and already commences to sink in the gelatin; on the third day liquefaction has reached the walls of FIG. 233. FIG. 224. FIG. 223. — Bacillus mesentericus vulgatus, from a culture in bouillon, x 1,000. (VignalO FIG. 224.— Bacillus mesentericus vulgatus, from an agar culture. X 1,400. (Vignal ) the test tube, and from the line of puncture a branching, filamentous growth is given off; liquefaction extends downward from the surface, and after the tenth day the bacterial growth is seen suspended in the liquefied gelatin. Upon the surface of agar a mycelial-like, branching growth develops along the line of inoculation. Upon potato, at the end of twenty-four hours, a whitish-gray, shining layer, about three millimetres broad, is developed; at the end of forty-eight hours this has extended over the entire surface. 367. BACILLUS MESENTERICUS VULGATUS. Synonym. — Potato bacillus. First found upon potatoes — a common and widely distributed species ; found in milk by Loftier, in the Freiburg water supply by Tils, in the ali- mentary tract of man by Vignal. etc. Morphology. — Thick bacilli with round ends, from 1.2 to 3.5 ju. long; often united in pairs, or in chains containing several elements. Biological Characters. — An aerobic, liquefying bacillus. Forms spheri- cal spores. Grows rapidly at the room temperature— also in the incubating oven. Upon gelatin plates the colonies are at first almost transparent, blu- ish white, later with an opaque, white centre; the superficial colonies may attain a diameter of nearly one centimetre; they are somewhat sunken in the liquefied gelatin ; under a low power they are seen to be granular and have rough margins ; liquefaction of the gelatin is rapidly induced. In 674 NON-PATHOGENIC BACILLI. gelatin stick cultures liquefaction occurs at first in funnel form along the line of inoculation, and rapidly progresses until the gelatin is entirely lique- fied; numerous grayish flocculi are seen in the liquefied gelatin, and a deli- cate, grayish-white, wrinkled layer forms upon the surface; an abundant flocculent deposit collects at the bottom of the tube. Upon the surface of agar a dirty-white layer is developed. Upon potato a thick, wrinkled, white layer, extending over the entire surface, is quickly developed ; this penetrates the substance of the potato and is extremely viscid, stringing out into long threads when touched with the platinum needle. Spores are formed in the bacilli cultivated upon potato. Upon blood serum a white layer is developed and liquefaction of the medium occurs. Grows in bouil- lon containing one part in two hundred of hydrochloric acid (Vignal). In milk causes coagulation of the casein, which is subsequently dissolved and floats upon the surface as a slimy layer (Fliigge). 368. BACILLUS MESENTERICUS FUSCUS (Flugge). Found in the air, in hay dust, upon potato, and in water — a common and widely distributed species. Morphology. — Slender and short bacilli, often in pairs or in chains of four. Biological Characters. — An aerobic, liquefying, actively motile bacillus. Forms small, shining spores which are irregularly distributed in the rods. Grows at the room temperature. Upon gelatin plates forms spherical, whitish colonies, which under a low power are seen to have a well-defined contour; later these are surrounded by delicate offshoots, have a yellowish- brown color and a finely granular surface ; liquefaction of the gelatin quickly occurs. In gelatin stick cultures a whitish cloudiness is first seen along the line of puncture, and at the same time liquefaction commences near the surface in funnel form; this extends to the walls of the tube in from four to six days, and the liquefied gelatin contains numerous grayish- white flocculi. Upon potato, at the end of twenty-four hours, a smooth, yellowish layer is developed ; the surface of this soon becomes wrinkled and brown in color ; the growth is comparatively thin and does not penetrate deeply into the potato, as is the case with Bacillus mesentericus vulgatus ; it extends rapidly over the entire surface. 369. BACILLUS MEGATHERIUM (De Bary). First found upon the leaves of boiled cabbage. Morphology. — Bacilli with round ends, about 2.5 ft, thick and three to four times as long as broad ; often somewhat curved ; forms chains containing as many as ten elements; the protoplasm of the cells is granular; involution forms are common. Biological Characters. — An aerobic, liquefying, motile bacillus. The move- ments are peculiar, being slow and amoeboid in character. Forms long- oval spores which are nearly as long as the cells containing them, but not so broad. Grows best at the room tempe- rature— also in the incubating oven. Upon gelatinplates forms whitish, puiic- tiform colonies, which under the micro- scope are yellowish and irregular in form. The superficial colonies are some- times kidney-shaped or crescentic ; they cause the gelatin to be slowly liquefied. In gelatin stick cultures lique- FIG. 225.— Bacillus megatherium; a, chain of bacilli (x 250); 6, bacilli (x COO); c-f shows the development of (spores ; h-m shows the germination of spores; p, bacilli stained with solution of iodine. (De Bary.) NON-PATHOGENIC BACILLI. 675 faction occurs in funnelform along the upper portion of the line of inocu- lation and gradually extends downward; an abundant deposit is seen at the bottom of the tube, and the liquefied gelatin above is but slightly clouded ; no mycoderma forms upon the surface. Upon the surface of agar a whitish layer is developed which is easily separated from the culture medium. Upon potato a thick, cheesy, yellowish-white layer is quickly developed along the line of inoculation ; in potato cultures an abundant development of spores occurs and involution forms are common. 370. BACILLUS ALBUS PUTIDUS (De Bary). Found in water. Morphology. — Small bacilli, which grow out into filaments. Biological Characters. — An aerobic, liquefying, motile bacillus. Spore formation not observed. Grows rapidly at the room temperature. Upon gelatin plates forms thin, round colonies upon the surface, which under a low power are light-brown in color and are surrounded by a transparent aureole which at the end of four days has a diameter of five millimetres. In gelatin stick cultures development occurs both on the surface and along the line of puncture, producing rapid liquefaction of the gelatin; gelatin cul- tures give off an intense and disagreeable odor, like that of liquid manure. Upon the surface of agar a smeary layer is developed. Upon potato a slimy growth. 371. BACILLUS BRASSic^E (Pommer). Obtained from an infusion of cabbage leaves. Morphology. — Bacilli from 1.9 to 5.4/t Iongand0.91 tol 2 p thick; differ greatly in different culture media, forming sometimes twisted and tangled filaments, often spiral in form, and producing at times a network similar to that of Bacterium Zopfii ; the filaments are often segmented and slightly notched and bent at the points where the segments join. Biological Characters. — Anaerobic and facultative anaerobic, liquefy- ing, non-motile bacillus. Forms spores. Grows in the usual culture media at the room temperature. Upon gelatin plates forms colonies which resem- ble the mycelium of a mucor and cause liquefaction of the gelatin. In gela- tin stick cultures a branching, mycelial-like growth is seen along the line of puncture, in funnel form, and liquefaction of the gelatin quickly occurs. Upon the surface of agar a layer is formed consisting of spots surrounded by a dull cloudy appearance ; later these have a whitish or yellowish color ; under the microscope they are seen to consist of closely lying parallel fila- ments, which may run in a straight or serpentine direction, or may form circular and ellipsoidal figures. Along the line of puncture in agar cultures small, white colonies are developed, which are seen under the microscope to be made up of a confused mass of straight or curved, short filaments, and a network of filaments is given off from these. 372. BACILLUS BUTYRICUS OF HUEPPE. Found in imperfectly sterilized milk. Morphology. — Bacilli, often slightly curved, about 2.1 ft long and 0.38 M thick ; may grow out into filaments. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, actively motile bacillus. Forms, at 30° C., oval spores which are lo- cated in the centre of the rods. Grows at the room temperature — more rap- idly at 35° to 40° C. Upon gelatin plates deep-lying colonies of yellow color are developed, which cause rapid liquefaction of the gelatin and unite into coarsely granular, brown masses. In gelatin stick cultures liquefaction rapidly occurs along the entire line of puncture ; upon the surface of the G76 NON-PATHOGENIC BACILLI. liquefied gelatin a thin, whitish-gray, slightly wrinkled film is formed, and below this it is densely clouded and of a yellowish color. Upon the surface of agar a thin, smeary, yellowish layer is formed. Upon potato a fawn- colored, transparent layer, which is sometimes slightly wrinkled ; later the surface loses its transparency and becomes clouded. In milk coagulation occurs and the precipitated casein is subsequently dissolved; the milk ac- quires a bitter taste; produces butyric acid in milk. Bouillon cultures to which sulphuric acid has been added give off an acid distillate which has the odor of butyric acid. 373. BACILLUS GASOFORMANS (Eisenbergj. Found in water. Morphology. — Small bacilli. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, actively motile bacillus. Spore formation not observed. Grows rap- idly at the room temperature — not in the incubating oven at 37° C. Upon gelatin plates forms tolerably large, saucer-shaped cavities of liquefied gela- tin, the contents of which are finely granular and may contain gas bubbles. In gelatin stick cultures liquefaction rapidly occurs all along the line of puncture and gas bubbles form in the non liquefied gelatin. 374. BACILLUS CARABIFORMIS (Kaczynsky). Found in the stomach of dogs which had been fed exclusively on meat for three days. Morphology. — Short and slender bacilli. Biological Characters. — An aerobic, liquefying actively motile bacillus. Spore formation not observed. Grows rapidly at the room temperature. Upon gelatin plates foi-ms small colonies, from the centre of which are given off long processes with jagged outlines. In gelatin stick cultures liquefac- tion occurs along the line of puncture, and the liquefied gelatin has a green- ish-yellow color, while a whitish deposit accumulates at the bottom. Upon the surface of agar a yellowish-white layer. 375. BACILLUS GRAVEOLENS (Bordoni-Uffreduzzi). Found attached to scales of epidermis from between the toes of man. Morphology. — Bacilli 0.8 « long and of about the same breadth (micro- cocci ?) Biological Characters. — An aerobic, liquefying bacillus (?). Grows at the room temperature. Cultures have a disagreeable odor. Upon gelatin plates forms irregular, grayish-white spots which cause rapid liquefaction of the gelatin and give off a disagreeable odor like that from the feet; later the gelatin has a greenish-yellow color. Upon potato forms a grayish, stink- ing layer. Liquefies blood serum. 376. BACILLUS CAROTARUM (A. Koch). Obtained upon cooked carrots and sugar beets. Morphology. — Bacilli from 0.97 to 1.05 /u long; grows out into long, flexi- ble filaments — resembles Bacillus brassicae. Biological Characters. — An aerobic, liquefying, non-motile bacillus. Forms large oval spores. Grows best at 40° C. Upon gelatin plates forms round, white colonies upon the surface, which under a low power appear to be perforated with holes at the centre and are marked by fine lines. The deep colonies in the liquefied gelatin are spherical, with a sharply defined, smooth outline. In gelatin stick cultures a considerable growth occurs upon the surface ; very scanty development along the line of puncture. NON-PATHOGENIC BACILLI. 077 Upon the surface of cigar a white layer is formed. Upon potato a light- brown, circular layer is quickly developed; this at first has a dull and later a shining surface. 377. BACILLUS INFLATUS (A. Koch). Found as an accidental impurity — from the air ? Morphology. — Bacilli from 4.6 to 5,5 /* long and 0.6 to 0.8 // broad; often grow out into filaments. Biological Characters. — An aerobic, liquefying, actively motile bacillus. Forms two large spores in each rod. Grows at the room temperature. Upon gelatin plates forms spherical white colonies with folded margins, from which an outgrowth of delicate filaments is seen, similar to Bacillus alvei. In gelatin stick cultures development occurs all along the line of puncture, and short, hair-like filaments radiate into the gelatin, which is very slowly liquefied. Upon the surface of agar forms a thin, slimy, light-brown layer. In bouillon a thin, slimy, whitish film forms upon the surface, which sinks to the bottom when the tube is shaken. 378. BACILLUS RAMOSUS. Synonym.— "Wurtzel bacillus. Found in the soil and in water — common. Morphology. — Bacilli with round ends, about three times as long as broad — "about as long as Bacillus subtilis, but thicker " (Frankel) ; fre- quently united in long chains, or may grow out into filaments. Biological Characters. — Anaerobic, liquefy ing bacillus. Exhibits slight movements. Forms large oval spores which are located at the centre of the rods. Grows rapidly at the room temperature — also in the incubating oven. Upon gelatin plates, at the end of two days, veil-like, rapidly ex- tending, whitish colonies with ill-defined margins are developed; these re- semble the mycelium of a fungus ; under a low power the colonies are seen to consist of a network of twisted and interwoven filaments ; liquefaction oc- curs in the course of a few days. In gelatin stick cultures an outgrowth of branching filaments occurs along the line of puncture, looking ' ' like a sma1! fir tree turned upside down "; upon the surface a moist and shining, white layer is developed ; liquefaction soon occurs at the surface, and pro- gresses until the gelatin is entirely liquefied ; in old cultures a mycoderma is seen upon the surface, the gelatin below this is transparent, and at the bottom there is a deposit of crumbling, whitish flocculi. Upon the surface of agar a development of branching filaments occurs along the line of in- oculation; these form a moist- looking, grayish- white layer, which later be- comes thicker at the centre, and the root-like growth is only seen at the edges. Upon potato a whitish, smeary streak is developed along the line of inoculation ; the bacilli form numerous large oval spores when cultivated on potato. 379. BACILLUS SUBTILIS (Ehrenberg). Synonym. — Hay bacillus. A widely distributed species; found in hay infusion, in water, in the soil, etc. Morphology. — Bacilli with slightly rounded corners, from 4. 5 to 6 n long, and about three times as long as broad ; usually in chains consisting of several elements ; often grows out into very long filaments; possesses a ter- minal flagellum at each end of a single rod or at the two extremities of a chain. Biological Characters. — An aerobic, liquefying, motile bacillus. Forms large oval spores, which are located at the centre of the rods; these are about 1.2 long and 0.6 n broad. The movements are of a waddling charac- 58 678 NON-PATHOGENIC BACILLI. ter and not very rapid. Grows rapidly at the room temperature — better at 30' C. Development may occur at any temperature between 10° and 45° C. Spore formation is favored by a temperature of 30° C. The spores ger- minate most readily at 30° to 40° C. The exosporium is ruptured at one side of the long-oval spore, and the newly formed bacillus takes its exit from this opening in a direction perpendicular to the long axis of the reproductive element. The spores have great resistance to heat and to chemical agents. By boiling a hay infusion for a short time a pure culture may often be ob- tained, as other microorganisms present are killed, while the spores of Ba- cillus subtilis survive and subsequently germinate. Upon gelatin plates small, white colonies are first developed, which under the microscope are seen to be slightly granular, somewhat irregular in outline, and of a green- ish tint; development progresses very rapidly, and liquefaction of the sur- rounding gelatin is quickly induced, forming saucer-like cavities with gray- ish, translucent contents ; the central portion is white and opaque ; frequently 8 FIG. 226. — Bacillus subtilis; A, bacilli; B shows formation of spores; C shows the germina- tion of a spore, a, and development of a short rod, /, and subsequently of a longer filament, h. X 1,020. (Trazmowski.) a radiate appearance of the bacterial growth is observed ; under the micro- scope a dense, grayish-yellow central mass is seen, arid around this a tangled network of filaments and of rods undergoing the characteristic waddling movements ; at the -margins the filaments are seen to radiate into the non- liquefied gelatin, forming a crown-like aureole. In gelatin stick cultures liquefaction quickly occurs along the entire line of puncture ; later a dense, dry. and friable mycoderma forms upon the surface, and the gelatin below, which was at first filled with whitish flocculi, becomes clear as a result of their deposition at the bottom of the tube. Upon the surface of agar a wrinkled, white layer is developed which is easily lifted entire from the cul- ture medium. Blood serum is liquefied by this bacillus, and a wrinkled myco- derma forms upon the surface. Upon potato the entire surface is soon cov- ered with a cream-like, white layer, which in a short time contains an abun- dance of spores. NON-PATHOGENIC BACILLI. 079 380. BACILLUS SUBTILIS siMiLis (Sternberg). Obtained in cultures from the liver of a yellow-fever cadaver in Ha- vana, 1889. Morphology. — Bacilli with slightly rounded ends, from 2 to 4 ^ long and about 1 M thick ; grow out into jointed filaments. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile bacillus. Forms long-oval spores, which are centrally located and nearly as long as the cells in which they are developed. The motion is like that of Bacillus subtilis, viz. : a slow, to-and-fro, progressive movement. Upon gelatin plates the deep colonies, at the end of thirty-six hours at the room temperature, are spherical, finely granular, and pearl-like by reflected light; the superficial colonies have commenced to liquefy the gelatin at this time, and have a granular, white mass at the centre surrounded by a saucer- shaped cavity containing liquefied gelatin. In gelatin stick cultures lique- faction does not occur as rapidly as with Bacillus subtilis ; at the end of ten days at the room temperature the upper half of the gelatin is liquefied and small, pearl-like colonies are scattered along the line of puncture below; on the floor of the liquefied gelatin is a flocculent, white deposit and a thin my- coderma is seen upon the surface. On potato a dry, yellowish- white layer the size of a dime is formed at the end of forty -eight hours at 30° (J., and the bacilli, which grow out into long, jointed filaments, contain spores. Upon the surface of agar a thick, cream-white layer is formed in four or five days at the room temperature. In agar stick cultures there is a branch- ing growth along the upper portion of the line of puncture; in old agar cul- tures variously contorted involution forms are seen in the surface growth and but few spores are present. 381. BACILLUS LEPTOSPORUS (L. Klein). Obtained as an accidental contamination of a pure culture— from the air ? Morphology. — Resembles Bacillus subtilis; when cultivated at 35° C. forms short chains ; at 18° to 20° C. grows out into long, twisted and inter- laced filaments. Forms spores which are 0.6 u thick and 1.5/tlong; in vegetating, these first increase in thickness to 1 to 1.2 # — the thickness of the vegetative cells; the spores have a membranous envelope consisting of two layers, and are Fsurrounded by a jelly-like substance having a dull silvery lustre ; vegetation occurs at the same time fi'om both poles, and the mem- branous envelope is not ruptured and left intact after the emergence of the vegetative cell, as is the case with Bacillus subtilis, but is gradually dis- solved, or serves as the cell wall of the newly formed bacillus (?). Biological Characters. — Characters of growth in solid culture media not given. The motions are said to be peculiar, especially in filaments made up of four, eight, or sixteen elements ; one end of the chain is jerked hither and thither with a whip-like, convulsive motion, by which the elements are thrown into various and constantly changing angular figures. 382. BACILLUS SESSILIS (L. Klein). Found in the blood of a cow supposed to have died of anthrax. Morphology. — Resembles Bacillus subtilis, but is distinguished from this species by the germination of the spores. Biological Characters. — An aerobic, non-motile bacillus. In alkaline bouillon at 28° C. a diffuse cloudiness is seen on the second day, and on the fourth day a mycoderma is developed upon the surface, which contains spore- bearing bacilli. The spores resemble those of Bacillus subtilis, but the ger- mination of these reproductive elements is quite different and resembles more that of Bacillus butyricus. The vegetative cell emerges from a rup- ture at one of the poles of the exosporium, and a second rod appears to fol- 080 NON-PATHOGENIC BACILLI. low the first, pushing it before it; this appearance is, however, due to binary division of the vegetative cell before it has completely emerged from the spore membrane. Characters of growth in solid media not given. Not pathogenic for guinea-pigs. 383. BACILLUS ALLANTOIDES (L. Klein). Obtained as an accidental contamination from a culture of Bacillus me- gatherium— from the air ? Morphology. — Bacilli about 0.5 /< thick and three to four times as long as thick; form chains of four to eight elements, the members of which are rather widely separated from each other, but are firmly bound together by a jelly-like membrane which is somewhat narrower than the rods. The rods exhibit an intermittent to-aiid-fro movement ; subsequently they undergo segmentation into spherical elements which are surrounded by a jelly-like material and form sausage-shaped zoogloea masses. Characters of growth in solid media not determined. 384. BACILLUS OF SCHEUELEN. Found in cancerous tissues by Scheurleii ; upon the skin of healthy per- sons by Bordoni-Uff reduzzi (Bacillus epidermidis) ; in scales of epidermis from the nipple and in the mammae of healthy women by Rosenthal. Morphology. —Bacilli from 1.5 to 2.5 ju long and 0.5 u- broad. Biological Characters.— Anaerobic, liquefying, motile bacillus. Forms long-oval spores. Grows very slowly at the room temperature — best at 29° C. In gelatin stick cultures, at the end of eight to fourteen days, a funnel- sliaped cavity is formed near the surface, which is covered by a wrinkled, membranous layer, while but little liquid gelatin is contained in it. Upon the surface of agar, at 39° C., at the end of twelve hours a dull, fissured, colorless layer is developed. Upon potato, at the end of twelve to twenty- four hours in the incubating oven, the whole surface is covered with a yel- low, wrinkled layer ; the potato beneath this has a dirty-pink color. 385. BACILLUS LACT1S ALBUS (Loffler). Found in milk. Morphology. — Bacilli which average 3.4 « in length and 0.96 /* in thick- ness ; in milk grow out into long filaments ; resembles the bacillus of an- thrax. Biological Characters. — Anaerobic, liquefying, motile bacillus. Forms large spores. Grows slowly in the usual culture media at the room tempera- ture. In gelatin stick cultures liquefaction occurs slowly, and upon the surface of the liquefied medium a whitish layer, made up of interlaced fila- ments, is seen. Upon the surface of agar forms a tolerably thick layer with thinner margins. Upon potato a thin, dry, white layer. In milk causes coagulation and subsequent solution of the casein, and produces leu- cin and ty rosin. 38G. BACILLUS LIODERMOS (Loffler). Synonym. — Gummibacillus. Found in milk Morphology.— Resembles Bacillus mesentericus vulgatus— small, thick rods with round ends, in pairs or in jointed filaments. Biological Characters. — An aerobic, liquefying, motile bacillus. Forms spores. Grows rapidly at the room temperature. In gelatin stick cultures causes rapid liquefaction in funnel form ; the liquefied gelatin is slightly clouded and is soon covered with a whitish mycoderma. Upon the surface NON-PATHOGENIC BACILLI. 681 of agar a whitish, rosette-like layer is developed. Upon potato forms a transparent, gum-like layer, which later is thrown into thick, soft folds, similar to the growth of Bacillus mesentericus vulgatus. In milk causes co- agulation of the casein, winch at 30° C. is precipitated as a whitish, cloudy sediment, above which a clear serum is seen ; the casein is subsequently peptonized. 387. BACILLUS ULNA (Cohn). First described by Cohn, and subsequently found by Prazmowski in a solution of egg albumin. Morphology. — Bacilli of 1.5 to 2.2 ju in breadth and of various lengths — at least 3 M ; forms long, jointed filaments; forms spores which are 2.5 to 2.8 n long and 1 /* broad. Biological Characters. — An aerobic, motile bacillus which is said to grow only in albuminous solutions (?), in which it develops as cloudy masses wrhich collect at the surface and form a thick, dry mycoderma consisting of long, interlaced filaments in bundles and irregular aggregations ; does not give on a putrefactive odor. Imperfectly described. 388. BACILLUS ULNA OF VIGNAL. Found by Vignal in the salivary secretions of healthy persons, and sup- posed to correspond with Bacillus ulna of Cohn. Morphology. — Straight bacilli with round ends, about 2 u long ; often united in pairs, in which the elements are strongly adherent; rarely in chains containing more than two segments. Biological Characters . — An aerobic, liquefying bacillus. Spore forma- tion not observed. Motility not mentioned. Upon gela- tin plates, at the end of twenty-four hours, small, gray- ish superficial colonies are formed ; the centre of these is thicker than the periphery; under the microscope the peripheral zone is seen to be made up of fine interlaced filaments ; by the second or third day the colonies have increased considerably in size, and a small, grayish- white, opaque mass is seen, which is surrounded oy an extended zone of liquefied gelatin ; this is seen to con- sist of four secondary zones : next the central mass the gelatin is almost transparent, outside of this is a granular FIG. 227. — Bacillus zone, outside of this a grayish, less granular zone, and ulna of Vignal, from a finally an outer zone which is almost transparent. In culture in nutrient gelatin stick cultures, at the end of forty-eight hours, agar. x i.soo. (Tig- liquefaction in funnel form has occurred along the line nai.) of puncture ; the liquefied gelatin is transparent and con- tains suspended opaque, white flocculi, which accumulate at the bottom ; by the fourth day the gelatin is completely liquefied as far as the inoculating needle penetrated, and a whitish film is seen upon the surface. Upon the surface of agar a very adherent, white layer is developed which presents punctiform and linear depressions ; the agar below acquires a slightly brown- ish color. In bouillon the liquid remains transparent and acquires a yel- lowish tint, while a tolerably thick, fragile, smooth, white mycoderma is formed upon the surface ; rather a scanty white deposit accumulates at the bottom. Grows in acid bouillon— 1 :2,000 of hydrochloric acid. Upon po- tato a thin, grayish-white layer is formed at the end of forty-eight hours, upon the surface of which fine, slightly elevated lines cross each other in all directions ; sometimes this forms quite regular hexagonal figures Blood serum is liquefied by this bacillus. All the cultures have a disagreeable odor, similar to that given off by Bacillus pyogenes fcetidus. 682 NON-PATHOGENIC BACILLI. 389. BACILLUS LIQUEFACIENS (Eisenberg). Found ill water. Morphology. — Short, rather thick rods with round ends. Biological Characters. — An aerobic, liquefying, actively motile bacil- lus. Spore formation not observed. Grows rapidly at the room tempera- ture— not at 37° C. Upon gelatin plates forms round colonies with a smooth margin, which in the middle are white and slimy ; liquefaction com- mences in saucer shape and progresses rapidly; after a time a putrefactive odor is given off. In gelatin stick cultures development is rapid and lique- faction occurs in the form of a funnel, often like an air bubble; the line of puncture is filled with a whitish, granular mass. Upon the surface of agar forms a dirty-white layer. Upon potato a pale-yellow growth. 390. BACILLUS MAIDIS (Cuboni). Obtained from corn which had been soaked in water for eight hours at 30° C., and in the faeces of individuals suffering from pellagra. Morphology. — Bacilli with square ends, from 2 to 3 n long; solitary, in pairs, or in chains of three elements — seldom more ; resembles Bacillus me- sentericus f uscus in morphological and biological characters. Biological Characters. — An aerobic, liquefying, actively motile bacil- lus. Forms large oval spores located at the centre of the rods. Grows in the usual culture media at the room temperature — best at 26° to 30° C. Pro- duces in saccharine solutions acetic and butyric acids. Upon gelatin plates, at the end of twenty-four to thirty-six hours, grayish-white, puiictiform col- onies are developed below the surface, which have a yellowish color. The superficial colonies are thin and veil-like ; under a low power they are seen to be finely granular and have an irregularly folded margin ; later liquefac- tion commences and they have a radiate, finely striped margin ; the lique- faction progresses rapidly, forming shallow, saucer-like cavities. In gelatin stick cultures liquefaction occurs along the line of puncture within twenty- four hours in the form of a funnel or cylindrical tube, and rapidly extends to the walls of the test tube at the surface. Upon the surface of agar, at 34° to 36° C., a thin, dry, wrinkled layer covers the entire surface within twen- ty-four hours ; this is white or yellowish-white and easily detached. Upon potato a white, somewhat granular, and later finely wrinkled layer, which acquires a yellowish-brown color. Blood serum is liquefied by this bacillus. 391. PROTEUS SULFUREUS (Lindenborn). Found in water. Morphology. — Bacilli of various lengths, the average length being about 1.6 fJ- and the breadth 0.8 /*; often in long chains or filaments. Resembles Proteus vulgaris, and is perhaps identical with this species (Eisenberg). Biological Characters. — An aerobic, liquefying, motile bacillus. Spore formation not observed. Grows rapidly at the room temperature. Forms sulphuretted hydrogen. Upon gelatin plates forms white colonies, from which filamentous outgrowths upon the surface of the gelatin are given off ; these form "swimming islands"; later liquefaction occurs in the form of broad funnels with grayish -white contents. In gelatin stick cultures devel- opment occurs along the line of puncture, and liquefaction in funnel shape near the surface. Upon the surface of agar a thick, grayish-white layer is developed. Upon potato a slimy, grayish- white layer, which later acquires a brownish color. In milk an alkaline reaction is produced in the course of several weeks, and the casein is peptonized without previous precipitation ; the milk acquires a bitter taste and a yellowish color. 392. BACILLUS THERMOPHILUS (Miquel). Found in the contents of sewers, in the alimentary tract of man and ani- mals, and in the soil. NON-PATHOGENIC BACILLI. 683 Morphology.— Bacilli about 1 u thick, which form filaments of various lengths. ^Biological Characters. — Anaerobic, non-motile bacillus. Forms spores, which are located at the extremities of the rods. No growth in gelatin at the room temperature. Grows at temperatures between 42° and 72° C. . but not below 42° or above 72° ; grows best at 65° to 70° C. Upon the surface of agar, at 42° to 45° C., a white, disc shaped, prominent layer is formed — more abundant growth at 50° to 65° C. In bouillon, at 50° C., development occurs upon the surface in the form of a mycoderma which is easily broken up, and the liquid below is diffusely clouded. 393. BACILLUS TUMESCENS (Zopf). Found upon beets. Morphology. — Short bacilli, about 1.17 Abroad; grow out into irregu- larly bent and twisted filaments; resembles Bacillus megatherium. Biological Characters. — An aerobic, liquefying, motile bacillus. Move- ments slow. Forms oval spores. Grows rapidly at temperatures above 20° C. Upon gelatin plates forms round, superficial colonies which have a FIG. 228.— Bacillus buccalis maximus, after treatment with iodine solution. X 1,800. (Miller.) brownish-yellow color ; after several days the margins are no longer well defined, but have a fringed appearance, and liquefaction commences. Upon potato a thick, white, viscid layer, with somewhat folded margins, is de- veloped ; later this extends over the entire surface. 394. BACILLUS BUCCALIS MAXIMUS (Miller). Found in the mouth of man — common. Morphology. — "Isolated bacilli or threads, but much oftener tufts of threads, parallel to or crossing each other, from 30 to 150 /* long, and dis- tinctly articulated. The rods are from 2 to 10 /* long, sometimes even longer, and from 1 to 1.3 /* broad. This bacterium is therefore the largest occurring in the mouth; it has a very 1'egular contour and usually the same thickness throughout. Not all of the cells of this bacterium show the iodine reaction — a statement which applies equally well to most bacteria that turn blue on the addition of iodine. The majority of them, however, respond very distinctly to the test, becoming stained brown-violet, either through- out or only in isolated places." Biological Characters not determined. 084 NON-PATHOGENIC BACILLI. 395. LEPTOTHRIX BUCCALIS OF VIGNAL. Found by Vignal in the healthy human mouth — rare. Supposed by Vignal to be identical with Leptothrix buccalis of Robin ; but as the charac- ters of the microorganism receiving this name were not definitely deter- mined by Robin, such identification is impossible. It seems more probable that the large bacillus described by Miller (Bacillus buccalis maximus), which is found very commonly in the human mouth, but has not been cul- tivated, is the leptothrix of Robin. Morphology. — Bacilli from 1 to 1.5 IJL in breadth and varying greatly in length — from 1.6 to BO M; often united in long chains, or in filaments which are seen to be segmented when treated with staining, agents. This lepto- thrix is characterized by the presence of transverse partitions in the interior of the rods, seen only in stained preparations ; these are best seen in old rods, which are less deeply stained than those of more recent development; the partitions in these are more deeply stained than the remaining portion of the filament. Biological Characters. — An aerobic, liquefying bacillus. Spore forma- tion not observed. Grows very slowly in the usual culture media at tempe- ratures above 20° C. Upon gelatin plates, by the third or fourth day, small, round, grayish-white, projecting colonies are formed ; by the fifth or sixth day an irregular border of rounded festoons is developed; this is semi-trans- parent and presents depressions and ridges which are radial or parallel with the margin; the prominent centre remains opaque and of a grayish- white color; later this border extends considerably and the gelatin below it becomes liquefied. In gelatin stick cultures, at the end of forty-eight hours, a small mass is developed at the point of puncture, about two milli- metres in diameter, and a slender line of growth is visible along the track of the needle below ; by the fourth day the surface growth has a diameter of five to six millimetres, and below it a little transparent, liquefied gelatin is seen in a cup-shaped cavity ; this increases slowly in dimensions and the liquefied gela- tin remains clear, while a bluish mycoderma is seen upon the surface; the growth along the line of puncture remains scanty, but is a little more abun- dant above in contact with the cup shaped cavity; by the eighth day this ex- tends to the walls of the tube; by the twelfth day the cup form has disap- peared and the gelatin is liquefied to a depth of about one centimetre ; it is still covered with a bluish membranous layer, and a scanty whitish deposit of leptothrix filaments is seen at the bottom of the liquefied gelatin Upon the surface of agar, at the end of twenty-four hours at 36° to 38° C., a slightly wrinkled, transparent, white layer is developed; later this is still more wrinkled, dry, and of a transparent yellow color; it is easily broken up. In neutral bouillon a slight cloudiness is produced and a scanty white precipitate is formed at the bottom of the tube; no mycoderma develops upon the surface. Upon potato a layer is developed which is of a dirty-white color at the centre and of a purer white near the margins. 396. BACILLUS b OF VIGNAL. Obtained quite frequently by Vignal in cultures from healthy buccal se- cretions. Morphology. — Bacilli with square ends, straight or slightly curved, about 0.5 n in diameter and varying greatly in length — from 1.5 to6.5//; often united in chains. Biological Characters. — An aerobic, liquefying bacillus. Spore forma- tion not observed. Motility not mentioned. Grows rather slowly at the room temperature— more abundantly at 37° C. Upon gelatin plates, at the end of twenty-four hours at a temperature of 18' to 20° C., prominent, small, grayish-white colonies are developed upon the surface; at the end of forty-eight hours a collarette with irregularly festooned margins is devel- oped around this central mass ; this is thinner and much more transparent XOX-PATHOGENIC BACILLI. 685 than the central portion of the colony ; under a low power it is seen to be formed of an innumerable series of skein-like bundles, arranged side by side and more or less twisted, which proceed from the central mass. In gelatin stick cultures, at the end of forty-eight hours, a small, flat mass is developed at the point of puncture, and a scanty growth is seen along the line of inoculation. On the fourth day the superficial growth covers the entire surface, it is translucent by transmitted light and white by reflected light; by the sixth day the gelatin is liquefied to a depth of one centimetre below the superficial growth ; the liquefied gelatin remains transparent ; upon the surface is seen a white, membranous layer, and at the bottom a rather scanty white deposit ; liquefaction slowly extends downward, and by the twelfth day has reached a level corresponding with the bottom of the line of puncture. Upon the surface of agar, at the end of twenty-four hours at 36° to 38° C., a dull- white layer, having a thickness of about one milli- metre, is developed ; this is easily broken up with the platinum needle. In neutral bouillon a slight cloudiness is quickly produced, a thin film forms upon the surface, and a scanty white precipitate at the bottom of the tube. Upon potato, at the end of forty-eight hours, a layer the size of a five-franc piece is developed, which has a pale-pink color and a rough surface — resem- bling a lichen. Blood serum is liquefied rather rapidly, and acquires a brownish color, while an abundant white precipitate accumulates at the bottom of the tube. 397. BACILLUS / OP VIGNAL. Found by Yignal in the salivary secretions of healthy persons. Morphology. — Bacilli with slightly rounded ends, from 0.8 to 1.2 u in length when cultivated upon agar, and from 1.4 to 2.4 u when cultivated in neutral bouillon ; usually solitary, occasionally united in short chains. Biological Characters. — An aerobic, liquefying bacillus. Spore forma- tion not observed. Motility not mentioned. Grows rather slowly at the room temperature — more rapidly at 37° C. Upon gelatin plates, at the end of forty- eight hours, small, projecting, opaque, white colonies are developed ; at' the end of four days the colonies are seen as conical, opaque, white masses, di- vided into about twenty segments by grooves which start from the summit. In gelatin stick cultures a small but prominent white mass is seen at the point of puncture, and a scanty line of development along the track of the inoculating needle; on the fourth day the surface growth has extended nearly to the walls of the tube, and just below this some fine branches are given off from the line of growth ; the sixth day the entire surface is covei'ed and the gelatin below is liquefied for a short distance; the eighth day the liquefaction has extended downward, and the solid gelatin below has a clouded appearance owing to the development of a quantity of small, white colonies ; by the twelfth day the liquefied gelatin has a depth of about two centimetres, a shining, white mycoderma is seen upon the surface, a white deposit at the bottom, and below this numerous small colonies in the solid gelatin. Upon the surface of agar very adherent, white colonies are formed, which later extend to form a transparent, white membrane. In neutral bouillon a slight cloudiness is produced, and a very scanty, whitish deposit is seen at the bottom of the tube. Does not develop well in acid bouillon. Upon blood serum a whitish layer is formed, which later becomes semi-transparent and causes a slow liquefaction of the medium. Uponpo- tato, at the end of forty-eight hours, a layer is developed which has a vel- vety appearance in the centre, and a yellowish or brownish-white color ; by the end of forty -eight hours this layer is as large as a five-franc piece. 398. BACILLUS BUCCALIS FORTUITUS. Synonym. — Bacillus./, Vignal. Found by Vignal in the salivary secretions of healthy persons. 59 686 NON-PATHOGENIC BACILLI. Morphology. — Bacilli with square ends, from 1.4 to 3/< long; often united in pairs, the elements of which may be joined at an angle of greater or less degree. Biological Characters. — An aerobic, liquefying bacillus. Spore for^ mation not observed: Motility not mentioned. Grows at the room tempera- ture in the usual culture media. Upon gelatin plates, at the end of forty- eight hours, small, round colonies are developed, which increase considerably in thickness and diameter, and by the fourth or fifth day have caused lique- faction of the sm*roundiiig gelatin. In gelatin stick cultures, at the end of forty-eight hours, a small mass has formed at the point of inoculation, and a scanty line of development is seen along the track of the inoculating needle ; by the fourth day the growth has extended over the entire surface and pre- sents a decided prominence at the centre ; the gelatin below is liquefied and remains transparent, with some opaque, white flocculi in suspension ; by the twelfth day the liquefaction extends to a depth of two centimetres, and a yellowish- white, abundant deposit is seen at the bottom. Upon the surface of agar at 36° to 38° C., small, white, opaque colonies are developed, which present a small, nipple-like projection at the centre. In neutral bouillon a diffuse cloudiness is produced ; a thick, dull- white layer forms upon the sur- face, and an abundant dull-white deposit is seen at the bottom of the tube. Does not grow well in acid bouillon. Upon potato a rather thick growth is developed, which extends slowly and acquires a slightly pinkish tint. . 399. BACILLUS HAVANIENSIS LIQUEFACIENS (Sternberg). Obtained in cultures from the sui'face of the body of patients in the char- ity hospital at Havana. Morphology. — Bacilli with round ends, about 0.8 /* thick and varying greatly in length — from 1.2 to 5 /< ; solitary, in pairs, or may grow out into long filaments. Biological Characters. — An aerobic, liquefying, motile bacillus. Spore formation not observed. Grows at the room temperature — better at 37° C. Upon gelatin plates, at the end of twenty-four hours at 22° C., round colo- nies are developed which have a milky opacity and are surrounded by a transparent marginal zone with irregular margins; under the microscope these colonies are seen to be finely granular ; at the end of twenty-four hours liquefaction commences. In gelatin stick cultures liquefaction occurs along the entire line of puncture; at the end of four days the liquefied gela- tin is clouded throughout ; in old cultures it is quite transparent, and a slight flocculent deposit is seen at the bottom of the tube. Upon the surface of agar, at the end of two weeks, a thin, pale-brown layer is developed. No growth upon potato. Not pathogenic for rabbits. . 400. BACILLUS LIQUEFACIENS COMMUNIS (Sternberg). Obtained in cultures from the faeces of yellow-fever patients at Decatur, Ala. (1888). Morphology. — Bacilli with round ends, from 1 to 2 {t long and about 0.7 /< thick ; solitary or in pairs. Biological Characters. — Anaerobic and facultative anaerobic, liquefy- ing, actively motile bacillus. Grows in the usual culture media at the room temperature, also at comparatively low temperatures, and in th i incubating- oven at 37° C. Spore formation not observed. Grows in an acid medium — 1 : 500 of hydrochloric acid. In gelatin stick cultures liquefaction occurs rapidly in the form of a purse. On potato, at the end of t\vo weeks, an ir- regular, corrugated layer is developed which has a pinkish color. Not pathogenic for rabbits. NOX-PATHOGENIC BACILLI. G87 E. Strictly Anaerobic Bacilli. •401. BACILLUS MUSCOIDES (Liborius). Found in the soil by inoculations in mice, also in old cheese, and in the excrement of cattle. Morphology.— Bacilli about 1 ju thick, with slight inclination to form fila- ments. Biological Characters. — An anaerobic, non-liquefying, motile bacillus. Forms short-oval spores, which are located at the ends of the rods. In gela- tin and in agar forms colonies which give off delicate, branching, moss-like offshoots. In gelatin stick cultures a delicate, branching growth is given off from the lower two-thirds of the line of puncture. FIG. 229.— Bacillus muscoides; colony in nutrient gelatin, x 80. (Liborius.) 402. BACILLUS SOLIDUS (Luderitz). Obtained from garden earth by inoculation into mice and guinea-pigs. Morphology. — Bacilli of from 1 to 10 n in length — average 4 5 ju— and 0. 5 n thick ; the longer bacilli consist of two segments, but regular filaments are not formed. Biological Characters. — An anaerobic, non-liquefying, motile bacillus. Movements tolerably active, pendulous and progressive. In old gelatin cultures some of the bacilli contain, at one or both ends, small refractive bodies which are probably spores. Grows at the room temperature. In nutrient gelatin containing grape sugar, at the end of two days, punctiform colonies are developed which later attain the size of a poppy seed ; they are spherical and have smooth outlines; the gelatin is not liquefied, but gas bub- bles are formed, and a disagreeable odor is given off by the cultures, resem- bling decomposing perspiration from the feet. In the absence of grape sugar the development is scanty and there is no development of gas. In nutrient agar the colonies are but little larger, they are transparent, and under a low power resemble little flocculi of cotton. In blood serum devel- opment occurs only in the middle and lower part of the line of puncture. G88 NON-PATHOGENIC BACILLI. In bouillon, when oxygen is excluded, an abundant development occurs at 37° C. at the end of twenty-four hours; the bouillon becomes clouded and gives off stinking gases. 403. BACILLUS POLYPIFORMIS (Liborius). Found in the soil by inoculations in mice, in the excrement, of cows, etc Morphology. — Slender bacilli of various lengths, a little more than 1 n thick ; do not form filaments. Biological Characters. — An anaerobic, non-liquefying bacillus ; ex- hibits very slight independent motion. Forms long-oval spores, which oc- cupy one-half to two-thirds of the length o£ the rods, and the diameter of which is a little more than that of the bacilli. The colonies in gelatin con- sist of small, yellowish masses with irregular, broad, flap-like projections ; under the microscope these are seen to consist of variously twisted and bent outgrowths, varying in thickness, which are given off in all directions like the tentacles of a polyp; later these outgrowths increase in thickness. In agar white colonies of irregular form, the size of a pin's head, are developed ; under a low power these appear as finely granular, brownish, mulberry -like FIG. 230.— Bacillus polypiformis; colony in nutrient gelatin, x 80. (Liborius.) masses. In blood serum a diffuse cloudiness is developed at the bottom of the line of puncture. The addition of two per cent of sugar to culture media favors the growth of this bacillus ; no gas is developed as a result of its growth in such a medium. 404. BACILLUS BUTYRICUS (Prazmowski). Synonyms. — Bacillus amylobacter; Clostridium butyricum. Found in putrefying vegetable infusions, in old cheese and milk, in the soil, etc. Morphology. —Bacilli with round ends, from 3 to 10 n long and about 1 u thick; frequently associated in chains ; also in filaments not apparently seg- mented. The rods assume a spindle or tadpole form when spore formation is about to occur, and may then be from 1.8 to 2.6 /Uhick; the spores are oval, about 2 to 2.5 // long and 1 /u broad, and are located centrally or at one extremity. When the spore vegetates a rupture of the exosporium occurs at one of the poles, and the bacillus grows out in the direction of the long axis of the reproductive element ; the empty spore case retains its form after the vegetative cell is extruded. Biological Characters. — An anaerobic, motile bacillus. Forms large oval spores. Grows at the room temperature, in the absence of oxygen. The NON-PATHOGENIC BACILLI. 689 spores are destroyed by a temperature of 100° C. maintained for five minutes. In solutions containing- starch, sugar, dextrin, or salts of lactic acid this ba- cillus produces a considerable quantity of butyric acid, and at the same time carbon dioxide and hydrogen are given off. The most favorable tempera- ture for its development is from 35 J to 40° C. According to Fitz, it does not cause the coagulation of sterilized milk, but the casein is slowly peptonized. This bacillus also causes the decomposition of cellulose, producing hydrogen and carbon dioxide, or methane, carbon dioxide, and sulphuretted hydrogen, according to the composition of the culture medium (Tappeiner). It appears to be the bacillus which usually gives rise to butyric acid fermentation in milk which has been kept for some time, and also in cheese ; this occurs in milk after the lactic acid fermentation, which is due to aerobic bacilli, and especially to Bacillus acidi lactici. The characters of growth in solid media have not been determined. A peculiar staining reaction occurs when this D FIG. 231.— Bacillus butyricus; A, single bacilli; B, chains and filament; C, spore formation, showing " clostridium form " ; D, germination of a spore — a to i. X 1,020. (Prazmowski.) bacillus from cultures containing starch or cellulose is treated with iodine solution ; the protoplasm of the cells acquires a blue or dark-violet color, the younger cells being of a pure blue ; in some cases an oblique zone of blue only is seen, in others the entire cell is stained. 405. CLOSTRIDIUM FCETIDUM (Liborius). Obtained from garden earth by inoculations in mice, etc. Morphology. — Bacilli of various lengths and about 1 u thick ; sometimes grow out into filaments. Forms large oval spores, located centrally or at one end of the rod ; these are of greater diameter than the bacilli before spore formation, and cause the rods to have a spindle (clostridium) form, or oc- casionally an expanded extremity ; resembles Bacillus butyricus. Biological Characters. — An anaerobic, liquefying, actively motile ba- cillus. Forms spores. Grows in the usual culture media, in the absence of 690 NON-PATHOGENIC BACILLI. oxygen, at the room temperature. Upon cigar plates, in the absence of air, small, yellowish-white colonies are developed which are irregular in form and vary considerably in size; these are surrounded by outgrowths which are more compact and less branched than similar colonies of the bacillus of malignant oedema. In nutrient gelatin irregular, spherical colonies are de- veloped, which rapidly cause liquefaction of the surrounding gelatin, and spherical cavities filled with a deeply clouded liquid are formed. In blood serum a homogeneous cloudiness is seen in the vicinity of the line of punc- ture, and at the lower part of this a few isolated, irregularly branching col- onies are developed. A considerable amount of gas is formed in the cultures in various media ; this appears as scattered bubbles, and also causes a split- ting-up of the culture medium ; in gelatin cultures liquefaction gradually extends upward until it reaches the surface. The gases evolved have an ex- tremely disagreeable odor; they are produced more abundantly in culture media containing sugar. 406. BACILLUS LIQUEFACIENS MAGNUS (Luderitz). Found in garden earth by inoculations in mice and guinea-pigs. Morphology. — Bacilli with slightly rounded ends, straight or slightly curved, from 3 to 6 u long and from 0.8 to 1.1 /z thick; may grow out into long, flexible filaments. Biological Characters. — A a anaerobic, lique- fying, motile bacillus. Grows rapidly at the room temperature, in the absence of oxygen, in the usual culture media. Forms long-oval spores, from 1 to 2 /* long and 0.8 n broad; these are located at or near the middle of bacilli from 4 to 6 /* long — not in the long fila- ments unless these are segmented. When culti- vated in a medium containing grape sugar, the spore-bearing bacilli are stained violet with iodine solution — sometimes pale and in places only, at others throughout. In gelatin cultures development is already evident, at the end of twenty -four hours, one or two centimetres below the surface, as scattered punctiform colonies ; at the end of two days these are one to two milli- metres in diameter and have smooth margins, with transparent, dull-white contents; at the end of three or four days the gelatin is usually entirely liquefied ; the liquefaction extends upward, and the liquefied gela- tin, which at first is clouded, becomes clear, while a slimy, whitish deposit is seen at the bottom of the tube. In cultures containing grape sugar some gas is developed ; this has a disagreeable odor like that of old cheese. In gelatin stick cultures liquefaction begins along the line of puncture, from 1 to 1.5 centimetres below the surface, within forty-eight hours; and a sau- sage-shaped collection of liquefied gelatin, of a dull-white, or silver-gray color, is seen. In long agar cultures development is rapid, and colonies are seen nearer the surface than in similar gelatin cultures — often within five milli- metres; the young colonies have a delicately branched, moss-like appear- ance; in older colonies the branching is coarser. In stick cultures in blood serum, in the incubating oven, at the end of twenty four hours a simple line of growth is seen along the lower portion of the track of the inoculat- ing needle ; later numerous lateral offshoots give the growth a brush-like appearance; the blood serum is soon liquefied and foul-smelling gases are given off. Not pathogenic for mice or for guinea-pigs. 407. BACILLUS LIQUEFACIENS PARVUS (Luderitz). Obtained from garden earth by inoculations in mice and guinea-pigs. FIG. 232. — Bacillus liquefa- ciens magnus ; young colonies in nutrient gelatin, x 60. (Lude- ritz.) NON-PATHOGENIC BACILLI. GUI Morphology. — Bacilli from 2 to 5 jn long and 0.5 to 0.7 /* thick; often grow out into long, flexible filaments. Biological Characters. — An anaerobic, liquefying, non-motile bacilhis. Grows rapidly at the room temperature, in the absence of oxygen, in the usual culture media. In bouillon cultures the thickness of many of the rods is increased to 1 or 1.2 /*, and in these small, round, refractive bodies are seen at the ends, or in a linear series, which are probably spores. The addition of two per cent of grape sugar to culture media is favorable to the growth of this bacillus. In gelatin cultures development occurs at a distance of one to two centimetres below the surface ; at the end of two days, at 20° C., punctiform colonies are developed; these, when not too close together, attain a diameter of 2 to 2.5 millimetres; they are filled above with trans- parent liquid gelatin and below with a whitish mass of bacteria. In gelatin stick cultures a row of spherical colonies is developed along the line of puncture ; these increase in diameter from above downward. In nu- trient agar opaque colonies are developed within 0.5 to 1 millimetre of the surface ; these are tabular, almond- shaped, or whetstone-shaped, and have a tolerably smooth contour at first ; later they are irregular in out- line. Blood serum is slowly liquefied by this bacillus. In agar cultures a few gas bubbles are developed, and also in blood serum. In bouillon a decided putrefac- tive odor is developed.. Not pathogenic for mice. FIG. 233.— Bacillus li- quefaciens parvus; col- ony in nutrient agar. X 60. (Luderitz.) 408. BACILLUS RADIATUS (Liideritz). Obtained from garden earth by inoculations in mice and guinea-pigs. Morphology. — Bacilli with round ends, from 4 to 7 /* long and about 0.8 H thick ; often grow out into long filaments, which are seen to be composed of separate segments. Biological Characters. — An anaerobic, liquefying, motile bacillus. Move- ments are less active than in Bacillus liquefaciens magnus, and only ob- served in specimens from a recent culture. Spores are developed in the sin- gle rods only— not in the filaments; they are from 1.2 to 2 n long and 0.8 to 0.9 n thick, and are centrally located in the rods; the spore-bearing bacilli are somewhat thicker than others, but do not acquire a spin- dle shape. Grows in the usual culture media at the room temperature when oxygen is ex- cluded. The addition of two per cent of grape sugar is favorable to the development of this bacillus. In test-tube cultures in nu- trient gelatin, at 22° C., colonies are developed to within one or two centimetres of the sur- face ; when these are numerous the gelatin is penetrated throughout, below the limit men- tioned, with numerous glistening threads, and within two or three days is liquefied; some gas accumulates beneath the layer of solid gelatin above ; later liquefaction extends to the surface, and the liquefied gelatin, which at first is clouded, gradually becomes transparent from a deposition of the suspended bacilli. When only a few colonies are developed the growth resembles that of the mycelium of a fungus, the margin consist- ing of interlaced filaments, while the centre shows commencing liquefac- tion; new centres of development are formed by the filaments wnich pene- trate the gelatin, and this is soon completely permeated and at the same FIG. 234. — Bacillus radiatius; young colony in nutrient gelatin. X 60. (Luderitz.) 002 ^OX-PATHOGENIC BACILLI. time liquefied ; a similar appearance is observed when gelatin plates are pre- pared arid kept in an atmosphere of hydrogen. When successive cultures are made in gelatin tubes the bacillus gradually loses its vigor of growth and the colonies have a different appearance ; the thread-like outgrowths are no longer seen, or are developed to a slighter extent, and the spherical or balloon-shaped colonies are filled with a clouded liquid, while a thick sedi- ment is formed. In gelatin stick cultures development occurs along the line of puncture to within one or two centimetres of the surface; at the end of two days numerous branching filaments are given off, which give the growth the appearance of a hairy caterpillar. In nutrient agar stick cul- FIG. 285. FIG. 236. FIG. 287. FIG. 238. FIG. 239. FIG. 235.— Bacillus liquefaciens magnus; stick culture in nutrient gelatin. (Luderitz.) FIG. 236.— Bacillus radiatus ; stick culture in nutrient gelatin. (Luderitz.) FIG. 237.— Bacillus spinosus ; stick culture in nutrient gelatin. (Luderitz ) FIG. 238.— Bacillus liquefaciens parvus; stick culture in jiutrient gelatin. (Luderitz.) FIG . 239.— Clostridium f oetidum ; culture in nutrient agar, under oil. (Liborius.) tures growth occurs to within one centimetre of the surface; it is branching in character and resembles that of Bacillus liquefaciens magnus, but the filaments are more delicate; later the principal branches are thicker, and they are surrounded by a denser mass of fine filaments, among which some thick outgrowths are also seen. Blood serum is rapidly liquefied by this NON-PATHOGENIC BACILLI. 693 bacillus. The gases which are developed in cultures containing sugar have a disagreeable odor like that of cheese and onions ; cultures in blood serum give off a putrefactive odor. Not pathogenic for mice. 409. BACILLUS SPINOSUS (Liideritz). Obtained from garden earth by inoculations into mice and guinea-pigs. Morphology. — Bacilli with round ends, straight or curved, about 0.6 n thick and of various lengths — usually from 3 to 8 // ; may grow out into long, segmented, flexible filaments. Spores are formed in the single rods only and are long- oval in form ; they are located toward one or the other ex- tremity of the rod, which is from 1 to 1.2 fi broad at the point where they are developed, and has more strongly rounded or pointed ends than those not containing spores. Biological Characters. — An anaerobic, liquefying, motile bacillus. Grows in the usual culture media, with the addition of two per cent of grape sugar, at the room temperature, in the absence of oxygen. In gelatin tube cultures, at the end of twodays at 20° C., colonies are formed in the deepest portion of the gelatin to within 3 or 3.5 centimetres of the surface; these are irregular in form and the size of a poppy- or hemp-seed ; they contain liquefied gelatin, and as they increase in size the gray, shining contents are seen to contain a radiating, whitish growth ; this is most marked in the deep-lying colonies, in which the radiating growth extends to the non- liquefied gelatin around the colony; later the colonies become confluent and the liquefaction slowly extends upward. In agar tubes opaque colonies are developed which may attain a dia- meter of four millimetres ; under the microscope they are seen to be made up of numerous inter- FIG. 240.-Bacillus spinosus; laced filaments which in old colonies are only colony in nutrient agar, one seen in the marginal zone ; or they may be com- day old. x 60. (Luderitz.) posed of thick, knotty masses. Blood serum is liquefied by this bacillus. Gas is formed in all of the cultures, and in those containing sugar it has an odor which is compared to a mixture of Swiss cheese and fermented raspberry juice. Not pathogenic for mice or guinea- pigs. 410. BACILLUS ANAEROBICUS LIQUEFACIENS (Sternberg). Obtained in anaerobic cultures from the contents of the intestine of a yellow-fever cadaver. Morphology. — Slender bacilli, about 0.6 /* in diameter and three to five times as long as broad; often in pairs: grows out into long filaments. Biological Characters. — An anaerobic, liquefying, non-motile bacillus. Forms spores. Grows in the usual culture media at the room temperature, when oxygen is excluded. In gelatin roll tubes (Esmarch's) filled with hydrogen, granular, white colonies are developed, around which the gelatin is liquefied. In a long stick culture in nutrient agar it grows along the line of puncture nearly to the surface. Pathogenic power not tested. 60 X. NOX-PATHOGENIC SPIRILLA. 411. SPIRILLUM SPUTIGENUM (Miller). Found in the mouths of healthy individuals, especially along the margin of inflamed gums in neglected mouths— common. Moi'phology. — Curved rods which resemble the "commas" of Spirillum choleras Asiaticae ; these are frequently united in S-form or in short, spiral filaments — actively motile. This spirillum was supposed by Lewis to be identical with Koch's " comma bacillus," but Miller has shown that it differs essentially from this, inasmuch as it fails to grow in the usual culture media. He says : ' 'Although I obtained nearly pure culture material in several cases and used all possible kinds of nutrient media, I did not succeed in producing the slightest growth of this fungus." 412. SPIRILLUM DENTIUM. Synonyms. — Spirochaete denticola; Spirochaete dentium. Found in the mouths of healthy individuals, ' ' under the margins of the Fl°- 841- FIG. 242. FIG. 243. FIG. 241.— "Spirochsete dentium." x 1,100. (Miller.) FIG. 242.—" Spirochsete plicatilis (6), Vibrio rugula (a), and other bacteria." X 500. (Fluggej FIG. 243.— "Spirillum dentium." x 500. (Flugge.) ms, when they are covered with a dirty deposit and slightly inflamed " Morphology.— Long, flexible, spiral filaments of unequal thickness and irregular spiral windings; from eight to twenty-five V long. Biological Characters.— Not known, as all attempts to cultivate this spirillum have been unsuccessful. 413. SPIRILLUM PLICATILE. %no?i2/?n.— Spirochaete plicatilis. Found in swamp water containing decomposing algae. NON-PATHOGENIC SPIRILLA. 695 Morphology. — Long, flexible, spiral filaments. The spiral curves are close and regular, but the extremely long filaments present secondary, wave- like curves which are not uniform. The ends of the filaments are blunt. They may attain a length of one hundred to two hundred jn ; the movements are extremely active. Biological Characters not determined. 414. VIBRIO RUGULA (Mliller). Found in swamp water, in faeces, and in tartar from the teeth. Morphology. — Kod-shaped cells from 6 to 8 /* long and from 0.5 to 2.5 /* thick, slightly bent or with a flat spiral curve; sometimes united in long chains. Spores are developed at one extremity of the slightly curved rods ; they are spherical, and the end of the rods containing them presents a sphe- rical enlargement, giving them a comma-like appearance ; terminal flagella have been demonstrated by Koch. a b c FIG. 244.— Vibrio rugula; a, young rods; 6, thicker rods; c, spore-bearing rods. (Prazmowski.) X 1,020. Biological Characters. — The earlier investigators did not determine the characters of growth of Vibrio rugula, but Vignal (1886) has succeeded in cultivating a strictly anaerobic microoganism, obtained by him from the human mouth, which he has described under the same name, although he is not entirely satisfied that it is identical with Vibrio rugula of Prazmowski and previous observers. The biological characters, as determined by the au- thor named, are as follows: An anaerobic, liquefying, motile "vibrio." Movements rotary and progressive. Forms terminal spores, which are rather pear-shaped than round. Upon gelatin plates, in the absence of oxygen, it forms spherical, opaque, yellowish colonies; about the third day the ori- ginal colony is surrounded by a zone of transparent, liquefied gelatin. In gelatin stick cultures, in an atmosphere of hydrogen, development occurs along the line of puncture within twenty-four hours, and a small mass is formed at the point of puncture; at the end of forty-eight hours liquefaction occurs in funnel form ; the liquefied gelatin is clouded, white, and opaque ; later it becomes transparent. In long stick cultures in nutrient gelatin, without exclusion of the air, development occurs only at the bottom of the line of puncture. Upon the surface of agar a white, slightly wrinkled pel- licle, covering the entire surface, is formed in an atmosphere of hydrogen. Development occurs in neutral or in acid bouillon, which becomes diffusely clouded, and from which an abundant white sediment is deposited. Upon, the surface of blood serum a white layer is developed and liquefaction of the medium occurs. Upon potato a wrinkled, white layer is rapidly developed; 696 NON- PATHOGENIC SPIRILLA. later tli is acquires a yellowish tiut; the growth penetrates the potato to a considerable depth. Much gas is formed in all of the cultures, and they give off a strong faecal odor. According to Prazmowski, Vibrio rugula causes an energetic decomposition of cellulose. 415. SPIRILLUM VOLUTANS (Ehrenberg). Found in swamp water, etc. Morphology. — Spiral filaments with round and somewhat pointed ends, from 1.5 to 2 /u thick and 25 to 30 //long; each filament consists of two and one-half to three and one-half — rarely six or seven — spiral turns which are from 9 to 13 p long; a single long, whip-like flagellum at each extremity ; the protoplasm contains numerous opaque granules. Exhibits active rotary and progressive movements — sometimes motionless. Biological Characters not determined. FIG. 245. FIG. 246. FIG. 245.— Spirillum volutans. X 600. (Cohn.) FIG. 246.— Spirillum sanguineum. X 600. (Cohn.) 416. SPIRILLUM SANGUINEUM. Synonym. — Ophidomonas sanguinea. Found in brackish water containing putrefying marine algae. Morphology. — Rigid spiral filaments with round ends, 3 n or more thick, and having two to two and one-half spiral turns, each 9 to 12 /* long ; the protoplasm, contains numerous refractive red granules. FIG. 247. Fio. 848. FIG. 247.— Spirillum serpens. X 650. (Flugge.) FIG. 248.— Spirillum tenue. X 650. (Flugge.) FIG. 249.— Spirillum uudula. X 650. (Flugge.) FIG. 849. 417. SPIRILLUM SERPENS (Muller). Synonym. — Vibrio serpens. Found in stagnant water, vegetable infusions, etc. Morphology. — Rigid filaments having two or three wave-like undulations, from 11 to 28 n long and 0.8 to 1.1 u thick; sometimes united in chains. Actively motile and often associated in closely crowded swarms. Biological Characters not determined. NON-PATHOGENIC SPIRILLA. 697 418. SPIRILLUM UNDULA (Ehrenberg). Found in putrefying animal and vegetable infusions. Morphology. — Rigid, spiral filaments, from 8 to 12 /* long and from 1.1 to 1.4 H thick. Each spiral turn has a length of 4 to 5 jn and each filament from one-half to three turns. A long, whip-like flagellum may be demon- strated at each extremity. The movements are rotary and rapidly progres- sive— darting. Biological Characters riot determined. 419. SPIRILLUM TENUE (Ehrenberg). Found in putrefying vegetable infusions, etc. Morphology. — Slender spiral filaments, from 4 to 15 ft long. The height and length of a single turn are from 2 to 3 u, and each filament has from one and a half to five turns. Often associated in closely crowded swarms. Motions rotary and progressive — extremely rapid. Biological Characters not determined. 420. SPIRILLUM LINGU/E. Synonyms. — Vibrio lingualis; Zungenbelag vibrio (Weibel). Obtained from deposit upon the tongue, by inoculation in a mouse. Morphology. — Curved rods of the size of the cholera spirillum ; sometimes in S- shape. Grow out into longer or shorter wavy filaments, the 'extremi- ties of which are sometimes enlarged— button-like. Involution forms are common. Stains by Gram's method, and is differentiated by this from other ' ' vibrios " described by Weibel. Biological Characters. — An aerobic and facultative anaerobic, non. liquefying, non-motile spirillum ("vibrio"). Spore formation not determined. Grows at the room temperature in the usual cul- ture media. Upon gelatin plates forms dirty -white colonies, which at the end of a week may attain a diameter of one milli- metre. Under a low power the margin of the deep colonies is seen to consist of fine, white, interlaced filaments, with irregular offshoots. The margin of the superficial colonies has a greenish-yellow shimmer, and thread-like offshoots are given off in a tangential direction ; the contour is round. In gelatin stick cultures a delicate, white, Fm. 250.— Spirillum linguae. (Weibel.) veil-like stripe is developed along the line of puncture, and no growth occurs upon the surface. Upon the surface of agar a dirty-white, finely granular — not slimy — layer is developed. In bouillon a flocculent deposit collects at the bottom of the tube, and the liquid above is slightly clouded. The flocculi in bouillon cultures consist of closely interlaced filaments, and frequently shorter rods or fragments are so placed as to give the impression that they are bud-like offshoots from the longer filaments. Not pathogenic for Avhite mice. 421. SPIRILLUM NASALE. Synonyms. — Vibrio nasalis; Naseiischleimvibrio (Weibel). Found in nasal mucus. Morphology. — Curved rods with rounded ends, about as thick as the an- 098 NON-PATHOGENIC SPIRILLA. thrax bacillus, and two to five times as long as thick ; the amount of curva- ture varies considerably, from nearly straight to semicircular, and short rods may be quite straight. In preparations from agar or gelatin cultures long, closely wound spiral filaments are sometimes seen, or the filaments may be wavy or made up of curved segments. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, non-motile spirillum. Grows slowly at the room temperature — better at 37° C. Spore formation not determined. Upon gelatin plates, at the room temperature, grows very slowly ; by the fifth day the colonies may have a diameter of 0.3 millimetre, and finally of 0.6 millimetre at the out- side; under a low power they are seen to be finely granular, and yellowish- brown by transmitted light ; they are spherical in form, with sharply defined margins. In gelatin stick cultures a delicate, veil-like growth is seen along the track of the needle ; no growth upon the surface. Upon the surface of agar a dirty- white, slimy layer is developed at 37° C. No growth upon po- tato. 422. SPIRILLUM a OF WEIBEL. Synonym. — Vibrio saprophiles a (Weibel). Found in putrefying hay infusion and in slime from sewers. Morphology. — Curved rods, with pointed ends, about 0.6 n thick at the centre, and averaging 3 /* in length ; often two are united in S-form — longer %$iimf ~'- t'&i?je~' FIG. 251. FIG. 252. FIG. 251.— Spirillum « of Weibel. (Weibel.) FIG. 252.— Spirillum ft of Weibel. (Weibel.) chains are not common; grow out into spiral filaments; involution forms common. Biological Characters. — An aerobic, non-liquefying, actively motile spirillum. Grows rather slowly at the room temperature. Spore formation not determined. Potato cultures give off a strong odor of ammonia. Upon gelatin plates the deep colonies, by the third day, are from 0.2 to 0.3 milli- metre in diameter; later they may attain a diameter of 0.6 millimetre; under a low power they are seen to be spherical and yellowish-brown in color ; the opaque centre is surrounded by concentric rings. The superficial colonies are flat white or yellowish discs, irregular in outline, with a finely granular structure ; the centre is greenish-yellow and opaque, and the color fades out toward the periphery; at the end of a week they may attain a dia- meter of two millimetres. In gelatin stick cultures a veil-like, white growth is seen along the line of puncture; later this has a dirty yellowish-red color ; upon the surface a whitish layer gradually extends from the point of punc- ture, and beyond this a transparent, whitish film covers the surface. Upon the surface of agar a cream-like, yellowish-white layer is developed, and the agar beneath is clouded to a depth of one to two millimetres. Upon po- tato, at the end of twodays, an abundant slimy layer is developed; this lias a yellowish-red to chocolate-brown color, and resembles the growth of the bacillus of glanders. NON-PATHOGENIC SPIRILLA. 699 423. SPIRILLUM ft OF WEIBEL. Synonym.— Vibrio saprophiles ft (Weibel). Found in putrefying hay infusion. Morphology.— Slender, curved rods, of about the thickness of the tubercle bacillus, and averaging 2u in length; the ends are blunt; frequently two elements are united in S-form, but long filaments do not occur; involution forms common. Biological Characters.— An aerobic, non-liquefying, actively motile spirillum ("vibrio"). Spore formation not determined. Grows rather slowly at the room temperature. Upon gelatin plates the colonies never ex- ceed 0.3 millimetre in diameter; they are spherical, and by transmitted light have a yellowish-brown color. In gelatin stick cultures the growth is similar to that of the preceding species—Spirillum a. In agar stick cul- tures no growth occurs along the line of puncture; on the surface a cream - like, yellowish-white, viscid layer is formed, which cannot be raised with- out bringing away some of the culture medium. Upon potato a thin a FIG. 253. FIG. 254. FIG. 253.— Spirillum y of Weibel. (Weibel.) Fio. 254.— Spirillum aureum. (Weibel.) shining, varnish-like layer of a dirty brownish-green color and a viscid, •dry consistence, which is with difficulty removed by the platinum needle. 424. SPIRILLUM Y OP WEIBEL. Synonym. — Vibrio saprophiles y (Weibel). Found in the slime deposited in sewers. Morphology. — Curved rods with round ends, one-half larger than "Vib- rio saprophiles a. " of Weibel ; S-shaped forms are seen, but spiral filaments are rare; involution forms very common in old cultures. Biological Characters.— -An aerobic, non-liquefying, actively motile spi- rillum ("vibrio"). Spore formation not determined. Grows rather quickly at the room temperature. Upon gelatin plates the deep colonies are white, and at the end of a week about 0.5 millimetre in diameter; under a low power they are seen to be spherical, granular, and orange- colored at the cen- tre, with a sharply defined, pale-yellow marginal zone. The superficial colo- nies are flat, dirty-white, slightly opalescent, with a more prominent white centre ; under a low power the "margin is irregular and notched, the mar- ginal zone white and marked by numerous fine, anastomosing furrows, next to this a light ochre-yellow zone marked by darker lines, and at the centre a golden-brown nucleus marked by delicate, dark, interlacing lines ; at the end of a week the superficial colonies may attain a diameter of five millimetre*. In gelatin stick cultures development occurs along the line of 700 NOX-PATHOGENIC SPIRILLA. puncture and to a moderate extent upon the surface. In agar stick cultures no growth occurs along the track of the inoculating needle ; on the surface a dirty-white, pap-like layer is developed. Upon potato the growth is sometimes dry, viscid, and dark-brown in color, or it may be of a mahogany- brown with a moist lustre. 425. SPIRILLUM AUREUM. Synonym. — Vibrio aureus (Weibel). Found in the air and in the slimy deposit in sewers. Morphology. — Curved rods with blunt ends, about one-half thicker than the Spirillum cholerae Asiaticae, and varying greatly in length; typical "commas" and S-forms are seen; also filaments of various lengths and sometimes regular spiral filaments which are extremely slender ; also invo- lution forms. Biological Characters. — An aerobic, non-liquefying, non-motile, chro- mogenic spirillum. Produces a golden or orange-yellow pigment. Spore formation not determined. Grows rather rapidly at the room temperature — also in the incubating oven. Upon gelatin plates the deep colonies, at the end of three days, have a diameter of 0.3 millimetre (in ten days of one millimetre); under a low power they are seen to be coarsely granular, spherical or whetstone-shaped, golden-yellow in the centre and later brown or black, with more transparent, golden-yellow margins. The superficial colonies are round, "with well-defined margins, granular, and of a pure golden-yellow color; at the end of three days they have a diameter of one millimetre and in ten days of three to four millimetres. In gelatin stick cul- tures a tolerably abundant, finely granular growth is seen along the line of puncture; upon the surface a rounded mass of a yellow-ochre color is de- veloped about the point of puncture. Upon the surface of agar a dirty- white layer extends over the entire surface ; later round, elevated, golden- yellow islands are seen, and finally a uniform pap-like layer two millimetres thick. Upon potato an abundant, thick, pap-like growth of a golden- yellow or orange-yellow color. 426. SPIRILLUM FLAVESCENS. Synonym. — Vibrio flavescens (Weibel). Found in the slimy deposit of sewers. Morphology. — The same as Spirillum aureum. Biological Characters. — An aerobic, non-liquefying, non-motile, chro- mogenic spirillum. Produces a dirty yellowish-green pigment. Grows rather rapidly at the room temperature. Upon gelatin plates the colonies resemble those of Spirillum aureum, except that the yellow color is developed later and the shade is paler, duller, and less pure — upon a transparent back- ground a dirty yellowish-green. In gelatin stick cultures a finely granu- lar line of growth is seen along the track of the needle, and along the mar- gin of this in old cultures are coarser granular masses; upon the surface a pale-yellow, flat layer with flap-like margins slowly extends from the point of puncture. Upon agar a dirty-white color is developed, which gradually extends to the walls of the test tube; elevated, round, yellow masses are de- veloped in this, which increase in diameter, become confluent, and finally form a thick and uniform pap-like layer. Upon potato an abundant dull- yellow, pap-like layer. 427. SPIRILLUM PLAVUM. Synonym. — Vibrio flavus (Weibel). Found in the slimy deposit in sewers. Morphology. — The same as Spirillum aureum. NON-PATHOGENIC SPIRILLA. 701 Biological Characters. — The same as Spirillum aureum, but the pigment produced is ochre-yellow. Upon a dark background the colonies appear grayish-yellow, on a white ground straw-yellow ; under a low power the deep colonies are first pale-yellow then golden-yellow, and of the same tint throughout, without being darker in the centre ; they are finely granular, with a net-like marking. The superficial colonies under the microscope are pale-yellow with dull-gray spots; on the margin the color usually remains white ; upon the surface of agar an ochre-yellow layer; the same on potato. 428. SPIRILLUM CONCENTRICUM (Kitasato). Found in putrefying blood. Morphology. — Short spirilla with pointed ends, with two to three spiral turns which are 3.5 to 4 ju in length and 2 to 2.5/< in diameter — i.e., each complete spiral ; the filaments are a little thicker than the cholera spirillum ; in bouillon they grow out into long spirals having from five to twenty turns. Biological Characters. — An aerobic, non-liquefying, actively motile spirillum. Grows best at a temperature of 20° to 23° C. Spore formation not observed. Upon gelatin plates the colonies, by transmitted light, are seen, to be made up of concentric rings, which from the centre to the peri- phery are alternately opaque and transparent ; the contour is round, and from the margin numerous small, spiral outgrowths are given off. In gela- tin stick cultures development occurs principally upon the surface as £ cloudy layer penetrating the medium to a depth of one millimetre. Upon the surface of agar a diffuse growth which adheres firmly to the culture medium. Upon potato no growth occurs. In bouillon a diffuse cloudiness is slowly developed; later the medium becomes clear and an abundant, slimy deposit is seen at the bottom of the tube. 429. SPIRILLUM RUBRUM (Von Esmarch). Obtained from the putrefying cadaver of a mouse. 'Morphology.— Spirilla about twice as thick as the cholera spirillum and having from one to three spiral turns ; in bouillon grows out into very long, spiral filaments. Biological Characters. — An aerobic and .facultative anaerobic, non- liquefying, actively motile, chromogenic spirillum. Produces a wine-red pigment in the absence of oxygen only; on the surface of culture media the trowth is colorless. According to Lofner, this spirillum has numerous short agella. In old cultures unstained, slightly refractive bodies are seen in the filaments, which appear to be spores. Grows very slowly — best at 37° C. Upon gelatin plates small, slightly granular colonies with a tolerably smooth contour are developed at the room temperature; these are at first gray and bluish-red, later wine-red in color. In gelatin stick cultures closely crowded, isolated, spherical colonies are developed along the line of punc- ture ; the growth, except upon and near the surface, has from the outset a beautiful wine-red color. Upon the surface of agar a grayish- white layer of limited extent is developed ; later this has a pink color. Upon potato deep-red colonies the size of a hempseed are developed. 130. SPIRILLUM OF SMITH. Found in the intestine of swine. Morphology. — Comma-shaped rods and spiral filaments of one and a half to two or even as many as ten spiral turns ; have terminal flagella. Biological Characters. — An aerobic, non-liquefying, motile spirillum. Grows at the room temperature. Upon gelatin plates, at the end of thirty- six to forty-eight hours, small, spherical, finely granular colonies are de- veloped, which have a brownish color; and upon the surface small, round colonies with a somewhat irregular contour. In roll tubes, when the colo- 61 702 NON-PATHOGENIC SPIRILLA. nies are not crowded, they may have a diameter of 0.3 to 0.5 millimetre, and at the end of several days appear to be made up of concentric zones ; at the end of several weeks they have the appearance of the end of a tree trunk which has been sawed off and shows many concentric rings of growth, which are not very clearly defined. Under certain circumstances outgrowths occur around some of the deep colonies, which give them the appearance of a raspberry. The superficial colonies may attain a diameter of three to five millimetres ; they remain flat and circular in outline and acquire a slight yellowish color. Upon agar plates the deep colonies have a diameter of 0.5 to 0.7 millimetre ; those upon the surface are flat, round discs of a gray color and smooth appearance ; they may attain a diameter of five millimetres. In neutral bouillon containing one-fourth to one per cent of peptone, at 36° C., development is abundant, and the culture liquid in a few days is densely clouded ; examined in a hanging-drop culture, they appear a little larger than the spirillum of cholera and exhibit very active movements. They grow in milk without producing any perceptible change in this fluid. The cultures acquire a slightly alkaline reaction. Upon potato, at 37° C., a thin, yellow layer is developed in the course of a few days. Not pathogenic for guinea-pigs or pigeons. 431. SPIRILLUM OP MILLER. Synonym. — Miller's bacillus. Obtained by Miller (1884) from carious teeth. Morphology. — Straight or slightly curved rods, frequently in pairs in form of a letter S or of an O ; also in homogeneous or segmental spiral filaments. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile spirillum. Spore formation not observed. Grows at the room temperature in the usual culture media. No growth upon the surface of gelatin cultures, which are liquefied. Upon agar the growth is similar to that of the SDirillum of Finkler and Prior. Growth upon potato not charac- teristic. XI. LEPTOTRICHE^E AND CLADOTRICHE^E. ZOPF and other systematic botanists place among the bacteria cer- tain microorganisms which are more interesting to the student of general biology than to the pathologist, but which require description in a manual of bacteriology. In our descriptions of the species in- cluded by Zopf in the Leptotrichese and Cladotrichese we shall follow the author named. Four genera are included in the LEPTOTRICHE^E, viz.: Crenothrix, Beggiatoa, Phragmidiothrix, and Leptothrix. The CLADOTRICHE^E are included in a single genus : Cladothrix. 432. CRENOTHRIX KUHNIANA (Rabenhorst). Synonym. — Brunnenfaden. Very common in running or in stagnant water. It sometimes develops so abundantly in reservoirs and conduits of water that the water supply is unfit for drinking or for certain industrial purposes. Morphology, — In different stages of development appears as cocci, short rods, and long filaments. The cocci are small spheres of from 1 to 6 p in diameter ; the cell wall of these becomes gelatinous and they multiply by binarv division, the gelatinous capsule of the daughter cell remaining en- closed in that of the mother cell ; later they are set free by the solution of this gelatinous envelope ; the zooglcea formed by these cocci are irregular in form and may attain a diameter of one centimetre or more. These zooglcea sometimes accumulate in enormous masses in reservoirs of water; at first they are colorless, but later they are colored by hydrated oxide of iron and appear brick-red, olive green, dark-brown, or brownish-black. When culti- vated in swamp water these cocci grow out into rods, which by binary divi- sion produce filaments ; these are seen projecting in all directions from the zoogloea masses. When these reach a certain age they become segmented, and the segments, enclosed in a common sheath, acquire a rusty-red or dark-brown color from being impregnated with oxide of iron ; the rod-shaped segments break up into spherical bodies which are comparatively large (" macrococci ") ; some broad filaments, however, contain disc-like seg- ments, which break up into smaller cocci. The rod-shaped and spherical segments escape from the ruptured extremity of the common sheath. Some- times the sheath becomes prematurely gelatinous, and the cocci and rods remain in situ and germinate ; in this case they break through the gelati- nous walls, and the original filament is seen to be surrounded by a brush- like outgrowth of filaments. In these secondary filaments, as well as in the primary, there is a distinct differentiation of the two extremities, growth occurring at the free extremity and not at the base. The filaments are some- times wavy or even spiral in form. Biological Characters not well determined, owing to the difficulty of cul- 704 LEPTOTRICHE^J AND CLADOTRICHE^E. tivating this microorganism in artificial media. It is aerobic. Genuine spore formation has not been demonstrated. The BEGGIA.TOA are distinguished by the presence of grains of sulphur in the vegetative cells; these are seen as highly refractive granules with a dark contour. They are widely distributed, and are found both in salt and fresh water containing decomposing animal or vegetable material ; in sul- phurous waters they are especially abundant, and accumulate upon the muddy bottom, or upon organic substances undergoing decomposition, as a milk-white, gray, pink, or purple layer; the bottom of ponds or of small bays is often colored red by an extended and abundant growth of these wa- ter bacteria. Meyer has shown that they are able to decompose sulphate of FIG. 255. Fjo> 256. FIG. 255.— CrenothrixKuhniana; a-e, cocci in various stages of division; /, zooglcea of cocci (X 400); g, zoogloea of various forms (natural size); h, colony of short filaments, composed of rod-shaped cells, and developed from a group of cocci; i-r, filamentous forms, partly spirally bent and of different thicknesses. (Zopf .) FIG. 256.-Beggiatoa alba (x 400); a and 6, filaments having an evident difference between base and extremity, being segmented and free from sulphur grains below; 2-5, fragments of filaments of various thicknesses and containing a greater or less number of sulphur grains; 6-8, filaments stained with methyl violet, showing segmentation into rods and cocci-like elements; 9, cocci; 10, development of a rod, c, from a coccus, a (X 600). (Zopf.) soda in organic solutions suitable for their growth. Like the previously de- scribed genus (Crenothrix), spherical, rod-shaped, filamentous, and spiral forms are included in the life history of the species. The filaments show a differentiation as to base and free, growing extremity, but, unlike the Creno- thrix, the segments into which the filaments divide are not included in an external sheath. The filaments are flexible and exhibit a gliding movement; LEPTOTRICHE.^E AND CLADOTRICHEJE. 705 they are able to multiply abundantly in. thermal sulphur waters having a temperature of 55° C. and above. 433. BEGGIATOA ALBA (Vauch.). This is an extremely common and widely distributed species ; found es- pecially in thermal waters and in the refuse waters from sugar refineries and factories of different kinds. Morphology, — The filaments differ greatly in diameter as well as in length — from 1 to 5 M in diameter; the young and slender filaments often contain hut few grains of sulphur or none at all; the older filaments usually con- tain a considerable number of fine or coarser granules of sulphur. The filaments, which are attached to some substance, are usually seen to be seg- mented, even without the use of reagents, at least near the base where the grains of sulphur are less numerous or absent ; the free ends, containing numerous grains of sulphur, may not appear to be segmented, but when, they are stained with one of the aniline colors, or treated with hot glycerin, segmentation becomes apparent. In the thicker threads the segments divide into thin discs, and these again, under certain circumstances, divide, in a di- rection parallel with the long axis of the filament, into quadrants, each of which later becomes a spherical or ellipsoidal coccus; these usually contain one or several large grains of sulphur. These cocci remain attached for a time, then under favorable conditions become separated and enter upon the " swarm stage " of their existence, during which they are endowed with ac- tive movements. They come to rest upon some filament of an alga or other substance, which may be completely covered, and has a dark color as a re- sult of their presence. They multiply by binary division and form zooglcea masses of irregular form. Under certain circumstances they form straight or curved rods, which may also exhibit active movements. When these come to rest they grow out into long filaments, which may be straight or spirally curved. Fragments of the spiral filaments may, under certain cir- cumstances, become actively motile and resemble genuine spirilla; their movements are due to the presence of terminal flagella. The diameter, length, and the height of the spiral turns vary greatly. The straight fila- ments also show a decided tendency to break up into longer or shorter frag- ments, but these do not exhibit active motions; they, in common with the fragments of spiral filaments, are, however, flexible and exhibit " crawling " motions. 434. BEGGIATOA ROSEO-PERSICINA (Zopf). Synonyms. — Clathrocystis roseo-persicina (Cohn) ; Ophidomonas san- guinea (Ehrenberg) ; Bacterium rubescens (Lankester). Found upon the surface of putrefying animal or vegetable material in fresh or salt water. Morphology. — Presents the same developmental forms as Beggiatoa alba, viz., cocci, rods, filaments, and spiral threads. The filaments correspond with those of Beggiatoa alba, but are distinguished by their red or violet color. The cocci, formed in the filaments, when set free undergo binary division and form characteristic zoogloea of very various forms ; these were formerly described by Cohn under the name of Clathrocystis roseo-persi- cina. These zoogloea may be spherical, oval, irregularly branched, etc. ; at one time they may contain but a little, and at another considerable gela- tinous intercellular material. Rods are developed from the cocci under cer- tain circumstances, which may be straight or curved. In suitable media both the cocci and the rods, after the intercellular substance is dissolved, may become actively motile — " swarm stage." The short rods grow out into filaments, and these may, under certain circumstances, become spiral in form, either partially or entirely; the spiral filaments may break up into onotile fragments which correspond with Ophidomonas sanguinea of Ehren- 706 LEPTOTRICHE.E AND CLADOTRICHEJE. berg (Spirillum sanguineum, No. 416). The red pigment produced by this species is known as bacterio-purpurin. It is insoluble in water, alcohol, chloroform, ammonia, or acetic acid, and is changed by hot alcohol into a brown, and by chloroform into an orange-brown, substance. 435. BEGGIATOA MIRABILIS (Colin). Found in sea water, upon the surface of putrefying animal and vegetable substances, as a white layer; common upon the coasts of Denmark and Nor- way (Warming). ^Morphology. — This species is distinguished by the considerable thickness of the filaments, which may be as much as 30 n ; these undergo segmenta- tion into cylindrical masses, which again break up into thin discs, and these are supposed to divide into cocci-like elements such as Zopf has described in the other species of this genus. The complete life history of this species has not been determined. Like the other species described, the cells contain granules of sulphur. 436. PHRAGMIDIOTHRIX MULTISEPTATA (Engler). Found in sea water, attached to crabs Morphology. — Filaments from 3 to 6 jit in diameter, made up of thin disc- like segments, the diameter of which is four to six times less than the thick- ness; these cylindrical segments undergo segmentation into halves and quadrants, and finally into still smaller fragments which become rounded and resemble cocci ; these probably are set free, but this has not been ob- served; apparently they grow out first into very thin filaments which subse- quently increase in diameter. The genus to which this species belongs is distinguished from Beggiatoa by the absence of sulphur grains, and from Crenothrix by the fact that the segments are not enveloped in an exterior sheath, as well as by the comparative thinness of the cylindrical segments. LEPTOTHRIX BUCCALIS. — According to Zopf, the leptothrix so common in the human mouth presents the same variety of forms as has been ascribed to the Beggiatoa and Crenothrix. We cannot accept this as established, and have described Leptothrix buccalis among the bacilli (No. 395). "Vignal claims to have cultivated this species, but, in view of the failure of Miller and others to obtain it in cultures, it appears doubtful whether the microorganism described by him under this name corresponds with the Leptothrix buccalis of Robin (see page 683). 437. CLADOTHRIX DICHOTOMA (Cohn). Found in stagnant and running water — very common. It is frequently associated with Beggiatoa and is common in the refuse water of factories, especially of sugar factories. In Russia it is often found in abundance in the water supply of towns. It may readily be obtained from the surface of pu- trefying algae or animal substances immersed in river or swamp water. Morphology. — According to Zopf, the cocci-like reproductive elements grow out into rods, and these into fine filaments, from which later pseudo- ranches are given off. This apparent branching of the filaments is the dis- tinguishing generic character of Cladothrix. Under a sufficiently high power the branched appearance is seen not to be a true dichotomous ramifica- tion, but to result from the growing out in a lateral direction of a detached segment. These long filaments break up again into long rods, these into shorter rods, and finally into cocci. The sheath of the filaments is often col- ored yellow, rusty-red, olive-green, or dark-brown by oxide of iron. The segments are forced from the free extremity of the common sheath by the LEPTOTKICHE^E AND CLADOTRICHEJE. 707 growth and binary division of those lying deeper, or by their own motility — "swarm stage"; they may emerge from the sheath either solitary or in chains of several elements. Sometimes the cocci-like forms germinate within the common sheath and grow out through its walls into filaments. Fragments of the filaments are sometimes seen to exhibit peculiar gliding movements; again they may exhibit very active movements as a result of the development of terminal flagella. The filaments are sometimes straight and sometimes twisted in spiral form, the spiral turns being sometimes quite flat and at others well-developed corkscrew-like convolutions. When the spiral filaments break up into motile fragments provided with flagella they FIG. 857. — Cladothrix dichotoma; A, branching plant with wavy (a) or spiral (6) filaments; B, spiral filament more highly magnified; C, long spirochsete-like filament; D, fragment with one extremity spiral; E, spiral filaments segmenting into rods (7>) and cocci (c); F, spirocheete form, a undivided, 6 dividing into long rods, c into short rods, d into cocci-like elements. (Zopf..) resemble genuine spirilla. According to Zopf, the so-called zoogloea rami- gera is one form of development of Cladothrix dichotoma. Biological Characters not determined. 438. CLADOTHRIX FOERSTERI. Synonym. — Streptothrix Foersteri (Cohn). Found by Grafe in the lachrymal ducts of the human eye. Morphology. — Forms cocci-like masses, rods, and leptotiirix filaments, which may be spirally curved. According to Zopf, this species closely re- sembles Cladothrix dichotoma. Biological Characters not determined. 708 LEPTOTRICHE^E AND CLADOTRICHE^E. 439. CLADOTHRIX INTRICATA (Russell). Obtained by Russell (1891) from mud at the bottom of the Gulf of Na- ples. Morphology. — Differs considerably, according to the age of the culture and the culture medium. In gelatin it forms long filaments made up of long and slender cells having homogeneous contents. In potato cultures the cells are shorter and with rounded ends; these elements divide into several short and thick separate cells, and many of these finally contain a slender, oblong spore. The filaments in agar and potato cultures may present the appear- ance characteristic of the genus Cladothrix, viz. , a false branching. Stains with the usual aniline colors. Biological Characters. — An aerobic, liquefying, slightly motile clado- thrix. Forms long-oval spores. Grows in the usual culture media at the room temperature. Upon gelatin plates colonies are developed in from twenty- four to thirty-six hours ; these resemble, to the naked eye, colonies of mould fungi ; under a low power the interior of the colony is seen to be made up of a thick network of filaments, from which a quantity of curled and inter- twined filaments extend in all directions. The outside filaments are often tolerably straight, but they soon become more or less spiral and intertwined, or are united into interwoven, braid-like masses which extend in various directions from the principal colony. Liquefaction of the gelatin quickly occurs, and the filaments form in the liquefied gelatin a felt-like mass. When a cover glass is placed over a young colony and a microscopical ex- amination is made with a tolerably high power, the straight filaments at the margin of the colony are seen to present pseudo-branches. In gelatin stick cultures development is rapid ; at the end of twenty-four hours the line of inoculation is marked by finely curled filaments, which extend horizontally into the gelatin in all directions, the growth being most abundant near the surface. The gelatin near the surface is soon liquefied, and the liquefaction gradually extends downward. Upon potato an irregular, dull-white mass is quickly developed which is not especially characteristic ; this ceases to ex- tend after three days. Upon agar a tolerably abundant but thin, dull white layer is developed, and fine filaments extend from this into the culture me- dium, giving the culture a characteristic appearance. An abundant devel- opment occurs in bouillon and a jelly-like mass accumulates at the bottom of the tube ; this is readily broken up by shaking the tube. XII. ADDITIONAL SPECIES OF BACTERIA, NOT CLASSIFIED. 440. NITROMONAS OF WINOGRADSKY. Obtained from the soil at Zurich by Winogradsky (1890), who says: " In speaking of a nitrifying ferment I do not wish to affirm that there is but a single species capable of exercising this function over the whole surface of the globe. That appeal's to me not at all probable. . . . But it is probable that there are but few. At Zurich, for example, up to the present time I have found but a single one." Morphology. — Ellipsoidal cells, more or less elongated, the youngest cells often nearly spherical; 0.9 to 1 n broad and from 1.1 to 1.8 M long; the longer cells already show the central constriction which precedes binary di- vision; sometimes among the oval cells spindle-shaped cells are seen, and oc- casionally this is the prevailing form. As a rule, the cells do not remain as- sociated after binary division has occurred, but occasionally a chain of three or four elements is seen. Irregular masses are formed in cultures, which are held together loosely by a gelatinous material. Biological Characters, — Does not grow in the usual culture media, but was cultivated by Winogradsky in a solution containing one gramme of potassium phosphate and one gramme of ammonium sulphate in one thousand grammes of pure water. To this solution he adds half a gramme to a gramme of basic magnesium carbonate. In testing the nitrifying power of the ferment the ammonium sulphate is added in separate portions, a standard solution of ten grammes in five hun- dred cubic centimetres of water being used. From two to five cubic centi- metres of this solution are added at intervals of twenty-four to forty-eight hours, according to the rapidity of the ferment action, and the absence of ammonia as a result of this action is determined by the use of Nessler's solu- tion. The cultures are kept at the room temperature. The nitromonas of Winogradsky does not form spores. It is sometimes seen to exhibit active movements, but is usually at rest. The researches of the author named show that it is capable of growing and of exercising its ferment action in solutions from which all organic matter has been excluded, and the conclusion is reached that it is able to assimilate the carbon required for its development from the carbonic acid set free in the culture liquid as a result of the action of the nitric acid formed by the nitrifying ferment upon the magnesium carbonate in suspension. Recently (1891) Winogradsky has succeeded in cultivating this nitrifying ferment in a solid medium containing soluble silicates, which form a gela- tinous mass (see page 47). The bacillo-cocci of G. and P. F. Frankland appear to be identical with the nitromonas of Winogradsky. The authors named, finding that the nitrifying ferment did not grow in nutrient gelatin, succeeded in isolating it in liquid cultures by the method of dilution. They describe the nitrifying organism obtained by this method as a bacillus which is but little longer than it is broad, and which they designate a " bacillo- coccus." In this country E. O. Jordan and Ellen H. Richards have also 710 ADDITIONAL SPECIES OF succeeded in isolating a nitrifying ferment by the method of dilution and by cultivation in a liquid containing certain salts dissolved in distilled water. Their culture medium was constituted as follows : Ammonium chloride (resublimed) , Sodium carbonate, Sodium phosphate, . . . Potassium sulphate, 1.9070 grammes. 3.7842 0.2000 " 0.2000 " " These salts were dissolved in such a quantity of redistilled water that the solution contained one hundred parts of nitrogen per one hundred thousand, and two equivalents of alkali. Ten cubic centimetres of this solution were mixed with one litre of redistilled water and then inoculated as desired . " The nitrifying ferment obtained by Jordan and Eichards is described as a short bacillus of a slightly oval shape, about 8 to 9 M broad and from 1.1 to 1.7 u long. These bacilli are associated in irregular zooglcea by a jelly-like material. The masses are found chiefly at the bottom of the flasks, as was the case with the nitrifying ferment isolated by Winogradsky. No inde- pendent movements were observed by Jordan and Richards, who state that they have not been able to determine in a definite manner whether the nitri- fying organism isolated by them is identical with that of Winogradsky and of the Franklands. They remark that their bacillus stains with some diffi- culty with the usual aniline dyes. 441. NITRIFYING BACILLUS OF WINOGRADSKY. Obtained by Winogradsky (1891) from the soil by cultivation in a gela- tinous medium containing soluble silicates. Morphology. — Small bacilli of somewhat irregular form, often pyriform ; the average length does not usually exceed 0.5 /u, and the breadth is quite variable, even in the same cell. The bacilli are grouped in irregular masses and united into a membranous pellicle by a gelatin- ous material. This pellicle in old cultures is seen attached to the bottom of the tube as a transparent membrane, which may be detached by agitation ; in more recent cultures it is firmly adherent and sa transparent as to be seen with difficulty ; it gives to the glass a feeble bluish-gray tint, and is not detached by repeatedly rinsing the tube with distilled water. The bacilli are somewhat difficult to recognize in stained preparations, owing to the stain- ing of the gelatinous intercellular sub- stance. Winogradsky recommends wash- ing the stained preparation with water at 50° to 60° C. to remove the color from the gelatinous material. The Biological Charactersof this bacillus have not been fully determined. In a gelatinous medium made by the addition of silicic acid to a culture liquid containing certain salts, yellowish-gray, lenticular colonies were developed, from which liquid cultures were subsequently made by Winogradsky. In these the liquid remained transparent, and no pellicle formed upon the sur- face, but a very transparent, gelatinous film was found attached to the bot- tom of the flask ; the bacillus above described was found to be present in this and to be the cause of the active oxidation of nitrites present in the culture medium. With reference to its ferment action Winogradsky says: "The oxidizing power of this imponderable quantity of a living ferment is aston- ishing. " When this bacillus is associated with the microorganism previously Fin. 258.— Nitrifying bacillus of Wino- gradsky. x 1,000. From a photomicro- graph. (VVinogradsky.) BACTERIA, NOT CLASSIFIED. 711 described (No. 440) in a solution containing salts of ammonia, the ' ' nitro- monas" takes theprecedence ; and, according to Winogradsky, when the oxi- dation of the ammonia is completed this ferment ceases to absorb oxygen and enters into a state of repose, while the nitrifying bacillus commences to multiply and to exercise its special ferment action. 442. 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. 443. BACILLUS THALASSOPHILUS (Russell). Obtained by Russell (1891) from mud at the bottom of the Gulf of Na- ples. Morphology. — A slender bacillus, varying greatly in length, which grows out into filaments which are not visibly segmented. Stains with ZiehFs solution when obtained from a recent culture, but not by Loffler's solution or fuchsin. Biological Characters. — An anaerobic, liquefying, motile bacillus.. Forms spores, which are very small and are located at the middle or the ends of the rods. Grows at the room temperature in the absence of oxygen. In nutrient gelatin, prepared with sea water, at the end of two or three days colonies appear at the bottom of the line of puncture in the form of small, clouded bubbles; later other colonies develop above these, forming- finally a long, irregular, grayish, semi-transparent, liquid, sac-like mass. At the upper portion of this sac gas accumulates. The gelatin is now rap- idly liquefied, except above, the liquefaction extending to the walls of the tube. Finally the entire amount of gelatin is liquefied, and becomes clear above from the deposition of the bacilli at the bottom of the tube, wrhere they form a thick, sticky mass. The cultures give off a penetrating, disagree- able odor. In gelatin between double plates colonies first appear at the- end of two or three days ; under a low power these show a very thin net- work of filaments, which penetrate the gelatin in all directions. Much gas is developed in the colonies, and when the upper plate is lifted an odor of skatol is given off. The colonies soon become confluent and the gelatin is entirely liquefied. In agar a scanty development occurs along the line of puncture to within two centimetres of the surface. 444. BACILLUS GRANULOSUS (Russell). Obtained by Russell (1891) from the mud at the bottom of the Gulf of Naples; common, even at a depth of 1,100 metres. Morphology. — In hanging-drop cultures this bacillus grows out into long, slender filaments, made up of tolerably large bacilli having a thick cell wall and finely granular protoplasmic contents. In older cultures the long fila- ments are broken up into shorter, irregular fragments, and the separate seg- ments are seen as short, thick rods with coarsely granular contents. Upon agar and potato the development of filaments is irregular and they break up into a grape-like mass. These masses, which might be taken for involution 712 ADDITIONAL SPECIES OF forms or degenerative products, are capable of forming spores. In potato cultures these masses are seen to consist of short, plump cells, each one of which contains a highly refractive spore. The segments of a filament divide as in Bacillus megatherium, without any external appearance of division, the new cells growing in breadth rather than in length ; as a result of unequal growth irregular masses are formed, in which later spores are developed. In anaerobic cultures the cells are usually solitary, or, at most, united in pairs. The protoplasm of young cells is fine and granular; in older cells, and espe- cially in unfavorable media, the contents consist of large, shining granules. Stains with the usual aniline colors and by Gram's method ; the granules are deeply stained by Kiihne's car bol-methyl- blue solution. Biological Characters. — An aerobic and facultative anaerobic, lique- fying, usually non-motile bacillus; in bouillon cultures kept at 37° C. a slight to-and-fro movement may be observed. Forms spores. Grows in the ^ FIG. 259. FIG. 260. FIG. 25D.— Bacillus thalassophilus; culture in nutrient gelatin at end of fourth day; a, drum- rstick forms from gelatin culture; e, bacilli containing spores. (Russell.) FIG. 2CO.— Bacillus granulosus; young surface colony upon gelatin plate; a, b, c, normal forms from recent gelatin culture; /, /', from an old gelatin culture; g-h, abnormal forms from potato culture— at A, spores. (Russell.) usual culture media at the room temperature. Upon gelatin plates the col- onies differ considerably in appearance, especially those upon the surface. At first they are usually thin, almost transparent, and leaf -like; under a low power these colonies are seen to be covered with irregular, concentric lines, which are formed by the parallel arrangement of the filaments, and under the microscope resemble the anastomosing "nerves " upon a leaf. Liquefac- tion of the gelatin quickly occurs. The deep colonies are not characteristic, and remain as small, round, shining, opaque masses. The colonies are vis- cid, drawing out into threads, or, when touched with the platinum needle, the entire colony may be picked up. In gelatin stick cultures liquefaction ioccurs near the surface, forming a shallow cavity, at the bottom of which BACTERIA, NOT CLASSIFIED. 713 the bacilli accumulate in zooglcea masses ; later the liquefaction extends to the deeper layers of the gelatin medium. Upon agar, in the course of two or three days at the room temperature, or in twenty-four hours in the incubat- ing1 oven, rather scanty and thin, whitish or yellowish colonies are developed, which remain separated where the culture medium is scanty, as at the upper part of oblique cultures, while below, where the culture medium is moist, development is more abundant. In bouillon an abundant development occurs, which causes a diffused cloudiness of the liquid and a considerable deposit at the bottom of the tube. The growth upon potato is quite charac- teristic. At the end of twenty- four hours at the room temperature a moist, white, shining patch is seen ; instead of extending over the surface, this in- creases in thickness, forming a thick, viscid, shining-white mass; later this loses its lustre, becomes dull and waxy in appearance, and acquires a brownish color. 445. BACILLUS LIMOSUS (Russell). Obtained by Russell (1891) from mud from the bottom of the Gulf of Naples ; very abundant in all of the specimens examined. FIG. 261. FIG. 262. FIG. 261.— Bacillus limosus; culture in nutrient gelatin made with sea water, at end of two days, and bacilli from a gelatin culture. (Russell.) FIG. 262.— Spirillum marinum; culture in nutrient gelatin made with sea water, at end of two days, and spirilla. (Russell.) Morphology. — Long and slender bacilli, 1.25 u broad and 3 to 4 n long; usually united in pairs, or in chains containing several elements. In hang- ing-drop preparations from potato cultures the cells are shorter and thicker than in gelatin cultures ; the ends are rounded, and the cell contents often appear finely granular. Biological Characters. — Anaerobic, liquefying, motile bacillus ; exhibits slow to-and-fro movements. Forms spores, which are located at one ex- tremity of the rods. Grows in the usual media at the room temperature — 714 ADDITIONAL SPECIES OF more luxuriantly in nutrient gelatin made with sea water. Upon gelatin plates colonies are developed in from twenty-four to thirty hours, which at first are almost transparent; under a low power these are seen to be surrounded by long, slender filaments which extend far out into the gelatin ; in its further development the central portion of the colony extends and includes the slender filaments, forming a round mass surrounded by little, thorn-like projections ; liquefaction commences at the centre, causing a depression of •the colony ; in old colonies the liquefied gelatin has a grayish color and a thin pellicle forms upon the surface ; below this swim flocculent masses, and around the margins the thorny appearance is preserved. In gelatin stick cultures containing sea water development is very rapid, and at the end of twenty-four hours a funnel-shaped cavity, containing liquefied gelatin which is clouded throughout, has been formed ; in the course of seventy hours the gelatin is entirely liquefied and light, flocculent masses collect in the lower portion of the tube, while upon the surface a thin pellicle is formed which is easily broken up. Upon agar the development is abun- dant, forming a moist, shining-white layer. In bouillon a dense cloudiness is developed, and a thick and very resistant layer is formed upon the surface, while a considerable deposit accumulates at the bottom of the tube. Upon potato a thin, grayish-white layer is formed, which covers the greater part of the surface. 446. SPIRILLUM MARINUM (Russell). Obtained by Russell from mud at the bottom of the Gulf of Naples, and from water from the same source ; not very abundant. Morphology. — Bods which are usually more or less curved, like the cholera spirillum ; usually in pairs, but several elements are sometimes united to form a spiral filament ; the rods are sometimes straight. Stains with the usual aniline colors. Biological Characters. — An aerobic, liquefying, actively motile spiril- lum. Spore formation not observed. The movements are sometimes rotary, and sometimes progressive without rotation. Grows in the usual culture media at the room temperature, but not in the incubating oven; grows •better in media made without the addition of sea water. Upon gelatin plates the colonies are first seen as round, granular masses, often presenting radial striations. When liquefaction of the gelatin commences the colonies have .a rougher appearance, and flocculent masses float in the shallow cavities containing liquefied gelatin. In gelatin stick cultures development is very rapid at first; the gelatin is liquefied and clouded, and a thin, semi-trans- parent membrane forms upon the surface; later a stratum of liquefied gelatin is seen above, while below the culture medium remains unaltered. Upon agar the growth is luxuriant and forms a moist, white, pus-like layer. Upon potato the growth is very characteristic ; at the end of twenty-four hours a reddish-brown, sharply defined layer is developed, and the potato changes its color in the vicinity of the line of inoculation ; the growth increases .in dimensions and forms a thick, wax-like mass, which after a time covers the greater portion of the surface ; it remains soft and does not penetrate the potato, which gradually acquires a dark greenish-gray color. In bouillon made with sea water development is abundant and causes the culture liquid to be densely clouded; a smooth, white layer forms upon the surface. __447. BACILLUS LITORALIS (RuSSell). Obtained by Russell (1891) from mud at the bottom of the Gulf of Na- ples, near the shore. Morphology. — Bacilli two to four times as long as broad. Stains with Loffler's solution, but not by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, lique- fying, motile bacillus. Spore formation not observed. Movements very BACTERIA, NOT CLASSIFIED. 715 irregular. Grows slowly in the usual culture media at the room temperature. Upon gelatin plates deep colonies are seen at the end of three days as well- defined, small, brown points. The superficial colonies are at first shining and opalescent ; under the microscope they are seen to be finely granular and have smooth margins; at the end of five to eight days the colonies in con- tact with the air commence to cause liquefaction of the gelatin ; liquefac- tion progresses so slowly that evaporation keeps pace with it, and the colo- nies slowly sink into the gelatin ; when the layer of gelatin is quite thin a ring of liquefied gelatin may surround the colony. In gelatin stick cultures a scanty gi-owth is seen along the line of puncture at the end of twenty-four hours. Upon the surface a thin, irregular, whitish layer commences to form around the point of inoculation at the end of three or four days; the gelatin is slowly liquefied below this, and as a result of evaporation a small •cavity is formed which is lined with a thin layer of bacilli. But little de- « 0 c Fia. 263. FIG. 264. FIG. 263.— Bacillus litoralis; A, colony upon gelatin plate, ten days old; B, gelatin stick culture ten days old; C, bacilli from hanging-drop culture. (Russell.) FIG. 264.— Bacillus halophilus; A, culture in nutrient gelatin at end of twenty -four hours; B culture in sea-water gelatin at end of twenty- four hours. (Russell.) velopment occurs along the line of inoculation below, but the growth often acquires a reddish-brown color, and the gelatin around it is stained brown — this color is only developed in the absence of oxygen. In streak cultures white, semi-transparent colonies are formed along the line of inoculation ; these become visible after the second day, extend slowly, and finally coal- esce; in the course of five to seven days the thicker white colonies com- mence to sink below the surface as a result of liquefaction and evaporation ; liquefaction now progresses more rapidly, and after a time the deposit at the bottom of the liquefied gelatin acquires a reddish-brown tint. No growth occurs upon potato. Upon agar a scanty, thin, moist-looking, grayish-white layer is formed. In bouillon a uniform cloudiness is developed and no pel- licle forms upon the surface. 716 ADDITIONAL SPECIES OF 448. BACILLUS HALOPHILUS (Russell). Obtained by Russell (1891) from water of the Gulf of Naples and from mud from the bottom. Morphology, — Recent cultures in nutrient gelatin made with sea water contain bacilli of from 1.5 to 3.5 ju. in length and 0.7// broad; these are often united in pairs. In older cultures the form quickly changes, and at the end of two days cells resembling those of a torula — "yeast-like" — are seen; these abnormal forms increase in number and variety as the culture becomes older ; some contain a granular protoplasm, and some are so transparent as to be easily overlooked. These cells, however, are motile and resemble the so-called monads. The variability of form is still greater in ordinary nutrient gelatin, which is a less favorable medium than gelatin made with sea water. Stains with difficulty, and not at all with Loffler's solution; does not stain by Gram's method; the protoplasm is irregularly stained by Ziehl's solution, while fuchsin solution stains it feebly but homogeneously. Biological Characters. — An aerobic, liquefying, actively motile bacil- lus. Spore formation not observed. Grows slowly in the usual culture media at the room temperature. In stick cultures in nutrient gelatin made with sea water, at the end of twenty-four to thirty-six hours, punctiform colonies are developed, which quickly coalesce ; liquefaction occurs along the line of growth and gas is developed ; sometimes this is so abundant that the liquefied gelatin is forced up over the surface of the culture as a foamy mass ; later the liquefied gelatin becomes transparent, and a fine deposit is seen at the bottom of the tube. In nutrient gelatin not made with sea wa- ter the growth is considerably slower; at first a white line is seen, extending only along a portion of the puncture; in the course of seventy hours a slender cavity is formed as a result of slow liquefaction and evaporation ; this gradually increases in length and gives to the cultures a characteristic appearance. In plates made with sea- water gelatin, spherical, grayish- white, semi-transparent colonies are developed; these cause liquefaction, and by evaporation sharply defined, deep funnels are formed in the gelatin. The cultures have a strongly alkaline reaction. 449. BACILLUS CAPSULATUS MUCOSUS (Fasching). Obtained from the nasal secretion in two cases of influenza. Morphology. — Bacilli from 3 to 4 ft long and 0.75 to 1 n 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. 450. BACILLUS OF POTATO ROT (Kramer). Obtained by Kramer (1891) from potatoes affected with wet rot — " Nass- faue. Morphology.— Bacilli with round ends, from 2.5 to 4 n long and 0.7 to BACTERIA, NOT CLASSIFIED. 717 0.8 /it broad; often united in chains; grow out into filaments; in old cultures thicker rods of an ellipsoidal form are seen, and spores are formed which en- tirely fill the cell containing them. Biological Characters. — An aerobic, liquefying, motile bacillus. Forms large oval spores. Grows in the usual culture media at the room tempera- ture. In gelatin stick cultures liquefaction occurs rapidly, forming a fun- nel, at the bottom of which the bacilli accumulate. In streak cultures upon gelatin, at the end of twelve hours, an elevated, dirty-white line of growth is seen along the impfstrich ; this extends latei-ally, and the margins are scal- loped so that the growth resembles a leaf. Upon nutrient agar small, dirty- white, slimy drops, with sharply defined margins, are developed. Gelatin cultures containing litmus or carmine are decolorized by this bacillus. In solutions containing dextrose it multiplies abundantly, producing carbon dioxide and butyric acid. In starch paste containing ammonium tartrate the starch is only dissolved to a slight extent, and no butyric acid is de- veloped. It has but slight action upon cellulose. Potatoes sterilized by steam and inoculated with a pure culture of the bacillus undergo changes corresponding with " Nassfaule " in these tubers. These consist of a decom- position of soluble carbohydrates (sugar), with a formation of carbon dioxide and butyric acid ; then of the intercellular substance and the cell membranes, producing an acid reaction in the contents of the tuber. The starch is not changed. The albuminous substances undergo putrefactive decomposition, with production of ammonia, methylamin, and trimethylamin. These bases first neutralize the butyric acid, and later cause an alkaline reaction of the affected portions of the potato. In milk coagulation of the casein occurs, but no putrefactive change is induced, even at the end of several weeks, as is the case with Bacillus butyricus (Hueppe). 451. BACILLUS VACUOLOSis (Sternberg). Obtained by Sternberg (1888) in cultures from the intestine and stomach of yellow-fever cadavers. Morphology. — Bacilli with round ends, which vary considerably in the r dimensions and are sometimes slightly curved; length from 1.5 to 5 >u, breadth about 1 /*. In stained preparations unstained places — vacuoles ? — are seen in the rods. In surface cultures upon agar various involution forms are seen, which also present vacuoles and which are usually considerably larger than the normal bacilli. The bacilli sometimes grow out into long, jointed filaments. Stains with the usual aniline colors. Biological Characters. — An aerobic, liquefying, motile bacillus. Forms large oval spores. Grows in the usual culture media at the room tempera- ture. In gelatin stick cultures liquefaction occurs slowly, near the surface, forming a cup-shaped cavity. The liquefied gelatin is quite viscid, and a cream- white layer of bacilli forms upon the surface of the liquefied medium. In nutrient agar the development along the line of puncture is scanty ; on the surface a cream-white layer is formed and the bacilli are united in long, jointed filaments. It is not always seen to be motile, but under certain cir- cumstances exhibits slowly progressive, to-and-fro movements, as if propelled by a flagellum. Does not grow in an acid medium. On potato a thin, cream-white layer is formed. Pathogenesis. — Not pathogenic for rabbits. Not tested upon other ani- mals. 452. BACILLUS OF DANTEC. Synonym. — Bacille du rouge de morue. Obtained by Dantec (1891), in association with other microorganisms, from salted codfish, to which it imparts a red color. 62 718 ADDITIONAL, SPECIES OF Morphology.— Bacilli with round ends, from 4 to 12 n long, and usually containing a spore at one extremity. Resembles the bacillus of tetanus, but is considerably thicker. Biological Characters. — An aerobic, liquefying, motile, chromogenic bacillus. Foi'ms spores. Grows in the usual culture media at the room temperature. Produces a red pigment. In gelatin stick cultures liquefac- tion in funnel form occurs along the line of puncture. In old gelatin liquefaction may not v* occur. Upon the surface of nutrient gelatin a s » » I red streak is developed along the line of in- t** s |l oculation, and the gelatin below this is very "* \ ,N' \ * v ^ slowly liquefied. The colonies in. gelatin plates •* V * '3 may attain a diameter of two millimetres; i ^ / ^ i \ they have a pale-red centre with a deeper red N %\ . »' o periphery, and are disc-shaped. In bouillon ~ A! * * i \ I development is abundant, but without the for- » I. ^ "S. mation of pigment. Does not grow well upon ° «.* x* ./ § potato. Pigment is formed more abundantly lx * ' * at 10° to 15 3 C. than at a higher temperature. Upon dried codfish it grows readily, forming a red pigment, especially upon the side which FIG. a65.-Bac.llus of Dantee. hag ^ exposed {o the ^.lt Not pathogenic. 453. BACILLUS (MICROCOCCUS ?) HAVANIENSIS (Sternberg). Morphology.— Short-oval bacilli, usually in pairs, about 0.4 to 0.5 u in diameter. The cells are so nearly spherical that the writer has been in doubt whether to describe this microorganism as a bacillus or as a micrococcus. Biological Characters. — An aerobic, non-liquefying, chromogenic ba- cillus(?). Grows slowly in the usual culture media at the room temperature. Upon gelatin plates forms small, spherical, translucent colo- nies of a beautiful blood-red color. In gelatin stick cultures a thick, opaque, carmine layer develops about the point of inoculation and slowly increases in thickness and circumference ; very scanty growth, without color, at the upper part of the line of puncture. Upon the surface of agar the growth is slow but continuous, and forms, at the room temperature, a thick, carmine layer along the line of inoculation ; this has wavy outlines and a glistening, varnished-like surface ; it gradually extends in thickness and breadth, which at the end of a month may bo FlG- 266. -Bacillus Havanien- five to six millimetres. Frequently this micro- sis- x 1,000. (Sternberg.) coccus fails to grow on potato, perhaps because of an acid reaction. But upon old and rather dry potato it sometimes de- velops, as it does on nutrient agar, forming a thick, irregular mass of a carmine color. The pigment is only formed in the free pi'esence of oxygen. 454. BACILLUS AMYLOZYMA (Perdrix). Obtained by Perdrix (1891) from the hydrant water of Paris — best from the deposit left in a Chamberlain filter through which this water has been passed. Morphology. — Bacilli with round ends, from 2 to 3 n longandO.5// broad ; usually in pairs, or in chains of several elements. Stains with the usual aniline colors. BACTERIA, NOT CLASSIFIED. 719 Biological Characters. — A. strictly anaerobic, non-liquefying, motile bacillus. Forms spores. Grows in the usual culture media in ail atmo- sphere of hydrogen at the room temperature. In nutrient gelatin colonies are developed at the end of five or six days ; these are small, white plaques surrounded by gas bubbles. Upon potato, in an atmosphere of hydrogen, whitish colonies are developed, around which the potato appears excavated; at the same time it is partly liquefied by the ferment action of the bacillus,' and the liquid collects at the bottom of the tube. When these potato tubes are opened there is a little explosion, due to escaping gas. The development of the bacillus is most rapid and its ferment action most energetic at a tem- perature of about 35° C It causes fermentation of sugar and of starch, but not of cellulose. It ceases to grow in cultures containing 0.10 to 0.12 per cent of acid (estimated in sulphuric acid), and, as it produces an acid reac- tion of the culture medium, it is necessary to add carbonate of lime to this to insure its continuous development. The acids formed from the fermenta- tion of sugar are acetic (at the outset) and butyric. In culture media con- taining starch, ethylic and amylic alcohol are produced, as well as butyric acid ; a considerable portion of the starch is converted into sugar, and hydro- gen and carbon dioxide are given off freely as a result of the fermentation. 455. BACILLUS RUBELLUS (Okada). Obtained by Okada (1892) from dust by inoculations in guinea-pigs. Not pathogenic, but found in association with pathogenic bacteria in the bloody serum effused about the point of inoculation— with dust from the streets. Morphology. — Resembles Bacillus cedematis maligni in its form and di- mensions. Bacilli with slightly rounded ends, usually in pairs or chains of three; in old bouillon cultures grows out into filaments 10 to 15 // long; these are often surrounded by a capsule-like envelope. The rods show a slight swelling when spore formation occurs, becoming spindle shaped or having an expanded extremity, according as the spore is formed in the middle or at one extremity. Flagella may be demonstrated by Loffler's method of staining; these are seen at one or at both extremities. Stains with the usual aniline colors and also by Gram's method. Biological Characters.— An anaerobic, chromogenic, liquefying, motile bacillus. Forms large oval spoi-es, which are located at one extremity or in the centre of the rods. In gelatin plates kept in an atmosphere of hydro- gen, dull-white, punctiform colonies are developed at the end of ten days at 15° to 18° C ; under a low power these are seen to be long-oval in form and surrounded by fine offshoots ; after a time the gelatin is liquefied and ac- quires a reddish color. In deep gelatin stick cultures development com- mences at the bottom of the line of puncture, where, at the end of ten days, small, dull-white, spherical or oval colonies are developed ; these are seen to be surrounded by radiating offshoots ; the colonies increase in size, and the gelatin is gradually liquefied in the lower two-thirds of the tube, while above it remains solid ; a flocculent deposit is now seen at the bottom of the tube, which has a reddish color; finally the upper portion of the gelatin is also liquefied and the whole of the fluid has a red color. Upon agar plates, at 37° C. , development is more abundant and rapid ; colonies are developed in twenty-four hours, which subsequently increase in size and acquire a red color. In deep stick cultures in agar, in the incubating oven, development occurs below at the end of twenty-four hours and gradually extends up- ward to near the surface. Gradually the upper portion of the growth ac- quires a red color, which increases in intensity, and the upper portion of the culture medium is after a time diffusely colored. In bouillon in an atmo- sphere of hydrogen, at 37° C. , development is abundant and rapid, and the medium acquires a red color. Not pathogenic. 720 ADDITIONAL SPECIES OF 456. BACTERIUM URE^E (Jaksch). Found in ammoniacal urine. Morphology. — Bacilli with round ends, about 2 M long and 1 M thick. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying bacillus. Spore formation riot observed. Grows very slowly at the room temperature. Changes urea into carbonate of ammonia. Upon gelatin plates forms small, almost transparent colonies, which at the end of ten days may have the diameter of a " pfennig." In gelatin stick cultures a thin, gray, branching growth is seen along the line of puncture. Old cultures have the odor of herring-brine. Imperfectly described. 457. SARCINA MOBILIS (Maurea). Obtained by Maurea (1892) from ascitic fluid which had been preserved a long time in a test tube. Morphology. — Micrococci having a diameter of 1.5 /* and associated in pairs or in tetrads. Stains with the usual aniline colors and also by Gram's method. Biological Characters. — An aerobic, liquefying, motile, chromogenic sarcina. Does not grow in the incubating oven at 37° C. Motions progres- sive, serpentine, or rotatory (?). Upon gelatin plates, at 15° to 20° C., puncti- form, white colonies are seen on the third day. By the seventh day lique- faction of the gelatin commences around the colonies and a brick-red pig- ment is formed. In gelatin stick cultures, at the end of five days, a scanty development is seen along the line of puncture and a more abundant growth upon the surface ; later the surface growth acquires a brick-red color ; in from fifteen to twenty days liquefaction has occurred in the form of a small funnel, and by the end of thirty days one-half of the contents of the tube is liquefied. In bouillon the fluid becomes clouded in two or three days, and later a yellowish-red deposit is seen at the bottom of the tube. In agar stick cultures a whitish layer is developed on the surface, which later ac- quires a brick-red color. In milk growth occurs without producing coagu- lation. No growth upon potato. In hay infusion sarcina-like packets are developed in abundance, as well as tetrads and diplococci. 458. BACILLUS STOLONIFERUS (Pohl). Obtained from swamp water (1892). Morphology. — Bacilli 1.2 ^ long and 0.8 fi broad. Biological Characters. — An aerobic, liquefying, motile bacillus. Spore formation not observed. In gelatin stick cultures liquefaction, in funnel form, commences at the end of twenty-four hours and progresses rap- idly. Upon the surface of agar a thick, white mass develops along the track of the inoculating needle. Upon potato small colonies the size of a pin's head are developed along the line of inoculation and extend over the entire surface. In milk a scanty development occurs ; the milk is not coagu- lated and no acid is formed. 459. BACILLUS INCANUS (Pohl). Obtained from swamp water. Morphology. — Bacilli 1.7 n long and 0.4 n broad. Biological Characters. — An aerobic, liquefying, slightly motile bacillus, In gelatin stick cultures growth occurs along the line of puncture and upon the surface as a grayish-white, elevated mass. At the end of forty-eight hours slight liquefaction is observed ; this progresses very slowly. Upon the surface of agar a grayish-white, granular mass is developed along the track of the inoculating needle. Upon potato a gray, viscid layer is formed, which BACTERIA, NOT CLASSIFIED. 721 quickly extends over the entire surface. Does not produce an acid reaction in milk. 460. BACILLUS INUNCTUS (Pohl). Obtained from swamp water. Morphology. — Bacilli 3.5 n long and 0.8 to 0.9 ju broad. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile bacillus. Spore formation not observed. Upon gelatin plates forms oval or round colonies with a smooth margin and oily, shining ap- pearance. In gelatin stick cultures grows along the line of puncture, and below the growth has a radiating appearance; upon the surface a thick, white, shining layer is quickly formed ; later liquefaction commences and progresses slowly. Upon the surface of agar white, cloud-like masses are formed along the streak. Upon potato a white, slimy mass is formed which soon covers the entire surface. In milk an acid reaction is produced by the growth of this bacillus, but no coagulation occurs. In Pasteur's solution con- taining cane sugar or starch an abundant development of alcohol occurs. 461. BACILLUS FLAVESCENS (Pohl). Obtained from swamp water (1892). Morphology.— Bacilli 2.1 to 2.2 /* long and 0.8 /* broad. Biological Characters. — An aerobic, non-liquefying, chromogenic, slightly motile bacillus. Spore formation not determined. Forms a yellow pigment. Upon gelatin plates, at the end of four days, colonies are visible ; these are yellow, granular, and attain the size of a pin's head. In gelatin stick cultures it grows along the line of puncture, and slowly extends over the entire surface. Upon the surface of agar small, solitary, yellow colo- nies are developed along the track of the needle. Upon potato development is somewhat more rapid, and at the end of four days the entire surface is covered with a slimy, yellow layer. Gelatin colored blue with litmus is de- colorized without the previous change of color to red. In milk an acid reac- tion is produced without coagulation. 462. BACILLUS BUTYRICUS OF BOTKIN. Obtained by Botkin. in anaerobic cultures from milk, from hydrant wa- ter, well water, garden earth, and dust. Morphology. — In agar and gelatin cultures containing one and one-half per cent of grape sugar the bacilli are 0.5 ju long and 1 to 3 n in diameter, with round ends ; they are often united in pairs or in chains of three ; in liquid media they are not so thick, and often attain a length of 10 ju. or more, without segmentation ; long chains may also be observed. The spores are located in the centre of the rods, or occasionally at one extremity ; they vary greatly in their dimensions; the diameter is usually about 1 >« and the length 2 to 3 //. In old cultures various involution forms are seen. Stains with the usual aniline colors. Biological Characters. — An anaerobic, liquefying, slightly motile ba- cillus. Forms large oval spores, which have a high resisting power for heat. To obtain pure cultures Botkin subjects milk containing this bacillus to the boiling temperature —in the steam 'sterilizer— for half an hour. The spores resist this temperature, and the flask containing the milk is hermeti- cally sealed and placed in an incubating oven at 37° C. At the end of twelve hours, as a rule, fermentation commences; the casein is coagulated and col- lects, together with the fat, at the upper part of the flask, while a clear, yel- lowish serum is seen below ; there is an abundant development of gas. The most favorable temperature for growth is 37° to 38° C., but development may occur at temperatures ranging from 18° to 42° C. ; at 18° development is very slow and usually no gas is formed. In culture media containing 722 ADDITIONAL SPECIES OF starch many of the bacilli, at the end of two or three days, contain granules which acquire a deep-blue color when treated with iodine solution. Upon agar plates (with 1.5 per cent of grape sugar), in an atmosphere of hydro- gen, colonies are developed in fifteen to eighteen hours, which under the microscope are seen to be round or elliptical in form and to consist of closely interwoven filaments ; as a rule, the margins are not well defined, and radiat- ing filaments are given off from the felt-like colony. In deep stick cultures in agar, growth commences at a depth of one and one-half centimetres below the surface; later, when the oxygen is displaced by the gases produced, it approaches the surface. The growth along the line of puncture has an ir- regular outline with wave-like projections. In gelatin (with 1.5 per cent of grape sugar) growth begins, in deep stick cultures, about two centimetres below the surface ; much gas is developed and the gelatin is quickly lique- fied. In bouillon (with 1.5 per cent of grape sugar) an abundant develop- ment occurs within twenty-four hours in an atmosphere of hydrogen. The fluid becomes clouded and much gas is developed; at the end of three days the fermentation ceases, the bouillon becomes clear, and a thick, white de- posit is seen at the bottom of the tube. In milk, contained in flasks com- pletely full, and from which the oxygen has been expelled by boiling, growth commences near the bottom, where at the end of fifteen hours a transparent layer of serum is seen, from which numerous gas bubbles are given off ; the fermentation progresses rapidly, and at the end of eighteen hours the coagulated casein and particles of fat have accumulated at the upper part of the flask; the pressure of gas is so great that many of the flasks are blown into fragments; at the end of a week development has ceased and the casein is almost entirely dissolved, the contents of the flask consisting of a transparent, yellowish fluid, with spongy masses of fatty material upon the surface and a flocculeiit, white deposit at the bottom. Upon potato, in an atmosphere of hydrogen, development occurs in the in- terior of the potato, but not upon the surface. Not pathogenic. Note. — Botkin is not able to identify the bacillus described by him with butyric-acid bacilli described by Pasteur, Cohn, Hueppe, and other authors, which for the most part have been cultivated only in liquid media. He re- marks that it is differentiated from Clostridium butyricum of Prazmowski (Vibrion butyrique of Pasteur) by the fact that it does not decompose cellu- lose or salts of lactic acid. It closely resembles a butyric-acid ferment de- scribed by Perdrix, but is differentiated from it by the fact that the bacillus of Perdrix does not liquefy gelatin. 463. UROBACILLUS PASTEURI (Miquel). Obtained by Miquel from decomposed urine. Morphology. — Bacilli differing greatly in dimensions in different media — the diameter may be as much as 1.2 ju. In urine to which two per cent of urea has been added it is usually seen in the form of short rods united in chains of two to six elements. In gelatin cultures containing urea the ba- cilli are from 4 to 6 M long and are usually in pairs. Biological Characters. — An aerobic, liquefying, motile bacillus. Forms spherical spores, usually solitary, and situated at one extremity of the rod. Grows at the room temperature in the usual culture media when these are made alkaline by the addition of ammonia, or when urea is added. In gela- tin plates containing urea minute colonies are developed within twenty- four hours and an ammoniacal odor is given off ; under a low power the colonies are seen to be spherical or oval, yellowish, and surrounded by dumbbell- shaped crystals ; at the end of eight days the gelatin commences to liquefy, and after a time has the consistence of castor oil. In ordinary flesh-peptone- gelatin no development occurs, unless the medium has a distinctly alkaline reaction. No growth occurs in the usual agar-agar medium, but when urea BACTERIA, NOT CLASSIFIED. 723 is added an abundant development occurs, and numerous very minute crys- tals are scattered through the culture medium. In urine alkaline fermenta- tion is quickly induced, and an abundant deposit of crystals of ammonio- magnesian. phosphate and of alkaline urates, together with the bacilli, accumulates at the bottom of the test tube ; this deposit acquires a blackish color. The most favorable temperature for the growth of this bacillus is 30° to 40° C. 464. UROBACILLUS DUCLAUXI (Miquel). Obtained by Miquel (1879) in the water of sewers, and subsequently in river water — very common and widely distributed. Morphology. — Slender filaments, from 0.6 to 0.8 in diameter and from 2 to 10 u long. In alkaline bouillon it may attain a diameter of 1 ft ; the ba- cilli are frequently united in chains. Biological Characters. — An aerobic and facultative anaerobic, motile, liquefying bacillus. The movements are comparatively slow. Forms spores located at the centre of the rods, which then are spindle-shaped. De- velops most rapidly at 40° C. No development occurs at 5° C., but at 8° to 10° the fermentation of a culture medium containing urea is accomplished in about a month, and at 20° in two or three days. Does not grow in ordinary bouillon or nutrient gelatin, but grows in the usual culture media when they are rendered alkaline by the addition of ammonia, or when urea is added. In nutrient gelatin containing urea numerous small, white colonies are de- veloped along the track of the needle, and a quantity of minute crystals are scattered through the culture medium. At the end of three or four months the gelatin medium is transformed into a transparent, ammoniacal, syrupy liquid. In alkaline gelatin not containing urea liquefaction does not occur. In bouillon made alkaline by the addition of ammonia, growth occurs, caus- ing the liquid to become clouded within twenty -four hours ; later an abun- dant sediment accumulates at the bottom of the tube, the liquid becomes vis- cid and gives off a disagreeable odor. 465. UROBACILLUS FREUDEXREICHI (Miquel). Obtained by Miquel from the air, from dust, from sewer water, etc. Morphology. — Closely resembles Urobacillus Pasteuri, but forms longer chains and has more active movements. The rods are from 1 to 1.3 n thick and have rounded extremities ; the length varies considerably, but in recent cultures is usually 5 to 6 ju. Under favorable conditions of temperature, 30° C. , actively motile filaments, consisting of six to ten elements, are de- veloped in alkaline bouillon ; upon solid media long filaments, composed of comparatively short elements and quite motionless, are developed. Biological Characters. — An aerobic, liquefying, motile bacillus. Forms spores. The most favorable temperature for the development of this bacillus is from 33° to 35 J C. As a ferment of urea it is ten times less active than Urobacillus Pasteuri. In nutrient gelatin, at 20° C , development occurs upon the surface as a milk-white growth, which is visible at the end of two days, and later forms a layer with irregular outlines having a diameter of three to four millimetres ; but little development occurs along the track of the inoculating needle ; liquefaction commences at the surface at the end of eight to ten days and progresses slowly ; at the end of thirty to forty days the gelatin is completely liquefied, it is of a pale-yellow color and quite viscid ; an abundant white deposit accumulates at the bottom of the tube. Upon gelatin plates (containing urea) small, spherical, white colonies are developed in two or three days ; these gradually increase in dimensions and are surrounded by an aureole of minute crystals ; later the development ceases and the bacilli are killed by an excess of carbonate of ammonia re- sulting from the decomposition of the urea. In neutral bouillon, at the end of two or three days, a slight cloudiness is developed, and later a scanty white deposit is seen at the bottom of the tube. 724: ADDITIONAL SPECIES OF 466. UROBACILLUS MADDOXI (Miquel). Obtained from sewer water and from river water — relatively rare. Morphology. — Bacilli with round ends, 1 u thick and 3 to 6 ft long ; in old cultures the bacilli are often seen as oval or spherical cells having a diameter of 6 to 8 /* ; in solid media the length is usually from 2 to 3 /*. Biological Characters. — An aerobic, liquefying, motile bacillus. Forms spores. Grows best at 38° C., but causes fermentation of urine at the end of several days at a temperature as low as 10° C. In nutrient gelatin it usually fails to grow, but occasionally a scanty development occurs along the track of the needle in the form of small, spherical colonies. In nutrient gelatin containing urea the line of inoculation is marked by numerous crys- tals, which are seen at the end of twenty hours, although the growth of the bacilli is scarcely apparent. In gelatin plates containing urea very small, spherical, opaque, whitish colonies, surrounded ^by crystals, may be seen under a low power. In nutrient agar containing urea, at 30° to 35° C., a layer is formed upon the surface, which is at first white and la.ter of a grayish- yellow color. In bouillon which is slightly alkaline it grows rapidly, and produces, at the end of two days, a dense turbidity of the culture liquid ; later an abundant glairy sediment accumulates at the bottom of the tube. Iri one litre of bouillon an amount of soluble ferment is produced by this bacillus which is capable of decomposing sixty to eighty grammes of urea in the course of two or three hours. 467. UROBACILLUS SCHUTZENBERGI (Miquel). Obtained by Miquel from river water and from the water of sewers. Morphology. — Small oval bacilli about 0. 5 M thick and 1 n long ; usually united in pairs. Biological Characters. — An aerobic, liquefying, motile bacillus. Spore formation not observed. In nutrient gelatin, at20°C., liquefaction in cup shape occurs at the surface and progresses rapidly, being complete by the end of ten days. In gelatin containing two per cent of urea liquefaction com- mences at the surface and numerous crystals are formed in the solid portion of the culture medium ; growth is soon arrested by the antiseptic action of the products developed. In gelatin plates small, translucent, spherical colonies are developed, which increase rapidly in size and acquire a milky appearance; when the colonies reach the surface liquefaction rapidly occurs ; when the gelatin contains urea the colonies cease growing after attaining a diameter of one to two millimetres, and they are surrounded by a zone of crystals. Upon nutrient agar, at 28° to 30° C. , development occurs in the form of a whitish layer with a slightly greenish tint. In bouillon, at the end of twenty-four hours, the medium becotnes clouded ; later a thin pellicle forms upon the surface and extends upward for a short distance upon the walls of the test tube; the bouillon remains clouded for several weeks. When urea is added to peptonized bouillon development is still more abun- dant, but ceases at the end of four or five days and the liquid becomes en- tirely transparent. 468. BACILLUS OF BOVET. Obtained by Bovet (1891) from the intestine of a woman who died of an acute enteritis with choleraic symptoms. Morphology. — Bacilli from 1 to 1.5 jit thick and 2 to 4 n long, isolated or in pairs. Stains with carbol-fuchsin solution. Biological Characters. — An aerobic, non-liquefying, motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature — more rapidly at 37° C. In nutrient gelatin, at 15° to 18° C., a grayish- white, somewhat translucent layer with irregular outlines is BACTERIA, NOT CLASSIFIED. T^o developed in two or three days ; along1 the line of the inoculating- needle in stick cultures a granular growth is seen which has a brownish-gray color. Upon agar the surface growth is similar to that upon gelatin and quickly covers the entire surface. When the culture medium contains sugar or gly- cerin lenticular bubbles of gas are formed in it. Upon potato a thick layer is developed resembling puree of peas, but not so decidedly yellow in color ; in old cultures the color is a dirty-gray. In anaerobic cultures a scanty de- velopment occurs. Pathogenesis. — Subcutaneous injections in rabbits or guinea-pigs gave a negative result. Iritraperitoneal injections in guinea-pigs produce peritoni- tis and death. 469. BACILLUS SCHAFFEKI (Freudenreich). Obtained by Freudenreich from cheese and from fermenting potato — fragments of raw potato in water. Morphology. — Bacilli about 1 n thick and from 2 to 3 // long; in old cul- tures long filaments are common — 20 to 25 fi. Stains well with the usual aniline colors; in gelatin cultui'es the rods are frequently only partly stained ; generally the central portion is stained while the poles remain unstained, but occasionally one-half is colored, or two stained portions may be separated by a clear space. Does not stain by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, non-li- quefying, motile bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates, at the end of two or three days, punctiform, yellowish colonies are developed ; under a low power these are seen to be granular, pale-yellow, and spherical or irregular in outline ; upon the surface the colonies are elevated, circular in outline, and porcelain- white in color; when widely separated they may finally attain the size of a two-franc piece — with very irregular margins ; under a low power these large colonies are seen to be granular, and to have a yellowish centre with pale margins. The colonies are not viscid and are easily detached from the surface with a platinum needle. In stick cultures in nutrient gelatin a layer is developed upon the surface which at first is nearly transparent ; later this has a grayish color and extends over the entire surface; the growth along the line of puncture extends to the bottom, but is not characteristic. Upon agar a grayish layer is developed, which later sometimes acquires a brownish color, especially along the line of puncture. Upon potato a moist, yellowish layer is developed ; this remains smooth and is without gas bubbles. In bouillon, at 373 C. , development occurs within five or six hours, causing turbidity ; when milk sugar is added to the pepton- ized bouillon there is a development of gas, and bubbles are given off in abundance when the culture is agitated. Culture media containing sugar acquire an acid reaction; at the end of several days a pellicle is formed upon the surface, which later falls to the bottom. In sterilized milk development is not abundant, but milk filtered through porcelain is a favorable culture medium; an acid reaction is produced, but coagulation, as a rule, does not occur— sometimes an imperfect coagulation occurs. This bacillus dies out in cultures in two or three weeks, and does not resist desiccation longer than forty-seven to fifty days. It is killed by a temperature of 70° C. maintained for fifteen minutes. According to Freudenreich, this bacillus closely re- sembles Bacillus coli communis, but is distinguished from it by the fact that it is actively motile, and by its ability to grow in solutions of milk sugar in the absence of oxygen, also by not being pathogenic for guinea-pigs when injected into the peritoneal cavity. 470. BACILLI OP GUILLEBEAU (a, 6, and C). Obtained by Guillebeau from the milk of cows suffering from mastitis, 720 ADDITIONAL SPECIES OF 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 «. Morphology. — Varies considerably in size, and may resemble a micrococ- cus in form ; usually 1 /* broad and 1 to 2 V long. Stains with the usual aniline colors, but rather feebly ; does not stain by Gram's method. Biological Characters. — An aerobic and facultative anaerobic, slightly motile, non-liquefying bacillus. Spore formation not observed. Grows in the usual culture media at the room temperature. Upon gelatin plates the deep colonies are spherical, granular, and yellowish in color; upon the sur- face they are round and granular at first, later they become opaque and re- semble a drop of wax. In gelatin stick cultures development occurs all along the line of puncture, and upon the surface a whitish layer is formed. Upon agar a grayish white layer is developed. Upon potato a thick, yel- lowish layer is formed; this is viscid and contains numerous gas bubbles. In milk coagulation is produced at the end of twenty-four hours, and an. abundance of gas is given off. In bouillon containing milk sugar it mul- tiplies abundantly and a large quantity of gas is liberated. Grows best at a temperature of 30° to 35° C. Thermal death-point 60° C. — fifteen minutes' exposure. BACILLUS b. Morphology. — Resembles bacillus a; bacilli from 1 to 2 /* long and about 1 p thick. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing, motile bacillus. Is differentiated from a by the fact that it causes lique- faction of nutrient gelatin after an interval of several weeks, and by the fact that the young colonies upon gelatin plates are quite viscid. Spore forma- tion not observed. Thermal death-point 80° C. — five minutes' exposure. An abundance of gas is given off from cultures containing milk sugar. BACILLUS C. Morphology. — Short bacilli ; often oval or even spherical in form ; about 1 n long. Biological Characters. — An aerobic, non-liquefying bacillus. Spore formation not observed. Upon gelatin plates colonies are developed which resemble those of bacillus a, but are more coarsely granular ; the colonies are very adherent and difficult to remove from the culture medium. Upon agar a viscous, white layer is developed. Upon potato the growth is of a yellowish- white color and similar to that of a and b, with gas bubbles; it is very adherent. In liquid media the growth of this bacillus causes the cul- ture liquid to become extremely viscous and almost gelatinous in consistence. In milk coagulation occurs at the end of sixty hours at 37° C., and the rnilk then loses its viscosity. 471. MICROCOCCUS FREUDENREICHI (Guillebeau). Obtained from milk which had undergone viscous fermentation. Morphology. — Micrococci having a diameter of 2 /*, solitary or in chains. Biological Characters. — An aerobic and facultative anaerobic, liquefy- ing micrococcus. Grows best at a temperature of 20° C. Upon gelatin plates made with milk serum, punctiform, granular colonies are developed at the end of thirty-six hours ; when touched with the platinum needle these may be drawn out into long threads; soon after the gelatin commences to liquefy and becomes very viscous. Upon potato the growth is sometimes BACTERIA, NOT CLASSIFIED. .727 in the form of a thin layer having numerous vacuoles ; sometimes a thick and shining1 layer is developed which has a pale sulphur-yellow color or is of a dull-brown mixed with yellow. In bouillon a cloudy opacity is first developed ; later a flocculent deposit is seen and the liquid above is limpid ; it is slightly viscid. In sterilized milk the viscosity produced is so great that, in old cultures, threads may be drawn out which are several metres in length. Non-sterilized milk becomes viscous at the end of five hours ; later it becomes acid, and at the end of several days the casein is coagulated and is seen as a granular precipitate, above which is a limpid and viscous serum ; at this time the milk has acquired a disagreeable odor. 472. BACTERIUM HESSii (Guillebeau). Obtained from the milk of a cow in the Alps, at an altitude of twelve hundred metres. Morphology. — Bacilli from 3 to 5 jn long and 1.2 fi thick (from potato cul- tures) ; shorter, nearly spherical elements are also seen in considerable num- bers, and occasionally long filaments ; the ends are round and stain more deeply than the central portion. Biological Characters. — An aerobic, liquefying, motile bacillus. Forms spores. Grows in the usual culture media at the room temperature. In nutrient gelatin containing serum from milk, colonies are developed upon plates at the end of a few hours ; these at first have well-defined outlines, but later are made up of interlacing filaments ; after liquefaction of the gela- tin the colony floats upon the surface; liquefaction progresses rapidly and the liquefied gelatin may be drawn out into threads. Upon potato a shin- ing layer is developed of a dull-white color, which later acquires a brownish tint. Bouillon without sugar is rapidly transformed into a viscous mass having an alkaline reaction. In milk an acid reaction is produced and the casein is precipitated at the end of two days. The viscosity produced in milk is less than that produced by the species previously described, and dis- appears at the end of two days in milk kept at a temperature of 35° C. In old cultures in bouillon a disagreeable odor is developed, said to resemble trimethylamin. 473. BACILLUS DENITRIFICANS. Obtained by Giltay and Aberson from the soil and from the air. Resem- bles Bacterium denitrificans of Gayou and Dupetit. Morphology.— Bacilli, usually in pairs, 1.5 n to 3 ft long and 0.5 M broad. Biological Characters. — Completely decomposes nitrates, producing ni- trogen monoxide as well as pure nitrogen. The reducing action of the fer- ment is favored by the presence of carbonate of lime in the culture medium. Grows in ordinary nutrient gelatin. Also cultivated in the following me- dium: Nitrate of potash two grammes, glucose two grammes, sulphate of magnesia two grammes, citric acid five grammes, monophosphate of potash two grammes, calcium chloride 0.2 gramme, two drops of a solution of per- chloride of iron, and one litre of water. 474. BACILLUS CYANO-FUSCUS. Obtained by Beyerinck from size and glue and Edam cheese. Morphology. — Bacilli from 0.2 to 0.6 /J. long and one-half as thick. Biological Characters. — An aerobic, chromogenic, liquefying, motile bacillus. Grows in the usual culture media at the room temperature. Spore formation not observed. When cultivated in a solution containing one-half per cent of peptone the culture medium acquires at first a green color, which later changes to blue, brown, and black; subsequently the color is almost entirely lost. In nutrient gelatin containing one-half per cent of peptone circular colonies are developed which are surrounded by a zone of black pigment and in which crystals of lime are formed. ADDITIONAL SPECIES OF 475. 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 spaces 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- liquefying, 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 be finely granular and to have a lemon-yellow color. Grows best in a slightly acid medium — very slowly at the room temperature. In gelatin stick cul- tures isolated colonies are formed along the line of puncture. Scanty growth 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 a longer or shorter course, from forty-eight hours to eight or ten days,1 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." 476. MICROCOCCUS AQUATILIS INVISIBILIS. Obtained by Vaughan from water. Morphology. — Oval cocci. Biological Characters. — An aerobic, non-liquefying micrococcus. Grows in the usual culture media at the room temperature, but feebly at 38" C. On gelatin plates deep brown colonies with smooth outline, spreading irregu- larly upon the surface. In gelatin tubes there is a scanty growth along the line of puncture and a spreading growth upon the surface. On agar a thin, white growth. The growth upon potato is invisible. Not pathogenic. 477. BACILLUS GRACILIS ANAEROBIESCENS. Obtained by Vaughan from water. Morphology. — "Bacilli three times as long as broad, often growing into long, slender rods." Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, actively motile bacillus. Grows rapidly in the usual culture media at the room temperature — feebly at 38° C. Upon gelatin plates brown- ish colonies are developed, " spreading irregularly." In gelatin tubes grows abundantly along the line of puncture and also spreads over the surface. On agar a thin, white layer is developed. On potato an abundant and promi- BACTERIA, NOT CLASSIFIED. 72!) nent yellowish -white layer. Grows in Parietti's solution, but not in Uffel- mann's gelatin. Forms gas abundantly in gelatin stick cultures. Not pathogenic. 478. BACILLUS FIGURANS. Obtained by Vaughan from water. Crooksbank has described a Bacillus flguraiis which appears to be identical with Bacillus mesentericus vulgatus. Morphology. — " Bacilli two to three times as long as broad, but showing marked variation in form. Sometimes they appear as very short bacilli, while at other times they grow into long threads." Biological Characters. — An aerobic, liquefying, sluggishly motile ba- cillus. Spore formation not mentioned. Grows rapidly in the usual cul- ture media at the room temperature— feebly at 38° C. " On gelatin plates the deep colonies are spherical and smooth; the superficial growth forms curved and interlacing lines, often presenting most grotesque figures. Plates may show no liquefaction after some days." in gelatin stick cultures does not develop along the line of puncture ; liquefies slowly, and sometimes the fluid is lost by evaporation as fast as it liquefies. After the gelatin has been liquefied half-way down the tube, the bacteria subside to the bottom and fur- ther liquefaction is very slow or does not occur. On agar a thin, white layer is formed upon the surface and a heavy deposit in the water of con- densation. On potato an abundant, faintly yellow, mucilaginous layer. Does not grow either in Parietti's solution or in Uffelmann's gelatin. Not pathogenic. A 479. BACILLUS ALBUS ANAEROBIESCENS. Obtained by Vaughan from water. Morphology. — " Bacilli two or three times as long as broad." Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, non-motile ("only an oscillation ") bacillus. Grows rapidly at the room temperature in the usual culture media — also at 38° C. Spore for- mation not mentioned. On gelatin plates forms smooth, spherical, yel- lowish or brownish colonies. In gelatin stick cultures grows both on the surface and along the line of puncture. On agar a thick, milk-white layer is developed. On potato a yellowish-white, glistening growth. Grows both in Parietti's solution and in Uffelmann's gelatin. Not pathogenic. 480. BACILLUS INVISIBILIS. Obtained by=Vaughan from water. Morphology. —Large bacilli with rounded ends, from two to five times as long as broad. Biological Characters. — An aerobic and facultative anaerobic, non- liquefying, motile bacillus. Spore formation not mentioned. Grows rap- idly in the usual culture media at the room temperature — also at 38° C. On gelatin plates pale-yellow, burr-like colonies, with irregular outlines and spreading slightly. In gelatin tubes grows abundantly along the line of puncture and also upon the surface, spreading slowly. On agar a thick, white growth with but little tendency to spread. On potato the growth is invisible. Grows both in Parietti's solution and in Uffelmann's gelatin. Not pathogenic. 481. 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 730 ADDITIONAL SPECIES OF 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. 482. 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. 483. 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 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. 484. BACILLUS VENENOSUS LIQUEPACIENS. 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 gelatin plates the deep colonies are finely granular, sphei'ical, and yellowish in 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 BACTERIA, NOT CLASSIFIED. 731 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 Ulfelmann's gelatin. Pathogenesis. — Pathogenic for mice, rats, guinea-pigs, and rabbits. 485. 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 anthracis, and varying in length — average length 3 to 6 /< ; 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 Ciiaracters. — 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 witli 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 a 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 any apparent effect, except in one instance in which a pregnant rabbit was killed, by the injection of one cubic centimetre, in twenty -one hours. If the animal is killed shortly after the injection the bacilli develop rapidly after death, with an abundant formation of gas in the blood vessels and organs, especially the liver. At temperatures of 18° to 20° C. the vessels, organs, and serous cavi- ties may be full of gas in eighteen to twenty-four hours, and at tempera- tures of 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 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. 486. BACILLUS OP CANON AND PIELICKE. TY>und 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 l>lood 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 732 ADDITIONAL SPECIES OF six to twenty hours. Some of the bacilli do not stain uniformly, but present the appearance of stained spots altei'nating 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. 487. BACILLUS SANGUINIS TYPHI. Obtained (1892) by Brannan and Cheesrnan 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 glyceriii-agar plates, arid 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 n 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 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 occui-s 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." BACTERIA, NOT CLASSIFIED. 733 488. MICROCOCCUS AGILIS CITREUS. Obtained by Menge (1882) from an infusion of peas — probably from the air. Morphology. — Micrococci, usually in pairs, but sometimes in short chains or irregular groups. Has a flagellum which is easily demonstrated by Lomer's method of staining, and which is about six times as long as the diameter of the micrococcus. Biological Characters. — An aerobic, non-liquefying, chromogenic, motile micrococcus. Forms a yellow pigment. Grows in the usual culture media at the room temperature. Upon gelatin plates a diffuse cloudiness of the gelatin occurs around the superficial colonies and extends over the plate, ex- cept at the numerous points where bundles of crystals are developed. In gelatin stick cultures a scanty growth occurs along the line of puncture, which is not colored ; upon the surface a round layer of an intense yellow color is slowly developed. Upon agar a pale and thin layer is developed along the line of inoculation by the end of the third day ; this increases in breadth and thickness and acquires a yellow color ; the growth upon agar FiG. 268. X 1,000. From a photomicro- FIG. 267. Fio. 267.— Bacillus gracilis cadaveris, from a gelatin culture. graph. (Sternberg.) FIG. 258. — Bacillus gracilis; colonies in gelatin roll tube, end of forty -eight hours. a photograph. (Sternberg ) X 12. From is extremely viscid and may be drawn out into long threads when touched with a platinum needle. In bouillon a diffuse cloudiness occurs and a yellow, viscid deposit accumulates at the bottom of the tube; there is no film formed upon the surface. Upon potato development is very slow, but after a time an abundant bright-yellow layer is formed, and around this the potato acquires a slightly bluish-gray color. Grows in milk without pro- ducing coagulation. Grows best at a temperature of 20° C. 489. BACILLUS GRACILIS CADAVERIS (Sternberg). Obtained (1889) from a fragment of liver, of man, kept for forty-eight hours in an antiseptic wrapping. Morphology. — Bacilli about 1 n broad and 2 /« long, associated in long chains. Biological Characters. — An aerobic and facultative anaerobic, non- motile, non-liquefying bacillus. Spore formation not observed. In gelatin 63 To! ADDITIONAL SPECIES OF BACTERIA, NOT CLASSIFIED. 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 five days a rather thick, white mass at the point of puncture, covering one-third of the surface, and closely crowded, opaque colonies at bottom of line of puncture, with slender, branching outgrowth 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 with irregular margins along the impfstrich. Cultures in bouillon have a milky opacity and a very disagree- able odor. Grows in aqua coco without formation of gas. Pathogenic for rabbits when injected into the cavity of the abdomen. XIII. BACTERIOLOGICAL DIAGNOSIS. THE researches made by bacteriologists during the past ten years show that there is an extensive bacterial flora, especially in water and in the soil, and that many of the species known are widely dis- tributed and may be recognized by their morphological and biologi- cal characters wherever they may be found ; but they also show that we cannot depend upon morphology alone for the differentiation of species, and that in many cases a careful study of the mode of growth in various culture media, and of pathogenic power by inocu- lations in the lower animals, shows slight differences in bacteria which resemble each other so closely in form and in certain biologi- cal characters that a less careful study would lead to the belief that they were identical. That there are true species among the bacte- ria, in the same sense as among the higher plants, is well established; but, as among the higher plants, it. is often difficult to determine whether the differences observed should be considered sufficient to justify the description of allied forms as distinct species, or whether they should simply be considered as varieties of a single species. For example, the well-known streptococcus of pus has morphologi- cal characters which enable us to distinguish it from many other bacteria, but streptococci have been obtained from various sources which present slight differences as to their growth in certain media and in their pathogenic power. The question arises as to whether these differences are to be considered "specific" or otherwise. If the differences noted are permanent, and enable a bacteriologist to distinguish one streptococcus from the other wherever it may be found, we are justified in considering them as two distinct micro- organisms. And the decision as to whether they are to be consid- ered as different species, or as varieties of a single species, need not detain us. As a matter of fact, nature is continuous, and specific lines are not sharply drawn except in systematic text books of bot- any, etc. The more complete our knowledge of any class of animal or vegetable organisms, the less sharply differentiated are the so- called species on account of the introduction of intermediate forms in a series having common characters. 730 BACTERIOLOGICAL, DIAGNOSIS. When the differences noted are not permanent in character, de- scription under a distinct name only leads to confusion. Among the bacteria, as among higher plants, such differences, constituting more or less permanent varieties, have been developed by cultivation under various conditions, and, without doubt, are constantly being devel- oped under natural conditions as a result of changes in the environ- ment of these minute plants. Thus we have artificial varieties of certain common chromogenic species in which no pigment is pro- duced, non-pathogenic varieties of pathogenic species, and asporoge- nous varieties of bacilli which usually form spores. The attempt to classify the bacteria in a systematic manner is attended with especial difficulties, owing to their simple structure, the comparatively slight morphological differences which they pre- sent, and also because of the tendency to variation in their biological characters above referred to. But bacteriologists are generally agreed upon the importance of the following characters for their differentiation, viz. : form — micrococci, bacilli, spirilla, polymor- phous ; relation to oxygen — aerobic, facultative anaerobic, strict anaerobic ; growth in nutrient gelatin — liquefy, do not liquefy, do not grow in nutrient gelatin at the "room temperature"; growth on potato ; groivth in milk — coagulate milk, do not coagulate, etc. ; color of growth — chromogenic, non-chromogenic ; spore forma- tion; independent movements ; pathogenic power. It is upon these characters that we must rely chiefly in our bacte- riological diagnosis of known species. But the student must remem- ber that the lines are not sharply drawn between the groups formed when we classify bacteria with reference to any one of these charac- ters. Thus we have microorganisms of this class which we find it difficult to classify as regards form, because they are not round, and yet are so slightly elongated in one diameter that it is difficult to consider them bacilli. If we follow Cohn and group these short- oval bacteria under the generic name Bacterium, we have not re- moved the difficulty, but have made two arbitrary and artificial lines instead of one. Again, liquefaction of gelatin is sometimes so slight, or occurs at so late a date, that it may be a question whether a mi- croorganism of this class should be included among the liquefying or non-liquefying bacteria. And our division with reference to the formation of pigment must, to a certain extent, be arbitrary ; for many species which are not decidedly chromogenic present under certain circumstances a slight tint of yellow, gray, brown, or pink. A slight yellow or a decided brown color is often developed in potato cultures of bacteria which upon other culture media present a col- orless growth, and which are generally included by bacteriologists among the non-chromogenic species. With reference to independent BACTERIOLOGICAL DIAGNOSIS. 737 movements, it must be remembered that some of the motile bacteria, under certain circumstances, do not exhibit active movements ; the question of motility must therefore not be hastily decided in the negative from a single examination. Spore formation also, in many cases, depends upon special conditions, and great care will often be required in determining this character, which, indeed, is still unde- termined for some of the best-known species. We have endeavored in the present volume to include all bacteria which have been described by competent bacteriologists with suffi- cient detail to permit of their recognition when carefully studied by the usual methods. But we have also included a considerable num- ber which are imperfectly described and which could not be identi- fied by the descriptions given. And no doubt a certain number of those which have been described under different names are in fact identical or simply varieties of a single species. The plan adopted of grouping the different bacteria described with reference to their mor- phological and biological characters will bring together those micro- organisms which are similar, and will, we trust, be of assistance to working bacteriologists in determining identity or non-identity. Im- perfect descriptions, if not completed by future researches, may be eliminated hereafter, but we have thought it best to give them a place in this Manual, as most of them are included in systematic works published abroad — e. g. , the micrococci found by Koch in his experimental study of traumatic infectious diseases (1878), Bien- stock's faeces bacilli, etc. LIST OF BACTERIA DESCRIBED. PART THIRD, SECTION IV. — PYOGENIC BACTERIA. 1. Staphylococcus pyogenes aureus (Rosenbach). Micrococcus of infectious osteomyelitis (Becker). 2. Staphylococcus pyogenes albus (Rosenbach). Staphylococcus epidermidis albus (Welch). o. Staphylococcus pyogenes citreus (Passet). 4. Micrococcus pyogenes tennis (Rosenbach). 5. Streptococcus pyogenes (Rosenbach). Micrococcus of erysipelas (Fehleisen). Streptococcus of pus. Streptococcus longus (Von Lingelsheim). 0. Micrococcus gonorrhceaa. Gonococcus (Neisser). PART THIRD, SECTION V. — BACTERIA IN CROUPOUS PNEUMONIA. 7. Bacillus of Friedlander. 738 BACTERIOLOGICAL DIAGNOSIS. Pneumococcus (Friedlander). Bacillus pneumonias (Fliigge). 8. Micrococcus pneumonias crouposae. Micrococcus Pasteuri (Sternberg). Micrococcus of sputum septicaemia (Frankel). Diplococcus pneumonias (Weichselbaum). Bacillus septicus sputigenus (Fliigge). Bacillus salivarius septicus (Biondi). Lancet-shaped micrococcus (Talamon). Streptococcus lanceolatus Pasteuri (Gameleia). PART THIRD, SECTION VI. — PATHOGENIC MICROCOCCI NOT DESCRIBED IN SECTIONS IV. AND V. 9. Diplococcus intercellularis meningitidis (Weichselbaum). 10. Staphylococcus salivarius pyogenes (Biondi). 11. Micrococcus of progressive tissue necrosis in mice (Koch). 12. Micrococcus of progressive abscess formation in rabbits (Koch). 13. Micrococcus of pysemia in rabbits (Kocl,i). 14. Micrococcus of septicaemia in rabbits (Koch). 15. Micrococcus salivarius septicus (Biondi). 10. Micrococcus subflavus (Fliigge). Yellowish-white micrococcus (Bumm). 17. Micrococcus of trachoma ? (Sattler). 18. Micrococcus tetragenus (Gaffky, Koch). 19. Micrococcus botryogenus (Rabe). Micrococcus of " Myko-desmoids " of the horse. Micrococcus askoformans (Johne). Micrococcus Johnei (Cohn). 20. Micrococcus of Manfredi. Micrococcus of progressive granuloma formation (Manfredi). "21. Micrococcus of bovine mastitis (Kitt). 22. Micrococcus of bovine pneumonia ? (Poels arid Nolen). 23. Streptococcus septicus (Fliigge). 24. Streptococcus bombycis. Microzyma bombycis (Bechamp). 25. Nosema bombycis. Micrococcus ovatus. Panhistophyton ovatum. 20. Micrococcus of Heydenreich. Micrococcus of Biskra button— " Clou de Biskra," Fr.; " Pen- desche Geschwur," Ger. 27. Micrococcus of Demme. Diplococcus of pemphigus acutus (Demme). BACTERIOLOGICAL DIAGNOSIS. 739 28. Streptococcus of Manneberg. 29. Micrococcus endocarditidis rugatus (Weichselbaum). 30. Micrococcus of gangrenous mastitis in sheep (Nocard). 31. Streptococcus of mastitis in cows (Nocard and Mollereau). 32. Diplococcus of pneumonia in horses (Schiitz). 33. Streptococcus coryzae contagiosa3 equorum (Schiitz). 34. HEematococcus bovis (Babes). 35. Micrococcus gingivse pyogenes (Miller). 36. Pseudodiplococcus pneumonise (Bononie). 37. Streptococcus septicus liquefaciens (Babes). 38. Micrococcus of Kirchner. 39. Micrococcus No. II. of Fischel. 40. Streptococcus of Bonome. 41. Micrococcus of Almquist. 42. Staphylococcus pyosepticus (Hericourt and Richet). 43. Streptococcus perniciosus psittacorum. Micrococcus of gray parrot disease (Eberth and Wolff). 44. Micrococcus of Forbes. PART THIRD, SECTION VII. — THE BACILLUS OF ANTHRAX. 45. Bacillus anthracis. Milzbrandbacillus, Ger. La bacteridie du charbon, Fr. PART THIRD, SECTION VIII. — THE BACILLUS OF TYPHOID FEVER. 43. Spirillum Obermeieri. Spirochsete Obermeieri. Spirillum of relapsing fever. 744 BACTERIOLOGICAL DIAGNOSIS. Recurrensspirochate. 154. Spirillum anserum (Sakharoff). 155. Spirillum cholerce Asiatics (Koch). Spirillum of Asiatic cholera. Comma bacillus of Koch. Bacille-virgule cholerigene. 15G. Spirillum of Finkler and Prior. Vibrio proteus. 157. Spirillum tyrogenum. Kasespirillen (Deneke). Cheese spirillum of Deneke. 158. Spirillum Metschnikovi. Vibrio Metschnikovi (Gameleia). PART FOURTH, SECTION VIII.— NON-PATHOGENIC MICROCOCCI. 159. Micrococcus flavus liquefaciens (Fliigge). 160. Micrococcus flavus desidens (Fliigge). 1G1. Micrococcus agilis (Ali-Cohen). 162. Micrococcus fuscus (Maschek). 1G3. Diplococcus citreus conglomeratus (Bumm). 104. Diplococcus citreus liquefaciens (Unna). 105. Diplococcus flavus liquefaciens tardus (Unna). 1GG. Diplococcus fluorescens foetidus (Klamann). 1G7. Diplococcus luteus (Adametz). IGfS. Diplococcus roseus (Bumm). 100. Micrococcus cremoides (Zimmermann). 170. Micrococcus roseus (Eisenberg). 171. Micrococcus aurantiacus (Colin). 17^. Micrococcus cerasinus siccus (List). 17o. Micrococcus versicolor (Fliigge). 174. Micrococcus of Dantec. 175. Micrococcus carneus (Zimmermann). 170. Micrococcus cinnabareus (Fliigge). 177. Micrococcus cereus albus (Passet). 178. Micrococcus cereus flavus (Passet). 170. Micrococcus citreus. Cremefarbiger micrococcus (List). 180. Micrococcus fervidosus (Adametz). 181. Micrococcus flavus tardigratus (Fliigge). 182. Micrococcus luteus (Cohn). 183. Micrococcus violaceus (Cohn). 184. Staphylococcus viridis flavescens (Guttmann). 185. Micrococcus ochroleucus (Prove). 18G. Micrococcus acidi lactici liquefaciens (Kreuger). BACTERIOLOGICAL DIAGNOSIS. 745 187. Micrococcus aerogenes (Miller). 188. Micrococcus albus liquefaciens (Von Besser). 189. Micrococcus fcetidus (Klamann). 190. Micrococcus radiatus (Fliigge). 191. Diplococcus albicans amplus (Bumm). 192. Micrococcus candicans (Fliigge). 193. Micrococcus candidus (Colin). 194. Micrococcus acidi lactici (Marpmann). 195. Micrococcus lactis viscosus (Conn). 196. Sphaerococcus acidi lactici (Marpmann). 197. Micrococcus aquatilis (Bolton). 198. Micrococcus concentricus (Zimmermann). 199. Micrococcus cumulatus tenuis (Von Besser). 300. Micrococcus plumosus (Brautigam). 301. Micrococcus rosettaceus (Zimmermann). 302. Micrococcus urese (Pasteur). 303. Micrococcus ureae liquefaciens (Fliigge). 304. Micrococcus viticulosus (Katz). 305. Diplococcus albicans tardissimus (Eisenberg). Milk-white diplococcus (Bumm). 306. Diplococcus albicans tardus (Unna). 307. Staphylococcus albus liquefaciens. White liquefying staphylococcus (Escherich). 308. Micrococcus ovalis (Escherich). 309. Diplococcus coryzse (Hajek). 310. Micrococcus Finlayensis (Sternberg). 311. Micrococcus of Freire. Cryptococcus xanthogenicus (Freire). 312. Streptococcus coli gracilis (Escherich). 313. Streptococcus acidi lactici (Grotenfeld). 314. Streptococcus giganteus urethras (Lustgarten). 315. Streptococcus albus (Maschek). 216. Streptococcus vermiformis (Maschek). 217. Streptococcus brevis (Von Lingelsheim). Streptococcus cadaveris (Sternberg). 218. Streptococcus Havaniensis (Sternberg). 219. Streptococcus liquefaciens (Sternberg). 230. Micrococcus tetragenus versatilis (Sternberg). 331. Pediococcus albus (Lindner). 332. Pediococcus acidi lactici (Lindner). 333. Pediococcus cerevisiss (Balcke). 334. Micrococcus tetragenus mobilis ventriculi (Mendoza). 335. Micrococcus tetragenus subflavus (Von Besser). 226. Sarcina aurantiaca. 746 BACTERIOLOGICAL DIAGNOSIS. 227. Sarcina lutea (Schroter). 228. Sarcina flava (De Bary). 229. Sarcina rosea (Schroter). 230. Sarcina alba (Eisenberg). 231. Sarcina Candida (Reinke). 232. Sarcina pulmonum (Hauser). 233. Sarcina ventriculi (Goodsirj. 234. Micrococcus amylovorus (Burrill). 235. Ascococcus Billrothii (Cohn). 236. Leuconostoc mesenteroides (Cienkowski). PART FOURTH, SECTION IX. — NON-PATHOGENIC BACILLI. A. Chromogenic, Non-Liquefying Bacilli. 237. Bacterium luteum (List). 238. Bacillus aurantiacus (Frankland). 239. Bacillus brunneus (Adametz). 240. Bacillus aureus (Adametz). 241. Bacillus flavocoriaceus (Eisenberg). Sulphur-yellow bacillus of Adametz. 242. Bacillus berolinensis Indicus (Classen). 243. Bacillus constrictus (Zimmermann). 244. Bacillus fluorescens aureus (Zimmermann). 245. Bacillus fluorescens longus (Zimmermann). 246. Bacillus fluorescens tenuis (Zimmermann). 247. Bacillus fluorescens non-liquefaciens (Eisenberg). 248. Bacillus fluorescens putidus (Fliigge). 249. Bacillus erythrosporus (Eidam). 250. Bacillus viridis pallescens (Frick). 251. Bacillus virescens (Frick). 252. Bacillus iris (Frick). 253. Bacillus fuscus (Zimmermann). 254. Bacillus rubefaciens (Zimmermann). 255. Bacillus striatus flavus (Von Besser). 256. Bacillus subflavus (Zimmermann). 257. Bacillus cyanogenus (Hueppe). 258. Bacillus fuscus limbatus (Scheibenzuber). 259. Bacillus latericeus (Eisenberg). Ziegelroter bacillus (Adametz). 260. Bacillus spiniferus (Unna). 261. Bacillus rubescens (Jordan). 262. Bacillus allii (Griffiths). BACTERIOLOGICAL DIAGNOSIS. 747 B. Cliromogenic, Liquefying Bacilli. 263. Bacillus fulvus (Zimmermann). 264. Bacillus helvolus (Zimmermann). 265. Bacillus ochraceus (Zimmermann). 266. Bacillus plicatilis (Zimmermann). 267. Bacillus janthinus (Zopf). Violet bacillus. 268. Bacillus violaceus Laurentius (Jordan). 269. Bacillus tremelloides (Schottelius). 270. Bacillus cuticularis (Tils). 271. Flesh-colored bacillus (Tils). 272. Bacillus arborescens (Frankland). 273. Bacillus citreus cadaveris (Strassmann). 274. Bacillus membranaceus amethystinus (Eisenberg). 275. Ascobacillus citreus (Unna). 276. Bacillus cceruleus (Smith). 277. Bacillus fluorescens liquefaciens (Fliigge). 278. Bacillus fluorescens liquefaciens minutissimus (Unna). 279. Bacillus fluorescens nivalis (Schmolck). 280. Bacillus lactis erythrogenes (Hueppe). 281. Bacillus glaucus (Maschek). 282. Bacillus lividus (Plagge and Proskauer). 283. Bacillus Indicus (Koch). 284. Bacillus prodigiosus. Micrococcus prodigiosus. Monas prodigiosa. 285. Bacillus mesentericus ruber. Rothen Kartoffelbaciilus (Globig). 286. Bacillus pyocyanus ft (Ernst). 287. Bacillus mycoides roseus (Scholl). 288. Bacillus rosaceum metalloides (Dowdeswell). 289. Bacillus viscosus (Frankland). 290. Bacillus violaceus. 291. Bacillus sulfureum (Holschewnikoff). 292. Bacillus rubidus (Eisenberg). 293. Bacterium termo of Vignal. 294. Bacillus buccalis minutus. Bacillus g of Vignal. 295. Bacillus of Canestrini. (Pathogenic for bees.) 748 BACTERIOLOGICAL DIAGNOSIS. C. Non-chromogetiic, Non-liquefying Bacilli. 296. Bacillus ubiquitus (Jordan). 297. Bacillus candicans (Frankland). 298. Bacillus albus (Eisenberg). 299. Bacillus acidi lactici (Hueppe). 300. Bacillus limbatus acidi lactici (Marpmann). 301. Bacillus lactis pituitosi. Bacillus der schleimigen Milch (Loftier). 302. Bacillus aerogenes (Miller). 303. Bacterium aerogenes (Miller). 304. Heliobacterium aerogenes (Miller). 305. Bacillus aquatilis sulcatus No. I. (Weichselbaum). 306. Bacillus aquatilis sulcatus No. II. (Weichselbaum). 307. Bacillus aquatilis sulcatus No. III. (Weichselbaum). 308. Bacillus aquatilis sulcatus No. IV. (Weichselbaum). 309. Bacillus aquatilis sulcatus No. V. (Weichselbaum). 310. Bacillus multipediculus (Fliigge). 311. Bacillus cystiformis (Clado). 312. Bacillus hepaticus fortuitus (Sternberg). 313. Bacillus intestinus motilis (Sternberg). 314. Bacillus caviee fortuitus (Sternberg). 315. Bacillus coli similis (Sternberg). 316. Bacillus filiformis Havaniensis (Sternberg). 317. Bacillus Martinez (Sternberg). 318. Bacillus epidermidis (Bizzozero). 319. Bacillus nodosus parvus (Lustgarten). 320. Bacillus hyacinthi septicus (Heinz). 321. Bacterium gliscrogenum (Malerba). 322. Bacillus ovatus minutissimus (Unna). 323. Capsule bacilli of Smith. 324. Bacillus putrificus coli (Bienstock). 325. Bacillus subtilis simularis No. I. (Bienstock). 326. Bacillus subtilis simulans No. II. (Bienstock). 327. Bacillus striatus albus (Von Besser). 328. Bacillus stolonatus (Adametz). 329. Bacillus ventriculi (Raczynssky). 330. Bacterium Zopfii (Kurth). 331. Bacterium Zurnianum (List). 332. Bacillus of Colomiatti. 333. Bacillus scissus (Frankland). 334. Bacillus No. I. of Fulles. 335. Bacillus No. II. of Fulles. 336. Bacillus phosphorescens gelidus (Forster). BACTERIOLOGICAL DIAGNOSIS. 749 337. Bacillus smaragdino-phosphoresceiis (Katz). 338. Bacillus argenteo-phosphorescens No. I. (Katz). 339. Bacillus argenteo-phosphorescens No. II. (Katz). 340. Bacillus argenteo-phosphorescens No. III. (Katz). D. Non-cliromogenic, Liquefying Bacilli. 341. Bacillus cyaneo-phosphorescens (Katz). 342. Bacillus argenteo-phosphorescens liquefaciens (Katz). 343. Bacillus phosphorescens Indicus (Fischer). 344. Bacillus phosphorescens indigenus (Fischer). 345. Bacillus circulans (Jordan). 346. Bacillus superficialis (Jordan). 347. Bacillus reticularis (Jordan). 348. Bacillus hyalinus (Jordan). 349. Bacillus cloacae (Jordan). 350. Bacillus delicatulus (Jordan). 351. Bacillus aquatilis (Frankland). 352. Bacillus diffusus (Frankland). 353. Bacillus liquidus (Frankland). 354. Bacillus vermicularis (Frankland). 355. Bacillus nubilus (Frankland). 356. Bacillus pestifer (Frankland). 357. Bacillus filiformis (Tils). 358. Bacillus devorans (Zimmermann). 359. Bacillus gracilis (Zimmermann). . 360. Bacillus guttatus (Zimmermann). 361. Bacillus implexus (Zimmermann). 362. Bacillus punctatus (Zimmermann). 363. Bacillus radiatus aquatilis (Zimmermann). 364. Bacillus vermiculosus (Zimmermann). 365. Bacillus aerophilus (Liborius). 366. Bacillus mycoides (Fliigge). 367. Bacillus mesentericus vulgatus (Fliigge). Kartoffelbacillus. 4f-« «. Bacillus mesentericus fuscus (Fliigge). Bacillus megatherium (De Bary). Bacillus albus putidus (De Bary). ,-, • Bacillus brassicse (Pommer). Bacillus butyricus of Hueppe. Bacillus gasoformans (Eisenberg). Bacillus carabiformis (Kaczynsky). Bacillus graveolens (Bordoni-Uffreduzzi). Bacillus carotarum (A. Koch). 64 750 BACTERIOLOGICAL DIAGNOSIS. 377. Bacillus inflatus (A. Koch). 378. Bacillus ramosus. Wurtzel bacillus. 379. Bacillus subtilis (Ehrenberg). 380. Bacillus subtilis similis (Sternberg). 381. Bacillus leptosporus (L. Klein). 382. Bacillus sessilis (L. Klein). 383. Bacillus allantoides (L. Klein). 384. Bacillus of Scheurlen. 385. Bacillus lactis albus (Loftier). 386. Bacillus liodermos (Loffler). 387. Bacillus ulna (Conn). 388. Bacillus ulna of Vignal. 389. Bacillus liquefaciens (Eisenberg). 390. Bacillus maidis (Cuboni). 391. Proteus sulfureus (Lindenborn). 392. Bacillus thormophilus (Miquel). 393. Bacillus tumescens (Zopf). 394. Bacillus buccalis maximus (Miller). 395. Leptothrix buccalis of Vignal. 396. Bacillus b of Vignal. 397. Bacillus / of Vignal. 398. Bacillus buccalis fortuitus. Bacillus j of Vignal. 399. Bacillus Havaniensis liquefaciens (Sternberg). 400. Bacillus liquefaciens communis (Sternberg). E. Strictly Anaerobic Bacilli. 401. Bacillus muscoides (Liborius). 402. Bacillus solidus (Liideritz). 403. Bacillus polypiformis (Liborius). 404. Bacillus butyricus (Prazmowski). Bacillus amylobacter. Clostridium butyricum. 405. Clostridium fostidum (Liborius). 406. Bacillus liquefaciens magnus (Liideritz). 407. Bacillus liquefaciens parvus (Liideritz). 408. Bacillus radiatus (Liideritz). 409. Bacillus spinosus (Liideritz). 410. Bacillus anaerobicus liquefaciens (Sternberg). PART FOURTH, SECTION X. — NON-PATHOGENIC SPIRILLA. 411. Spirillum sputigenum (Miller). 412. Spirillum dentium. BACTERIOLOGICAL DIAGNOSIS. 751 Spirochsete denticola. 413. Spirillum plicatile. Spirochcete plicatilis (Ehrenberg). 414. Vibrio rugula (Miiller). 415. Spirillum volutans (Ehrenberg). 416. Spirillum sanguineum (Warming). Ophidomonas sanguinea. 417. Spirillum serpens (Miiller). 418. Spirillum undula (Ehrenberg). 419. Spirillum tenue (Ehrenberg). 420. Spirillum linguae. Vibrio lingualis (Weibel). 421. Spirillum nasale. Vibrio nasalis. Nasenschleim vibrio (Weibel). 422. Spirillum a of Weibel. Vibrio saprophiles a. (Weibel). 423. Spirillum ft of Weibel. Vibrio saprophiles fi (Weibel). 424. Spirillum y of Weibel. Vibrio saprophiles y (Weibel). 425. Spirillum aureum. Vibrio aureus (Weibel). 426. Spirillum flavescens. Vibrio flavescens (Weibel). 427. Spirillum flavum. Vibrio flavus (Weibel). 428. Spirillum concentricum (Kitasato). 429. Spirillum rubrum (Von Esmarch). 430. Spirillum of Smith. 431. Spirillum of Miller. Miller's bacillus. PART FOURTH, SECTION XI. — LEPTOTRICHEJE AND CLADOTRICHE^E. 432. Crenothrix Kiihniana (Rabenhorst). 433. Beggiatoa alba (Vauch.). 434. Beggiatoa roseo-persicina (Zopf). « Clathrocystis roseo-persicina (Cohn). Ophidomonas sanguinea (Ehrenberg). Bacterium rubescens (Lankester). 435. Beggiatoa mirabilis (Cohn). 436. Phragmidiothrix multiseptata (Engler). 752 BACTERIOLOGICAL DIAGNOSIS. 437. Cladothrix dichotoma (Colin). 438. Cladothrix Foersteri. Streptothrix Foersteri (Cohn). 439. Cladothrix intricata (Russell). PART FOURTH, SECTION XII. — ADDITIONAL SPECIES OF BACTERIA, NOT CLASSIFIED. 440. Nitromonas of Winogradsky. 441 . Nitrifying bacillus of Winogradsky. 442. Streptococcus conglomeratus (Kurth). 443. Bacillus thalassophilus (Russell). 444. Bacillus granulosus (Russell). 445. Bacillus limosus (Russell). 44G. Spirillum marinum (Russell). 447. Bacillus litoralis (Russell). 448. Bacillus halophilus (Russell). 449. Bacillus capsulatus mucosus' (Fasching). 450. Bacillus of potato rot (Kramer). 451. Bacillus vacuolosis (Sternberg). 452. Bacillus of Dantec. 453. Bacillus Havaniensis (Sternberg). 454. Bacillus amylozyma (Perdrix). 455. Bacillus rubellus (Okada). 456. Bacterium urese (Jaksch). 457. Sarcina mobilis (Maurea). 458. Bacillus stoloniferus (Pohl). 459. Bacillus incanus (Pohl). 400. Bacillus inunctus (Pohl). 461. Bacillus flavescens (Pohl). 462. Bacillus butyricus of Botkin. 463. Urobacillus Pasteuri (Miquel). 464. Urobacillus Duclauxi (Miquel). 465. Urobacillus Freudenreichi (Miquel). 466. Urobacillus Maddoxi (Miquel). 467. Urobacillus Schiitzenbergi (Miquel). 468. Bacillus of Bovet. 469. Bacillus Schafferi (Freudenreich). 470. Bacilli of Guillebeau — a, b, c — (Freudenreich). 471. Micrococcus Freudenreichi (Guillebeau). 472. Bacterium Hessii (Guillebeau). 473. Bacillus denitrificans (Giltay and Aberson). 474. Bacillus cyano-fuscus (Beyerinck). 475. Bacillus pyogenes soli (Bolton). BACTERIOLOGICAL DIAGNOSIS. 753 476. Micrococcus aquatilis invisibilis (Vaughan). 477. Bacillus gracilis anaerobiescens (Vaughan). 478. Bacillus figurans (Vaughan). 479. Bacillus albus anaerobiescens (Vaughan). 480. Bacillus invisibilis (Vaughan). 481. Bacillus venenosus (Vaughan). 482. Bacillus venenosus brevis (Vaughan). 483. Bacillus venenosus invisibilis (Vaughan). 484. Bacillus venenosus liquefaciens (Vaughan). 485. Bacillus aerogenes capsulatus (Welch). 486. Bacillus of Canon and Pielicke. 487. Bacillus sanguinis typhi (Brannan and Cheesman). 488. Micrococcus agilis citreus (Menge). 489. Bacillus gracilis cadaveris (Sternberg). BACTERIOLOGICAL DIAGNOSIS. MICROCOCCI. I. Staphylococci — micrococci, solitary, in pairs, in irregular groups, and occasionally in short chains or in groups of four. A. Grow in nutrient gelatin at the room temperature (20° to 22° C.) and liquefy the gelatin. a. Chromogenic ; pigment yellow: Staphylococcus pyogenes aureus (1). Staphylococcus pyogenes citreus (3). Micrococcus flavus liquefaciens (159). Micrococcus citreus liquefaciens (164). Micrococcus flavus desidens (160). Micrococcus cremoides (169). Micrococcus of Almquist (41). Micrococcus Finlayensis (210). Staphylococcus salivarius pyogenes (10). a. Pigment red or pink : Micrococcus fuscus (162). Micrococcus roseus (170). Micrococcus agilis (161). I) Non-chromogenic : Staphylococcus pyogenes albus (2). Staphylococcus pyosepticus (42). Micrococcus of Freire (211). Micrococcus albus liquefaciens (188). Micrococcus of gangrenous mastitis in sheep (30). Micrococcus urese liquefaciens (203). Micrococcus aerogenes (187). 754 BACTERIOLOGICAL DIAGNOSIS. Micrococcus radiatus (190). Micrococcus foetidus (189). B. Grow in nutrient gelatin at the room temperature (20° to 22° C.), and do not liquefy the gelatin. a. Chromogenic ; pigment yellow : Micrococcus versicolor (173). Micrococcus aurantiacus (171). Micrococcus cereus flavus (178). Micrococcus flavus tardigratus (181). Micrococcus luteus (182). Micrococcus agilis citreus (488). a. Pigment greenish-yellow : Staphylococcus viridis flavescens (184). a. Pigment violet : Micrococcus violaceus (183). a. Pigment red or pink : Micrococcus carneus (175). Micrococcus cinnabareus (176). Micrococcus cerasinus siccus (172). b. Non-chromogenic : Micrococcus candicans (192). Micrococcus cereus albus (177). Micrococcus concentricus (198). Micrococcus fervidosus (180). Micrococcus of bovine pneumonia ? (22). Micrococcus acidi lactici (194). Micrococcus aquatilis (197). Micrococcug cumulatus tenuis (199). Micrococcus ureee (202). Micrococcus of bovine mastitis (21). Micrococcus salivarius septicus (15). Micrococcus gingivsB pyogeries (35). Micrococcus rosettaceus (201). Micrococcus aquatilis invisibilis (476). C. Do not grow in nutrient gelatin at the room temperature : Micrococcus endocarditidis rugatus (29). Nitromonas of Winogradsky (440). D. Biological characters not determined : Micrococcus pyogenes tenuis (4). Micrococcus of progressive abscess formation in mice(12) Micrococcus of pysemia in rabbits (13). Micrococcus of Forbes (44). Nosema bombycis (25). BACTERIOLOGICAL DIAGNOSIS. 755 II. Micrococci united in zooglcea masses by an intercellular substance: Micrococcus viticulosis (204). Micrococcus plumosus (200). Micrococcus candidus (193). Micrococcus amylovorus (234). Ascococcus Billrothi (235). Leuconostoc mesenteroides (236). III. Micrococci in pairs — diplococci, not forming chains. A. Grow in nutrient gelatin at the room temperature (20° to 22° C.), and liquefy the gelatin. a. Chromogenic ; pigment yellow : Diplococcus subflavus (16) — stains by Gram's method. Micrococcus botryogenus (19). Diplococcus flayus liquefaciens tardus (165). a. Pigment red : Diplococcus roseus (168). b. Non-chromogenic : Diplococcus albicans amplus (191). Micrococcus of Heydenreich (26). B. Grow in nutrient gelatin at the room temperature, and do not liquefy ; non-chromogenic. a. Stain by Gram's method : Micrococcus of Manfredi (20). Micrococcus of trachoma ? (17). b. Do not stain by Gram's method : Diplococcus of pneumonia in horses (32). Hsematococcus bovis (34). Diplococcus albicans tardissimus (205). c. Staining by Gram's method not determined : Diplococcus coryzse (209). C. Do not grow in nutrient gelatin at the room temperature ; non- chromogenic ; do not stain by Gram's method : Diplococcus intercellularis meningitidis (9). Micrococcus gonorrhoeas (6). Micrococcus of Kirchner (38). Micrococcus of Demme (27). D. Biological characters imperfectly determined : Micrococci of Disse and Taguchi (p. 404). IV. Micrococci which multiply by division in one direction only, forming diplococci and streptococci. A. Grow in nutrient gelatin at the room temperature (20° to 22° C.), and liquefy the gelatin. 756 BACTERIOLOGICAL DIAGNOSIS. a Chromogenic , pigment yellow : Diplococcus luteus (167). a. Pigment green : Diplococcus fluorescens fcetidus (166). b. Non-chromogenic. 1 Grow on potato : Micrococcus No. II. of Fischel (39). Micrococcus lactis viscosus (195). Streptococcus liquefaciens (219). Streptococcus of Manneberg (28). Streptococcus coli gracilis (212). Micrococcus Freudenreichi (471). Streptococcus albus (215). 2. Does not grow on potato : Streptococcus septicus liquefaciens (37). B. Grow in nutrient gelatin at the room temperature, and do not liquefy. a. Chromogenic ; pigment yellow : Micrococcus citreus (179). Micrococcus ochroleucus (185). b. Non-chromogenic. 1. Grow on potato : j Streptococcus brevis (217). ( Streptococcus cadaveris. Pseudodiplococcus pneumoniae (36). Streptococcus vermiformis (216). 2. Do not grow on potato • coagulate milk : Streptococcus pyogenes (5). Streptococcus of mastitis in cows (31). 3. Growth on potato not stated : Streptococcus coryzae contagiosae equorum (33). Streptococcus septicus (23). Streptococcus acidi lactici (213). Streptococcus conglomeratus (442). C. Do not grow in nutrient gelatin at the room temperature : Micrococcus pneumonias crouposae (8). Streptococcus of Bonome (40). Streptococcus giganteus urethra (214). D. Biological characters imperfectly known : Streptococcus perniciosus psittacorum (43). Streptococcus bombycis (24). Streptococcus Havaniensis (218). Micrococcus of progressive tissue necrosis in mice(ll). BACTERIOLOGICAL DIAGNOSIS. 757 V. Micrococci which multiply by division in two directions, forming diplococci and tetrads. A. Grow in nutrient gelatin at the room temperature, and liquefy the gelatin. a. Chromogenic ; pigment yellow : Diplococcus citreus conglomeratus (163). Micrococcus tetragenus versatilis (220). b. Non-chromogenic : Micrococcus acidi lactici liquefaciens (186). Pediococcus albus (221). B. Grow in nutrient gelatin at the room temperature and do not liquefy. Non-chromogenic : Pediococcus acidi lactici (222). Micrococcus tetragenus mobilis ventriculi (224). Pediococcus cerevisisB (223). Micrococcus tetragenus (18). C. Do not grow in nutrient gelatin at the room temperature : Micrococcus tetragenus subflavus (225). Micrococcus gonorrhoeas (6). VI. Micrococci which multiply by division in three directions, form- ing cubical ' ' packets " — sarcince. A. Grow in nutrient gelatin at the room temperature, and liquefy the gelatin. a. Chromogenic ; pigment yellow : Sarcina aurantiaca (226). Sarcina lutea (227). Sarcina flava (228). a. Pigment red : Sarcina rosea (229). Sarcina mobilis (457). b. Non-chromogenic: Sarcina alba (230). Sarcina Candida (231). B. GroAy in nutrient gelatin at the room temperature, and do not liquefy. Non-chromogenic : Sarcina ventriculi (233). Sarcina pulmonum (232). 758 BACTERIOLOGICAL DIAGNOSIS. BACILLI. I. Aerobic bacilli (many of the bacilli in this group are facultative1 anaerobics). A. Growin nutrient gelatin at the room temperature (20° to 22° C. ),. and liquefy the gelatin, a. Chromogenic ; pigment yellow : 1. Motile ; grow on potato : Bacillus ochraceus (365). Bacillus buccalis minutus (294). Bacillus plicatilis (266), Bacillus citreus cadaveris (273). Bacillus fulvus (263). Bacillus arborescens (272). 2. Non-motile or undetermined ; grow on potato : Bacillus cuticularis (270). Bacillus hydrophilus fuse us (81). Ascobacillus citreus (275). Bacillus helvolus (264). Bacterium termo of Vignal (293). Bacillus lactis erythrogenes (280). Does not grow upon potato ; phosphorescent : Bacillus argenteo-phosphorescens liquefaciens (342). a. Pigment greenish-yellow or green ; grow on potato. 1. Motile : Bacillus fluorescens liquefaciens (277). Bacillus fluorescens liquefaciens minutissimus (278), Bacillus fluorescens nivalis (279). Bacillus chromo-aromaticus (134). Bacillus viscosus (289). Bacillus pyocyanus (96). Bacillus pyocyanus ft (286). 2. Non-motile : Bacillus smaragdinus fcetidus (78). a. Pigment violet or blue ; grow on potato. 1. Motile : Bacillus violaceus (290). Bacillus violaceus Laurentius (268). Bacillus janthinus (267). Bacillus lividus (282). Bacillus cyano-fuscus (474). 2. Non-motile : Bacillus membranaceus amethystinus (274). Bacillus cceruleus (276). BACTERIOLOGICAL DIAGNOSIS. 759» a. Pigment red, pink, or brown. 1. Motile. f Form spores : Bacillus of Canestrini (295). Bacillus of Dantec (452). Bacillus mesentericus ruber (285). * Spore formation not observed : Bacillus rubidus (292). Bacillus sulfureum (291). Bacillus rosaceum metalloides (288). Flesh-colored bacillus (271). Bacillus Indicus (283). 2. Non-motile ; spore formation not observed : Bacillus mycoides roseus (287). Bacillus glaucus (28.1). Bacillus prodigiosus (284). Bacillus tremelloides (269). b. Non-chromogenic. 1. Motile. f Form spores : Bacillus subtilis (379). Bacillus subtilis similis (380). Bacillus of Scheuiieii (384). Bacillus Hessii (472). Bacillus circulans (345). Bacillus mycoides (366). Bacillus mesentericus vulgatus (367). Bacillus mesentericus fuscus (368). Bacillus tumescens (393). Bacillus alvei (140). Bacillus butyricus, Hueppe (372). Bacillus liodermos (386). Bacillus ramosus (378). Bacillus megatherium (369). Bacillus of potato rot (450). Bacillus maidis (390). Bacillus inflatus (377). Bacillus gracilis (359). Bacillus lactis albus (385). Vibrio rugula (414). Bacillus limosus (445). Urobacillus Maddoxi (466). Bacillus vacuolosis (451). Urobacillus Pasteuri (463). BACTERIOLOGICAL DIAGNOSIS. Urobacillus Duclauxi (464). Urobacillus Freudenreichi (465). * Spore formation not observed. x. Grow on potato : Bacillus radiatus aquatilis (363). Bacillus vermiculosus (364). Bacillus guttatus (360). Bacillus pestifer (356). Bacillus nubilis (355). Bacillus albus putidus (370). Bacillus punctatus (362). Bacillus hyalinus (348). Bacillus cloacae (349). Bacillus liquefaciens (389). Bacillus liquidus (353). Bacillus diffusus (352). Bacillus delicatulus (350). Bacillus foetidus ozaenoe (111). Bacillus septicus ulceris gangrsenosi (127). Bacillus albus cadaveris (129). Bacillus leporis lethalis (94). Bacillus liquefaciens communis (400). Bacillus reticularis (347). Proteus vulgaris (97)— polymorphous. Proteus septicus (101)— polymorphous. Proteus mirabilis (99)— polymorphous. Proteus of Karlinsky (98)— polymorphous. Proteus sulfureus (391)— polymorphous. Urobacillus Schiitzenbergi (467). Bacillus b of Guillebeau (470). Bacillus figurans (478). Bacillus venenosus liquefaciens (484). Bacillus phosphorescens Indicus (343). Bacillus litoralis (447). Bacillus halophilus (448). Bacillus stoloniferus (458). y. Do not grow on potato : Bacillus superficialis (346). Bacillus devorans (358). Bacillus aquatilis (351). Bacillus Havaniensis liquefaciens (399). Bacillus cyaneo-phosphorescens (341). Bacillus phosphorescens indigenus (344). BACTERIOLOGICAL DIAGNOSIS. 761 z. Growth on potato not determined : Bacillus gasoformans (373). Bacillus of Schou (114). Bacillus carabiformis (374). 2. Non-motile. f Form spores : Bacillus anthracis (45). Bacillus brassicaa (371). Bacillus carotarum (37G). Bacillus of Tricomi (128). Bacillus vermicularis (354). Bacillus aerophilus (365). Bacillus of Letzerich (109). Bacillus implexus (361). Bacillus filiformis (357). Bacillus granulosus (444). * Do not form spores, or undetermined : Bacillus ulna of Vignal (388). Leptothrix buccalis of Vignal (395). Bacillus/ of Vignal (397). Bacillus b of Vignal (396). Bacillus buccalis fortuitus (398). Bacillus varicosus conjunctivas (130). Bacillus pulpee pyogenes (124). Bacillus gingivse pyogenes (122). Bacillus graveolens (375). Pneumobacillus liquefaciens bo vis (120). Bacillus incanus (459). Bacillus inunctus (460). B. Grow in nutrient gelatin at the room temperature, and do not liquefy, a. Chromogenic ; pigment yellow. 1. Motile ; grow on potato ; spore formation not observed : Bacillus aurantiacus (238). Bacillus subflavus (256). Bacillus constrictus (243). Bacillus aureus (240). Bacillus fluorescens aureus (244). Bacillus heminecrobiophilus (148). Bacillus flavescens (461). 2. Non-motile ; spore formation not observed : Bacillus spiniferus (260) — grows on potato. Bacillus of cholera in ducks (62) — grows on potato. BACTERIOLOGICAL, DIAGNOSIS. Bacillus fuscus (253) — grows on potato. Bac. of Tizzoni and Giovannini (145) — grows on potato. Bacillus flavocoriaceus (241). Bacillus luteum (237). •3. Motility not determined ; grows on potato : Bacillus striatus flavus (255). •a. Pigment yellowish-green or green. 1. Motile ; grow on potato. f Form spores : Bacillus of Lesage (106). Bacillus erythrosporus (249). * Do not form spores : Bacillus fluorescens longus (245). Bacillus fluorescens tenuis (246). Bacillus virescens (251). Bacillus fluorescens putidus (248). Bacillus canalis parvus (136). Bacillus dentalis viridans (123). 2. Non-motile ; do not form spores ; grow on potato : Bacillus fluorescens iion-liquefaciens (247). Bacillus iris (252). -a. Pigment violet or blue ; grow on potato. 1. Motile. t Forms spores ( ?) : Bacillus cyanogenus (257). * Do not form spores, or undetermined. Bacillus viridis pallescens (250). Bacillus beroliniensis Indicus (242). Bacillus cyanogenus Jordaniensis (257). Usually Bacillus cuniculicida Havaniensis (93). j" not motile. Booker's bacilli, d, e, f, g, h, k, n (89). ) Motility not Bacillus cavicida (69). j stated- Bacillus of Bovet (468). Bacillus Schafferi (469). Bacillus a of Guillebeau (470). Bacillus c of Guillebeau (470). Bacillus gracilis anaerobiescens (477). Bacillus invisibilis (480). Bacillus venenosus (481). Bacillus venenosus brevis (482). Bacillus venenosus invisibilis (483). y. Do not g'row on potato : Bacillus aquatilis sulcatus No. IV. (308). BACTERIOLOGICAL DIAGNOSIS. Bacillus argenteo-phosphorescens No. 1 (338). Bacillus argenteo-phosphorescens No. 3 (340). z. Growth on potato undetermined : Bacterium Zopfii (330). Bacillus ventriculi (329). Bacillus of Utpadel (139). Bacillus cystiformis (311). 2. Non-motile, t Form spores : Bacillus of Colomiatti (332). Bacillus acidi lactici (Hueppe) (299). Bacillus subtilis simulans No. I. (325). Bacillus epidermidis (318). Bacillus coprogenes fcetidus (116). * Spore formation not observed. x. Grow on potato : Bacillus diphtherise (47). Bacillus diphtherise columbrarum (49). Bacillus septicsemise hsemorrhagicse (61). Bacillus of Tommasoli (113). Bacillus pyogenes soli (475). Bacillus pneumosepticus (79). Bacillus acidiformans (93). Bacillus lactis aerogenes (91). Bacillus capsulatus mucosus (449). Bacterium urese (456). Bacillus ubiquitus (296). Bacillus scissus (333). Bacillus of Gessner (133). Bacillus of purpura hsemorrhagica of Kolb (147). Bacillus of purpura hsemorrhagica of Babes (146). Bacillus albus anaerobiescens (479). Bacillus tenuis sputigenus (82). Bacillus No. II. of Roth (143). Bacillus of Schimmelbusch (110). Bacillus crassus sputigenus (71). Bacillus Ziirnianus (331). Bacillus of Fiocca (96). Bacillus of intestinal diphtheria in rabbits (51). Bacillus coli similis (315). Bacillus limbatus acidi lactici (300). Bacillus multipediculus (310). Bacillus No. II. of Fulles (335). Bacillus candicans (297). a — c3 o BACTERIOLOGICAL DIAGNOSIS. 765 Bacillus lactis pituitosi (301). Bacillus striatus albus (327). Bacillus of Belfanti and Pascarola (64). Bacillus hepaticus fortuitus (312). Bacillus phosphorescens gelidus (336). Bacillus smaragdino-phosphorescens (337). Bacillus of Friedlander (7). Bacillus of rhinoscleroma (58). Capsule bacilli of Smith (323). Bacillus capsulatus (80). Bacillus canalis capsulatus (135). Proteus hominis capsulatus (73). Proteus capsulatus septicus (74). y. Do not grow on potato : Bacillus erysipelatos suis (67). Bacillus gallinarum (77). Bacillus of grouse disease (76). Bacillus pseudotuberculosis (121). Bacillus of Okada (144). Bacillus filiformis Havaniensis (316). Bacillus nodosus parvus (319). Bacillus Martinez (317). Bacillus argenteo-phosphorescens No. II. (339). z. Growth on potato not determined : Bacillus septicus keratomalacise (125). Bacillus oxytocus perniciosus (117). Bacillus of acne contagiosa of horses (141). Bacillus endocarditidis capsulatus (105). Pseudo-diphtheritic bacillus (48). 3. Motility not determined : Bacillus septicus agrigenus (66). Bacillus ovatus minutissimus (322). C. Do not grow in nutrient gelatin at the room temperature, but have been cultivated in other media. 1. Motile: Bacillus mallei (56) — grows on potato. Bacillus of Lumnitzer (112) — forms spores. 2. Non-motile : Bacillus tuberculosis (53). Bacillus tuberculosis gallinarum (54). Bacillus of Demme (107). Bacillus of Nocard (60) — grows on potato. Bacillus sanguinis typhi (487). 3. Motility not determined : Bacillus necrophorus (115). 65 766 BACTERIOLOGICAL DIAGNOSIS. Bacillus of Kartulis (138). Bacillus septicus acuminatus (126). Nitrifying bacillus of Winogradsky (441). Bac. of Canon and Pielicke — measles bacillus ? (486). D. Growth in nutrient gelatin not determined, but have been cul- tivated in other media. a. Chromogenic. f Pigment green : Bacillus allii (262). * Pigment blue : Bacillus indigogenus (137). b. Non-chromogenic. 1. Motile. f Form spores : Bacillus leptosporus (381). Bacillus ulna (387). * Spore formation not observed : Bacillus allantoides (383). 2. Non-motile. f Forms spores . Bacillus sessilis (382). * Spore formation not determined : Bacillus coprogenes parvus (68). II. Strictly anaerobic bacilli. A. Grow in nutrient gelatin at the room temperature, and liquefy the gelatin. 1. Motile ; form spores : Bacillus tetani (149). Bacillus of symptomatic anthrax (152). Bacillus cedematis maligni (150). Bacillus spinosus (409). Bacillus rubellus (455). Bacillus butyricus of Botkin (462). Clostridium foetidum (405). Bacillus liquefaciens magnus (406). Bacillus radiatus (408). Bacillus thalassophilus (443). 2. Non-motile ; form spores : Bacillus liquefaciens parvus (407). Bacillus anaerobicus liquefaciens (410). B. Grow in nutrient gelatin at the room temperature, and do not liquefy. 1. Motile ; form spores : Bacillus polypiformis (403). BACTERIOLOGICAL DIAGNOSIS. 767 Bacillus solidus (402). Bacillus amylozyma (454). 2. Non-motile ; forms spores : Bacillus muscoides (401). 3. Non-motile ; does not form spores : Bacillus aerogenes capsulatus (485). C. Growth in nutrient gelatin not determined, but have been cul- tivated in other media. 1. Motile ; forms spores : Bacillus butyricus (404). 2. Non-motile ; spore formation not observed : Bacillus cadaveris (151). III. Have not been cultivated in artificial media : Bacillus leprse (cultivation claimed) (54). Bacillus diphtherise vitulorum (50). Bacillus of Lustgarten (57). Bacillus buccalis maximus (394). SPIRILLA. Aerobic spirilla (many of the species are also facultative anaerobics). A. Grow in nutrient gelatin at the room temperature, and liquefy the gelatin. 1. Motile ; grow upon potato ; spore formation not deter- mined : Spirillum cholerse Asiaticse (155). Spirillum of Finkler and Prior (156). Spirillum tyrogenum (157). Spirillum Metschnikovi (158). Spirillum of Miller (431). Spirillum marinum (446). B. Grow in nutrient gelatin, and do not liquefy : a. Chromogenic : f Pigment j^ellow ; non-motile : Spirillum flavum (427). Spirillum aureum (425). * Pigment yellowish-green ; non-motile : Spirillum flavescens (426). I Pigment red ; motile : Spirillum rubrum (429). b. Non-chromogenic : f Motile : x. Grow on potato : Spirillum saprophiles a (422). 768 BACTERIOLOGICAL DIAGNOSIS. Spirillum saprophiles 0 (423). Spirillum saprophiles y (424). Spirillum of Smith (430). y. Does not grow on potato : Spirillum concentricum (428). * Non-motile : Spirillum nasale (421). Spirillum linguae (420). C. Biological characters not determined. Pathogenic : Spirillum Obermeieri (153). Spirillum anserum (154). Non-pathogenic, or undetermined : Spirillum serpens (417). Spirillum sanguineum (416). Spirillum dentium (412). Spirillum tenue (419). Spirillum undula (418). Spirillum volutans (415). Spirillum sputigenum (411). Spirillum plicatile (413). BIBLIOGRAPHY. PART FIRST. I. HISTORICAL. 1. MULLER, O. F. Animalia infusoria fluv. et marina. 1786. 2. EHRENBERG. Die Infusionsthierchen als vollkommene Organismen. Leipzig, 1838. 3. DUJARDIN. Histoire naturelle des zoophytes. Paris, 1841. 4. ROBIN. Histoire des vegetaux parasites. 5. DAVAINE. Dictionnaire encyclop. des sciences med., art. Bacteries. 1868. 6. Recherches sur les maladies charbonneuses. Compt. rend. Acad. des Sc., t. Iviii., pp. 220, 351, 386, and lix., p. 393. 7. SPALLANZANI. 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Acad. des Sc., t. xc., 1880, pp. 239-248. 16. Le rouget du pore ; aveo la collaboration de MM. Chamberlain, Roux et Thuillier. Compt. rend. Acad. des Sc., t. xcv , p. 1120. 17. Nouveaux fails pour servir a la connaissance de la rage; avec la colla- boration de MM. Chamberlain, Roux et Thuillier. Bull. Acad. de Med., 2eme s., xi., pp. 1440-1445. 18. HOFFMANN. Mykologische Studien ilber Gahrung. Annalen der Chemie und Pharmacie, vol. cxv., 1860, p. 228. 770 BIBLIOGRAPHY. 19. TYNDALL. Essays on floating matter of the air. London, 1881. 20. Coim. Beitrage zur Biologic der Pflanzen, vol. ii., 1876, p. 263. 21. KOCH. Die ^Etiologie der Milzbrandkrankheit. Beitrage zur Biologic dcr Pflanzen, vol. ii., 1876. 22. - Ueber Desinfektion. Mitth. aus dem K. Gesundheitsamte, Bd. i., 1881. 23. Zur Untersuchung von pathogenen Organismen. Mitth. aus dem K. Gesundheitsamte, Bd. i., 1882, pp. 1-48. 24. Wundinfektionskrankheiten. Leipzig, 1878. 25. Die yEtiologie der Tuberculose. Berliner klin. Wochenschrift, xix., 1851, pp. 221-230. 26. Ueber die Cholerabakterien. Deutsche med. Wochenschrift, 1884. 27. WEIGERT. Berliner klin. Wochenschrift, Nos. 18 and 19, 1877. 28. OBERMEIER. Vorkommen feinster, eigene Bewegung zeigender Faden im Blute von Recurrenskranken. Berliner klin. Wochenschrift, 1873. 29. HANSEN. Bacillus leprse. Nord. med. Ark., Stockholm, xii., 1880, pp. 1-10. 30. NEISSER. Centralbl. filr die med. Wiss., 1879, No. 28. 31. EBERTH. Der Bacillus des Abdominaltyphus. Virchow's Archiv, Bd. Ixxxi. and Ixxxiii. 32. GAFFKY. Zur ^tiologie des Abdominaltyphus. Mitth. aus dem K. Gesund- heitsamte, Bd. ii., 1884. 33. STERNBERG. A fatal form of septicaemia in the rabbit produced by the sub- cutaneous injection of human saliva. Nat. Board of Health Bull. , Wash- ington, vol. ii., 1881, p. 781. 34. LOFFLER UND ScHUTZ. Ueber den Rotzpilz. Deutsche med. Wochenschrift, 1882, No. 52. 35. LOFFLER. Untersuchungen liber die Bedeutung fur die Entstehung der Diph- theritis bei Menschen, etc. Mitth. aus dem K. Gesundheitsamte, Bd. ii.r 1884. 36. ROSENBACH. Mikroorganismen bei den Wundinfektionskrankheiten des Men- schen. Wiesbaden, 1884. 37. NICOLAIER. Beitrage zur ^Etiologie des Wundstarrkrampfes. Inaug. Diss. Gottingen, 1885. (First publication in Deutsche med. Wochenschrift, 1884, No. 52.) 38. PFEIFFER. Vorlaufige Mittheilungen liber den Erreger der Influenza. Deutsche med. Wochenschrift, 1892, Nos. 2 and 3. II. CLASSIFICATION. 39. EHRENBERG. Die Infusionsthierchen als vollkommene Organismen. Leipzig 1838. DUJARDIN. Op. cit. (No. 3). DAVAINE. Op. cit. (No. 5). 40. HOFFMANN. Memoire sur les bacteries. Ann. des Sc. Nat. Bot., 5eme s., t. xi., 1869. 41. NAGELI. Die niederen Pilze. Munchen, 1877. Untersuchungen ilber niedere Pilze, Munchen, 1882. 42. SACHS. Lehrbuch der Botanik. Leipzig, 1874. (English edition, 1882.) 43. COHX. Beitrage zur Biologic der Pflanzen, Bd. i. (1873), ii. (1877), iii. (1879). 44. MAGNIN. Les bacteries. Paris, 1878. 45. ZOPF. Die Spaltpilze. Breslau, 1884. 46. DE BARY. Vergleichende Morphologic und Biologic der Pilze, Mycetozoen und Bakterien. Leipzig, 1834 ; Vorlesungen ilber Bakterien, 1885. BIBLIOGRAPHY. 771 47. HUEPPE. Die Methoden der Bakterien- Forschung, 3d ed., 1886. 48. FLUGGE. Die Mikroorganismen, 2d ed., Leipzig, 1886. 49. BAUMGARTEN. Lehrbuch der pathologischeu Mykologie. Braunschweig, 1890. III. MORPHOLOGY. See Bibliography previously referred to, Nos'. 39 to 49. IV. STAINING METHODS. 50. WEIGERT. Berliner klia. Wochenschrift, 1877, Nos. 18 and 19. 51. Zur Technik der ruikroskopischeii Bakterieuuntersuchungen. Virchow's Archiv, Bd. Ixxxiv., p. 275. 52. KOCH. Verfahren zur Untersuchung, zum Conserviren und Photographiren der Bakterien. Cohii's Beitrage zur Biologie der Ptianzen, Bd. ii., Heft 3. Also : Zur Untersuchung von pathogenen Organismen. Mitth. aus dem K. Gesundheitsamte, Bd. i., 1881. . 53. BUCHNER. Ueber das Verhalten der Spaltpilz-Sporen zu den Anilin-Farben. Miinchener arztl. Intelligenzbl., 1884, No. 33. HUEPPE. Op. cit. (No. 47). 54. NEISSER'S method of staining spores. Zeitschrift fur klin. Med., 1884, p. 1. 55. LOFFLER. Eine neue Methode zum Filrben der Mikroorganismen, im beson- deren ihrer Wimperhaare und Geisseln. Centralbl. fiir Bakteriol., Bd. vi., Nos. 8 and 9. Also: Weitere Untersuchungen, etc. Centralbl. fiir Bak- teriol., Bd. viii., No. 20. 56. KUHNE. Praktische Anleitung zum mikroskopischen Nachweis der Bakterien im thierischen Gewebe. Leipzig, 1888. 57. NEISSEK. Kleine Beitrage zur bakterioskopischen Technik. Centralbl. fiir Bakteriol., Bd. iii., Nos. 16 and 17, 1888. 58 EIIRLICH. Zeitschrift fur klin. Med., Bd. i., 1880 ; ibid., Bd. ii., p. 710. Also: Deutsche med. Wochenschrift, 1882, No. 19. 59. GRAM. Ueber die isolirte Farbung der Schizomyceten. Fortschr. der Med., Bd. ii., 1884, No. 6. 60. GOTTSTEIX. Ueber Entfarbung gef arbter Zellkerne und Mikroorganismen durch Salzlosungen. Fortschr. der Med., 1835, p. 627. 61. BAUMGARTEN. Ueber ein bequemes Verfahren Tubercle-Bacillen in phthisischen Sputis nachzuweisen. Centralbl. fiir die med. Wissensch., 1882, No. 25. 62. PLAUT. Fiirbuugsmethode z. Nachweis der Mikroorganismen, H84. 2d ed., 1885. 63. UNNA. Die Entwickluug der Bakterienfarbung. Centralbl. fiir Bakteriol., Bd. iii., 1888. 64. TRENKMANN. Die Farbung der Geisseln von Spirillen und Bacillen. Centralbl. fiir Bakteriol., Bd. viii., p. 385. 65. MOLLEK. Ueber eine neue Methode der Sporenfiirbung. Centralbl. fur Bak- teriol., Bd. x., 1891, p. 273. 66. HEIM. Die Neuerungen auf dem Gebiete der bakteriologischen Untersuchungs- methoden seit dem Jahre 1837. Centralbl. fur Bakteriol., Bd. x., 1891, pp. 260, 288, 323. 67. PREGL. Ueber eine neue Karbolmethylenblau-Methode. Centralbl. fiir Bak- teriol., Bd. x., 1891, p. 826. 772 BIBLIOGRAPHY. V. CULTURE MEDIA. 68. PASTEUR. Etudes sur la biere, 1867. 69. BREFELD. Methoden zur Uatersuchung der Pilze. Verhandl. des physik. med. Ges.- in Wlirzburg, N. P., Bd. viii., 1874-75. Also : Kulturmethoden zur Untersuckung der Pilze. Bot. Unters. tiber Schimmelpilze, Bd. iv., 1881. KOCH. Op. cit. (No. 23). HUKPPE. Op. cit. (No. 47). 70. Ueber die Verwendung von Eiern zu Kulturzwecken. Centralbl. fur Bakteriol., Bd. iv., p. 80. 71. Roux ET NOCARD. Sur la culture du bacille de la tuberculose. Annales de 1'Institut Pasteur, t. i., p. 19. 72. KARLINSKY. Eine Vorrichtung zum Filtriren vollstandig klaren Agar-Agars. Centralbl. fur Bakteriol., Bd. viii., p. 643. 73. KUHNE. Zeitschrift fur Biologic, Bd. xxvii., p. 172. 74. WINOGRADSKY. Annales de Hnstitut Pasteur, t. iv., p. 213. 75. SELESKIN. Die Kieselsauregallerte als Nahrsubstrat. Centralbl. filr Bakteriol., Bd. x., 1891, p. 209. 76. BOLTON. A method of preparing potatoes for bacterial cultures. Med. News, vol. i., 1837, p. 318. 77. RASKINA. Bereitung durchsichtiger fester Nahrboden aus Milch, etc. Abstract in Baumgarten's Jahresbericht, Bd. iii., p. 480. 78. VON ROZSAHEGYI. Ueber das Zilchten von Bakterien in gefiirbter Nahrgelatine. Centralbl. fur Bakteriol., Bd. ii., p. 418. 79. SPINA, A. Bakteriologische Versuche mit gefiirbten Nahrsubstanzen. Cen- tralbl. filr Bakteriol., Bd. ii., p. 71. 80. VON FREUDENREICH. Zur Bereitung des Agar-Agar. Centralbl. filr Bakteriol., Bd. iii., p. 797. 81. NOEGGERATH. Ueber eine neue Methode der Bakterienziichtung auf gefarbten Nahrmedien zu diagnostischen Zwecken. Fortschritte der Med., Bd i., p. 1. 82. VAN PUTEREN. Ueber Bereitung von festem Nahrboden aus Milch, etc. Ab- stract in Centralbl. fur Bakteriol , Bd. v., p. 181. 83. Roux. De la culture sur pomme de terre. Annales de 1'Institut Pasteur, vol. ii., 1888, p. 28. 84. PUCCINELLI. II f ucus crispus nella preparazione dei terrani nutritivi dei batteri. Bull. d. Reale Accad. Med. di Roma, 1890, fasc. iv.-v. 85. HELLER. Der Harn als bakteriologischer Nahrboden. Centralbl. fur Bakte- riol., Bd. ix., p. 511. 86. STERNBERG. Cocoanut water as a culture fluid. Med. News, Philadelphia, 1890, p. 262. 87. TISCHUTKLN. Eine vereinfachte Methode der Bereitung von Fleisch-Pepton- Agar. Centralbl. filr Bakteriol., Bd. ix., p. 208. 88. UNNA. Der Dampftrichter. Centralbl. far Bakteriol., Bd. ix., p 749. 89. VAN OVERBECK DE MEYER. Ueber die Bereitung des Niihragars. Centralbl. filr Bakteriol., Bd. ix., p. 163. 90. KAUFMANN. Ueber einen neuen Nahrboden f iir Bakterien. Centralbl. f ilr Bak. teriol.,Bd. x., 1891, p. 65. 91. SCHULTZ. Zur Frage von der Bereitung einiger Nilhrsubstrate. Centralbl. fur Bakteriol., Bd. x., 1891, p. 52. 92. LAGEUUEIM. Macaroni als fester Nahrboden. Centralbl. f iir Bakteriol., Bd xi., 1892, p. 147. BIBLIOGRAPHY. 773 VI. STERILIZATION OF CULTURE MEDIA. 93. KOCH TJND WOLFFHUGEL. Untersuchungen iiber die Desinf ektion mit heisser Luft. Mitth. aus dera K. Gesundheitsamte, Bd. i., 1881. 94. KOCH, GAFFKY TJND LOFFLER. Untersuchungen ilber die Disinfektion mit heisser Luft. Mitth. aus dem K. Gesundheitsamte, Bd. i., 1881. 95. STERNBERG. The thermal death point of pathogenic organisms. Am. Jour. Med. Sc., Philadelphia, xciv., 1887, pp. 146-160. 96. GLOBIG. Ueber Bakterien-Wachsthuin bei 50° to 70°. Zeitschrift fur Hygiene, Bd. iii., p. 294. Also : Ueber einen Kartoffelbacillus mit ungewohnlich widerstandsfahigen Sporen. Ibid., Bd. iii., p. 322. 97. MIQUEL. Les organismes vivants de 1'atmosphere, 1883, p. 182. 98. VAN TIEGHEM. Societe botanique de France. Bulletins, t. xxviii., p. 35. 99. HEYDENREICH. Sur la sterilisation des liquides au moyen de la marmite de papin. Compt. rend. Acad. desSc., vol. xcviii., 1884. 100. TYNDALL. Philos. Trans, of the Royal Soc., London, 1877. 101. PLADT. Zur Sterilisationstechnik. Centralbl. fiir Bakteriol., Bd. iii., 1888, p. 100. 102. VIQUER.YT. Ein einfacher kupferner Sterilisirungsapparat. Centralbl. fur Bakteriol., Bd. vi., 1889, p. 602. 103. DOR. De la sterilisation de 1'eau par le filtre Chamberlain. Lyon Medical, 1889, No. 23. 104. BITTER. Versuche uber das Pasteurisiren der Milch. Zeitschrift fur Hygiene, Bd. viii. 105. BUJWID. Eine einfache Filtervorrichtung zum Filtriren sterilisirter Fliissig- keit. Centralbl. filr Bakteriol., Bd. ix., p. 4. 106. D'ARSONVAL. Emploi de 1'acide carbonique liquefie pour la filtration et la sterilisation rapide des liquides organiques. Compt. rend. Acad. des Sc., t. cxii., 1891, p. 6f57. VII. CULTURES IN LIQUID MEDIA. 107. PASTEUR. Memoire sur la fermentation appelee lactique. Compt. rend. Acad. des Sc., t. xlv., 1857, p. 913. Etude sur la biere, 1876. 108. KLEBS. Ueber fraktionirte Kultur. Arch, fur exp. Path. undPharm., Bd. i., 1873. 109. LISTER. On the lactic fermentation and its bearings on pathology. Trans. Path. Soc. of London, vol. xxix., 1878. 110. BREFELD. Botanische Untersuchungen ilber Schimmelpilze, vol. iv., 1881, and vol. v., 1883. 111. DUCLAUX. Ferments et maladies. Paris, 1883. 112. STERNBERG. In Bacteria, Magnin and Sternberg, 1883, pp. 175-179. 113. HUEPPE. Die Methoden der Bakterien-Forschung, 3d ed., 1886. VIII. CULTURES IN SOLID MEDIA. 114. KOCH. Zur Untersuchung von path. Organismen. Mitth. aus dem K. Gesund- heitsamte, Bd. i., 1881. 115. Blutserum. Berliner klin. Wochenschrift, 1882, No. 15. 116. Mitth. aus dem K. Gesundheitsamte, Bd. ii., 1884. 117. SALOMONSON. Zur Isolation differenter Bakterien. Bot. Zeitung, 1879, No. 39. 118. Eine einfache Methode zur Reinkultur versch. Faulnissbakterien. Ibid., 1880, No. 28. 774 BIBLIOGRAPHY. 119. BO.NCM. Menschliches Blutserum als Nahrboden f ilr pathogene Mikroorganismen.. Deutsche med. Wochenschrift, 1885, p. 910. 120. ESMARCH, E. Ueber eine Modification dos Koch'schen Plattenverfahrens, etc.. Zeitschrift fur Hygiene, Bd. i., 1886, p. 293. 121. Die Bereitung der Kartoffel als Nahrboden f ilr Mikroorganismen. Cen- tralbl. filr Bakteriol., Bd. i., 1887, p. 26. 122. PETRI, R. J. Eine kleine Modification des Koch'schen Plattenverfahrens. Centralbl. filr Bakteriol., Bd. i., 1887, p. 279. 123. UNNA, P. G. Ueber eine neue Art erstarrten Blutserums und liber Blutserum- platten. Monatshefte filr prakt. Dermatol., Bd. v., 18S6, No. 9. 124. BOCKHART. Ueber eine neue Art der Zubereitung von Fleisch als fester Nahr- boden filr Mikroorganismen. Tagebl. d. 60. Versammlung deutscher Na- turforscher und Aerzte in Wiesbaden, 1887, p. 347. 125. HUEPPE. Ueber Blutserumkulturen. Centralbl. furBakteriol., Bd. i., 1887, p. 607. 126. WILFARTII, H. Ueber eine Modification der bakteriologischen Plattenkulturen. Deutsche med. Wochenschrift, No. 28, 1887. 127. SCHIMMELBUSCFI, C. Eine Modification des Koch'schen Plattenverfahrens. Fortschr. d. Med., Bd. vi., 1838, p. 616. IX. CULTIVATION OF ANAEROBIC BACTERIA. 128. PASTEUR. Comptes rend. Acad. des So., t. Hi., 1861, p. 344; ibid., t. Hi., p. 1260; ibid., t. Ivi., 1863, p. 416; ibid., t. Ivi., p. 1189. 129. GRUBER. Eine Mcthode der Ktiltur auagrobischer Bakterien. Centralbl. filr Bakteriol., Bd. i., 1886, p. 367. 130. Roux. Sur la culture des microbes anafirobies. Annales de 1'Institut Pasteur^ vol. i., 1887, p. 49. 131. BUCHNER. Eine neue Methode zur Kultur anaeTober Mikroorganismen. Cen- tralbl. furBakteriol., Bd. iv., 1888, p. 149. 132. FRANKEL, C. Ueber die Kultur anafirober Mikroorganismen. Centralbl. fur Bakteriol., Bd. iii., 1888, pp. 735 and 763. 133. BRAATZ. Eine neue Vorrichtung zur Kultur von Anaeroben im hangenden Tropfen. Centralbl. fur Bakteriol., Bd. viii., p. 520. 134. KITASATO. Ueber den Rauschbrandbacillus und sein Kulturverfahren. Zeit- schrift filr Hygiene, Bd. vi., p. 105. 135. BLUCHER. Eine Methode zur Plattenkultur anafirober Bakterien. Zeitschrift filr Hygiene, Bd. viii., p. 499. 136. NIKIFOROFF. Ein Beitrag zu den Kulturmethoden der Auagroben. 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Ein neuer Thermostat uud Thermoregulator zum sofortigen Ein- stellen und absoluten Constanthalten jeder beliebigen Temperatur nach Lautenschlager. Centralbl. fur Bakteriol., Bd. v., p. 707. 145. KRASILTSCHICK. Nouvelle etuve, chauffe au petrole, ft temperature reglable 8, volonte. Aunales de 1'Institut Pasteur, t. iii., 1889, p. 166. 146. ALTMANN. Thermoregulator neuer Konstructiou. Centralbl. fur Bakteriol., Bd. ix., p. 791. 147. DESPEIGNES. Nouveau regulateur pour etuve chauffe au petrole. La Province Med., 1890, p. 270. 148. Roux. Sur un regulateur de temperature applicable aux etuves. Annales de 1'Institut Pasteur, t. v., p. 158. 149. BABES. Ueber einige Apparate zur Bakterienuutersuchung. Centralbl. fur Bakteriol., Bd. iv., p. 19. XL EXPERIMENTS UPON ANIMALS. 150. STRAUSS, M. Sur une seringue hypodermique, facile a steriliser. Gaz. heb- dom. de Med. et de Chir., 1886, p. 115. 151. TURSINI. Siringa per richerche battcrioscopiche. Morgagni, 1886. 152. PETRI. Einfacher Apparat zum Einspritzen von Fliissigkeiten filr bakteriolo- gische Zwecke. Centralbl. filr Bakteriol., Bd. iv., p. 785. 153. STROSCHEIN. Eine Injectionsspritze fur bakteriologische Zwecke. Mitth. aus Dr. Brehmer's Heilanstalt f lir Lungenkranke, Wiesbaden, 1889. 154. BUCIINER. Einfacher Zerstaubungsapparat zu InhalatioDsversuchen. Cen- tralbl. fur Bakteriol., Bd. vi., No. 19. 155. CHEYNE. Report on the study of certain of the conditions of infection. British Med. Journ., July 31st, 1886. XII. PHOTOGRAPHING BACTERIA. 156. WOODWARD, J. J. Report to the Surgeon-General of the United States Army on the magnesium and electric lights as applied to photomicrography. Washington, 1870. 157. KOCH. Verfahren zur Untersuchung, zum Conserviren, und Photographiren der Bakterien. Cohn's Beitrage zur Biol. der Pflanz., Bd. ii., Heft 3, 1877. 158. STERNBERG. Photomicrographs, and how to make them. Boston, 1884, 204 pages, 20 plates. 159. Photomicrography by gaslight. Johns Hopkins University Circulars, vol. ix., No. 81, p. 72. 160. Cox, J. D. On some photographs of broken diatom valves, taken by lamp- light. Journ. R. M. S., London, 1884, p. 853. 161. CROOKSIIANK. Photography of bacteria. Illustrated by 86 photographs repro- duced by autotype. London, 1887. 162. ISRAEL. Ueber Mikrophotographie mit starken Objectivsystemen. Virchow's Arch., Bd. cvi., p. 502. 163. NEUIIAUSS. Anleitung zur Mikrophotographie filr Arzte, Botaniker, etc. Berlin, 1887. .776 BIBLIOGRAPHY. 164. NEUHAUSS. Die Entmckelung der Mikrophotographie, etc. Centralbl. filr Bakteriol., Bd. iv., pp. 81 and 111. 165. Verschiedenes ilber Mikrophotographie. Zeitschr. filr wiss. Mikroskopie und fiir mikrosk. Teclmik, Bd. v., p. 484. 166. GUNTIIER. Photogramme der pathogenen Mikroorganismen. Berlin, 1887. 167. KITT. Ueber Mikrophotographien. Oesterr. Monatschr. filr Thierheilk., 1888 p. 241. 168. FRANKEL UND PPEIFFER. Mikrophotographischer Atlas der Bakterienkunde. Berlin, 1889. 169. 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TEXT BOOKS previously referred to : Nageli (41), Sachs (42), Magnin (44), Zopf (45), De Bary (46), Hueppe (47), Flilgge (48), Baumgarten (49). 179. CORNEL ET BABES. Les bacteries. 3d ed., Paris, 1890. II. CONDITIONS OF GROWTH. 180. PASTEUR. Animalcules infusoire vivants sans oxygene libre, etc. Compt. rend. Acad. des Sc., lii., 1861. 181. BOLTON. Ueber das verhalten verschiedener Bakterienarten im Trinkwasser. Zeitschrift filr Hygiene, Bd. i., 1886, p. 76. 182. MIQUEL. Les organistnes vivants de 1'atmosphere, 1883, p. 182. 183. VAN TIEGHE.M. Societe botanique de France. Bulletins, t. xxviii., p. 35. 184. GLOBIG. Ueber Bakterien-Wachsthum bei 50° bis 70° . Zeitschrift fiir Hygiene, Bd. iii., p. 294. 185. FRISCH. Ueber den Einfluss niederer Temperaturen auf die Lebensfahigkeit der Bakterien. Sitzungsb. der K. Akad. Wissensch., Wien, xxxv., 1877, p. 257; ibid.,lxxx., 1879. BIBLIOGRAPHY. <77 186. Roux. Sur la culture du bacille de la tuberculose. 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Journal of the Chemi- cal Society, vol. xlix., pp. 172 and 432. "228. GIUNTI. Ueber die Wirkung des Lichtes auf die Essiggahrung. Le Stazioni Speriment. Agrar. Ital., xviii., p. 171. BUTYRIC ACID FERMENTATION. 229. PASTEUR. Animalcules inf usoires vivant sans oxyg^ne libre, et determinant des fermentations. Compt. rend. Acad. des Sc., t. lii., p. 861. 330. - — Etudes sur la biere. Paris, 1876, p. 282. 231. FITZ. Ueber Spaltpilzgahrungen. Ber. der deutschen chem. Gesellschaft, ix , p. 1348 ; x., p. 176 ; xi., pp. 42 and 1890 ; xiii., p. 1309 ; xv., p. 867. •232. GRUBER. Eine Methode der Kultur anaSrobischer Bakterien, etc. Centralbl. fur Bakteriol., Bd. ii., 1887, p. 367. BIBLIOGRAPHY. 779 233. LIBORIUS. Beitrage zur Kenntniss des Sauersloffbediirfnisses der Bakterien. Zeitschrift fiir Hygiene, Bd. i., 1886, p. 115. 234. PRAZMOWSKI. Untersuclmngen ilber die Entwickelungsgeschichte und Fer- ment wirkung einiger Bakterien. Leipzig, 1880. 235. BOTKIN. Ueber ein neues Bacillus butyricus. Zeitschrift fiir Hygiene, Bd. xi., 1892, p. 421. CAUCASIAN MILK FERMENT. 236. KERN. Ueber ein neues Milchferment aus dem Kaukasus. Bull, de la Soc. Imp. des Naturalistes de Moskau, 1881, No. 3. 237. Dispora caucasica. Biolog. Centralbl., Bd. ii., p. 137. 238. KRAUNHALS. Ueber das kumysabnliche Getrank Kephir. Deutsches Arch. fiir klin. Med., Bd. xxxv., 1884. AMMONIACAL FERMENTATION OF URINE. 239. PASTEUR. Compt. rend. Acad. des Sc., 1., 1860. •240. Ann. de Chim. et de Physiol., Ixiv., 1862. 241. Bull, de 1'Acad. de Med., 1876, No. 27. 242. PASTEUR ET JOUBERT. Compt. rend. Acad. des Sc., Ixxxiii., 1376. 243. BECHAMP. Sur les microzymas de 1'urine. Montp. Medical, 1874. Bull. Acad. deMed., 1874 (Jan. 20th). 244. GUBLER. Fermentation ammoniacale de 1'urine. Compt. rend. Acad. des Sc., 1874. 245. FELZ ET RITER. Etude sur 1'alcalinite des urines, etc. Journ. de 1'anat. et de physiol., 1874. 246. VON JACKSCH. Studien tlber den Harnstoffpilz. Zeitschr. fiir physiol. 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Richerche sul Gliscrobatterio. Estratto dal Rend. della Accad. delle Sci. Fis. e Nat. di Napoli, 18S8. 258. Archives italiennes de biol., x. 259. ADAMETZ. Ueber einen Erreger der schleimigen Milch, Bacillus lactis viscosus. Milch-Zeitung, 1889, No. 48, p. 941. (Abstract in Centralbl. fiir Bak- teriol., Bd. vii., p. 767.) 780 BIBLIOGRAPHY. 260. ADAMETZ. Untersuchungen iiber Bacillus lactis viscosus. Berliner landwirth- schaftliche Jahrbticher, 1891. 261. VAN LAER. Note sur les fermentations visqueuses. (Abstract in Centralbl. fiir Bakteriol., Bd. vii., 1890, p. 308.) 262. KRAMER. Studien iiber die schleimige Gahrung. Sitzungsber. derK. Akad. der Wiss. in Wien. Monatshefte filr Chemie, Bd. x., 1889, p. 467. CELLULOSE FERMENTATION. 263. TAPPEINER. Celluloseverdauung. Fortsclir. der Med., Bd. i., p. 151, undBd. ii., pp. 377, 416. 264. VAN TIEGHEM. Sur la fermentation de la cellulose. Bull, de la Soc. Bot. de France, 1879, p. 25. 265. Compt. rend. Acad. des Sc., 1884. FORMATION OF HYDROSULPHURIC ACID. 266. GAYOU. Sur les alterations des ceufs. Compt. rend. Acad. des .Sc., Ixxxv. , 1877. 267. HOLSCHEWNIKOFF. Ueber die Bildung von Scliwefelwasserstoff durch Bakte* rien. Fortschr. der Med., 1889, p. 201. 268. MIQUEL. Fermentation sulphydrique. Bull, de la Soc. Chim., xxxii., 1879, p. 127. 269. ROSENHEIM. Demonstration von Bakterien mit der Eigenschaft H2S zu ent- wickeln. Deutsche med. Wochenschr. , 1887, p. 530. PUTREFACTION. 270. DONNE. Recherches sur la putrefaction spontanee des oeufs couves. Compt. rend. Acad. des Sc., Iviii., 1864. 271. Note sur la putrefaction des O3ufs. Ibid., Ixv., 1867. 272. GAYOU. Recherches sur les alterations spontanees des oeufs. These. Ann. de 1'Ecole norm, sup., 1875. 273. GAUTIER ET ETARU. Sur le mecanisme de la fermentation putride. Compt. rend. Acad. des Sc., 1882. 274. KENCKI. Ueber den chemischen Mechanismus der Faulniss. Journ. f iir prakt. Chemie, Bd. xvii. 275. Ueber die Zersetzung der Gelatine und des Eiweisses bei der Faulniss mit Pancreas. Bern, 1876. 276. BERGMANN. Das putride Gift und die putride Intoxication. Dorpat, Ib66. 277. GAUTIER. Traite de chimie physiologique. Paris, 1873. 278. - - Bull, de 1'Acad. de Med., 1882. 279. JEANNERET. Untersuch. iiber die Zersetzung von Gelatine und Eiweiss. In- aug. Diss., Leipzig, 1877, and Journ. fur prakt. Chem., 1877. 280. SALKOWSKI. Zur Kenntniss der Eiweissfaulniss. Ber. der deutschen chem. Ges.. xii., pp. 106, 648; xiii., p. 189. 281. BRIEGER Zur Kenntniss der Faulnissalkaloide. Zeitschr. fiir physiol. Chem.,, Bd. vii., 1883. 282. Ueber Spaltungsprodukte der Bakterien. Ibid., Bd. viii., 1884; Bd. ix.,. Heft 1. 283. SALOMONSON. Die Faulniss des Blutes. Kopenhagen, 1877. 284. HAUSER. Ueber Faulnissbakterien. Leipzig, 1885. 285. TAPPEINER. Med. Centralbl., 1884. 286. PANUM. Das putride Gift, etc. Virchow's Archiv, Bd. lx., p. 328. BIBLIOGRAPHY. 7 SI 287. SELMI. Sulle ptoma'iue del alcaloi'di cadaverici. Bologna, 1878. 288. SCHRANK. Untersuchungen uber den im Hiihnerei die stinkende Faulniss her- vorrufenden Bacillus. Wiener med. Jahrb., 1888, p. 303. 289. STRASSMANN UNO STRECKER. Bakterien bei der Leichenfaulniss. Zeitschr. f ilr Medicinalbeamte, 1888. 290. TACKE. Ueber die Entwickelung von Stickstbff bei Faulniss. Landwirthsch. Jahrb., 1887, p. 917. 291. SANFELICE. Contribute alia b'ologia e morfologia dei batterii saprogeni aGrobi e anafirobi. Atti della Accad. Medic, di Roma, Annoxvi., Serie ii., vol. v. 292. FLUGGE. Die Mikroorganismen, pp. 493-500. 293. NENCKI UNO SIEBER. Zur Kenntniss der bei Eiweissgilhrung auftretenden Gase. Sitzungsber. der K. Akad. der Wissensch. in "Wien, 1889. 294. BOVET. Des gas produits par la fermentation anae"robienne. Ann. de Mi- crographie, t. ii., No. 7. SOLUBLE FERMENTS PRODUCED BY BACTERIA. 295. WORTMANN. Ueber das diastatische Ferment der Bakterien. Zeitschrift fur physiol. Chemie, Bd. vi., 1882. 296. 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Rend, della R. Accad. de Lincei, 1886. 317. LAURENT. Experiences sur la reduction des nitrates par les vegetaux. Ann. de llnstitut Pasteur, t. iv., 1890, p. 722. 318. WINOGRADSKY. Recherches sur les organismes de la nitrification. Ann. de 1'In- stitut Pasteur, t. iv., 1890, pp. 213, 257, and 760 ; t. v., 1891, pp. 92 and 577. 319. FRANKLAND, G. C. XJND P. F. Ueber einige typische Mikroorganismen im Wasser und im Boden. Zeitschrift fiir Hygiene, Bd. vi., p. 373. 320. JORDAN AND RICHARDS. Investigations upon nitrification and the nitrifying organism. Rep. Mass. State B. of H., 1890 (Purification of sewage and water, part ii., p. 865) PHOSPHORESCENCE. 321 . FISCHER. Bakteriologische Untersuchungen auf einer Reise nach Westindien. Zeitschrift fur Hygiene, Bd. ii., 1887, p. 54. 322. Ueber einen neuen lichtentwickelnden Bacillus. Centralbl. fiir Bak- teriol., Bd. iii., 1888, p. 105. 323. BEYERINCK. Le pholobacterium luminosum, bacterie lumineuse de la mer du nord. 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Zeitschr. filrklin. Med.,Bd. xviii., 1890. 483. BUCHNER. Ueber die bakterientodtende Wirkung des zellfreien Blutserums. Centralbl. fur Bakteriol., Bd. v., p. 817, and Bd. vi., p. 1. 484. Ueber die nahere Natur der bakterientodtenden Substanz im Blutserum. Centralbl. filr Bakteriol., Bd. vi., p. 561. 485. WURZ. De Faction bactericide du blanc d'oeuf. La Semaine medicale, 1890, p. 21. 486. PRUDDEN. On the germicidal action of blood serum and other body fluids. Medical Record, New York, 1890 (Jan. 25th). 487. FOKKER. Ueber die bakterienvernichtenden Eigenschaften der Milch. Fort- schr. der Medicin, Bd. viii.. p. 7. 488. HANKIN. A bacteria-killing globulin. Proc. Roy. Soc., London, 1890 (May 22d). 489. LEHMANN. Ueber die pilztodtende Wirkung des f rischen Harns des gesunden Menscheu. Centralbl. filr Bakteriol., Bd. vii., 1890, p. 457. 490. OGATA. Ueber die bakterienfeindliche Substanz des Blutes. Centralbl. fur Bakteriol., Bd. ix., p. 597. XIII. PRACTICAL DIRECTIONS FOR DISINFECTION. 491. REPORT OF COMMITTEE ON DISINFECTANTS, AM. PUBLIC HEALTH ASSOCIA- TION, in vol. xiii. of Reports and Papers of the A. P. H. A., 1887 ; also sep- arate volume published by the Association in 1888, 266 pages. 492. ROHE. Methods of practical disinfection. Rep. of Com. on Disinfectants, p. 208. 790 BIBLIOGRAPHY. 498. ROHE. Apparatus for the application of dry and moist heat in disinfection.. Op. cit., pp. 89-115, 37 illustrations. 494. HOLT. The quarantine system of Louisiana ; methods of disinfection practised. Rep. of Com. on Disinfectants, pp. 215-232, 9 illustrations. 495. RAYMOND. Experiments with sulphurous acid gas. Rep. of Com. on Dis- infectants, pp. 65-77. 496. VAUGIIAN. Considerations concerning the practical use of mercuric chloride as- a disinfectant. Rep. of Com. on Disinfectants, p. 47. 497. VON ESMARCH. Der Henneberg'sche Desinfektor. Zeitschr. fur Hygiene, Bd. ii., 1887, p. 342. 498. Der Keimgehalt der Wande und ihre Desinfektion. Ibid., Bd. ii., p. 491. 499. Die desinficirende Wirkung desstromendenuberhitztenDampfes. Ibid., Bd.iv., 1888, p. 197. 500. ' Nachtrag zu der Abhandlung "Die desinficirende "Wirkung des stro- menden iiberhitzten Dampfes." Ibid., p. 398. 501. AUBERT. Nouvelles experiences sur la disinfection des habitations privees ou publiques a 1'aide de 1'acide sulfureux, et sur Faction de cet agent sur les effets meublants. Bull. gen. de Therap., Paris, ex., 1886, pp. 397-408. 502. KOCH UNO WOLFFHUGEL. Ueber den "Werth der schwefligen Saure als Des- infektionsmittel. Mitth. aus dem K. Gesundheitsamte, Bd. i., p. 188. 503. CHAUTEMPS. L'organisation sanitaire de Paris. Hopitaux d'isolement, voi- tures d'ambulances, stations de desinfections. Rapport presente au Conseil municipal. Paris, 1888, 142 pages, 5 pi., 4to. 504. DOBROSLAVINE. Etuve selhydrique pour la disinfection. Revue d'Hygiene, Paris, 1886, viii., p. 487. 505. DUJARDIN-BEAUMETZ. Experiences sur la disinfection des locaux ayant ete occupes par les malades atteints d'affections contagieuses. Bull. Acad. de Med., Paris, xiii., 1884, p. 1261. 506. FLEISCHHAUER UND MITTENZWEIG Priifung des Desinfektions-Apparatus der Stadt Dilsseldorf. Vierteljahrsschr. fiir gerichtl. Med., Berlin, xliv., 1886, p. 120. 507. FORD. Report of the Sanitary Committee of the Board of Health of Philadel- phia on municipal disinfection of clothing, bedding, etc. Rep. Board of Health of Philadelphia, 1885, p. 298. 508. FORSTER. Wie soil der Arzt seine Hiinde reinigen ? Centralbl. fur klin. Med.,. Leipzig, vi., 1885, p. 297. 509. GUTTMANN. Desinfektionsversuche in den Apparaten der ersten Offentlichen Desinfektionsanstalt der Stadt Berlin. Vierteljahrsschr. filr gerichtl. Med., Berlin, xiv., 1886, p. 161. 510. Ueber Desinfektion von Wohnungen. Archiv filr path. Anat., Berlin, cvii., 1887, pp. 459-475. 511. HERSCIIER. Note sur une etuve locomobile it desinfection. Rev. d'Hygiene,, Paris, ix., 1887, p. 738. 512. HOFFMANN. Moderne Desinfektionstechnik mil besonderer Beziehung auf off- entliche Desinfektionsanstalten. Deutsche Vierteljahrsschr. filr offentliche Gesundheitspflege xix., 1887, p. 117. 513. KREIBOHM. Zur Desinfektion der Wohnrilume mit Sublimatdampfen. Zeit- schr. filr Hygiene, Bd. i., p. 363. 514. MERKE. Bemerkungen ilber den fur die Stadt Diisseldorf bestimmten Des- infektions-Apparat. Vierteljahrsschr. fiir gerichtl. Med., Berlin, xliv., 1886, p. 145. 515. Mittheilungen ilber Betriebsergebnisse der ersten offentlichen Desinfek- BIBLIOGRAPHY. 791 tionsanstalt der Stadt Berlin. Deutsche Vierteljahrsschr. fur offentliche- Gesundheitspflege, xix., 1887, p. 311. 516. PARSONS. Report on disinfection by heat. Rep. Med. Off. Local Govt. Board, London, 1885, p. 218. 517. PROUST. De la disinfection a bord. Bull. Acad. de Med., Paris, xviii., 1887, pp. 147-160. 518. DISINFECTION OP RAGS. Report of the Special Com. of the Am. Public Health Association, in annual volume of A. P. II. A. for 1886, vol. xii. 519. SMITH, W. M. Contagious diseases propagated by rags, and the necessity of disinfection. Sanitarian, New York, xv., 1885, pp. 481-524. 520. VALLIN. Quelques experiences sur les etuves a disinfection dans les hopitaux de Paris. Ann. d'Hygiene, Paris, xi., 1884, p. 255. 521. Traite des desinf ectants et de la desinfection. Paris, 1883, 808 pages, 8vo. 522. VINAY. De la valeur pratique des etuves a desinfection. Lyon Medical, 1886, pp. 545-560. 523. - — Ibid., 1887, pp. 67-75. 524. WALZ UND WINDSCHEID. Der neue Desinfektions-Apparat in Dusseldorf. Centralbl. fur allgemeine Gesundheitspflege, Bonn, v., 1836, p. 426. 535. _ — ibid., vi., 1887, p. 208. 526. WEISGERBER. Le lazeret des epidemics a Strasbourg, et le nouvel appareil a desinfecter de M. A. Koch. Revue d'Hygiene, Paris, viii., 1886, p. 497. 527. WEUNICH. Die neuesten Fortschritte in der Desinf ektions-Praxis. Wiener Klinik, xiii., 1887, pp. 337-358. 528. BEHRING. Ueber Desinfektion, Desinfektionsmittel und Desinfektionsmetho- den. Zeitschr. filr Hygiene, Bd. ix., pp. 395-475. 529. WASSILJEW. Die Desinfektion der Choleradejektionen in Hospitalern. Zeit- schr. fur Hygiene Bd. in., 1887, p. 237 530. KUMMELL. Wie soil der Arzt seine Hande desinficiren ? Deutsche med. Wocheusch., 1886, p. 555. 531. WOLFFHUGEL. Ueber Desinfektion mittels Hitze. Gesundheits-Ingenieur, 1887, No. 1. K32. Du MESNII,. La desinfection par la vapeur sous pression et les etuves loco- mobiles dans le departement de la Seine. Ann. d'Hygiene publique, 1888, No. 6. 533. Roux ET REYNES. Sur une nouvelle methode de desinfection des mains du chirurgieu. Compt. rend. Acad. des Sc , t. cvii., 1888, p. 870. 534. LANDSBEKG. Zur Desinfektion der menschlichen Haut mit besonderer Beriick- sichtigung der Hande. Breslau, 1SS8. 535. Zur Desinfektion der Hande des Arztes. Deutsche med.Wochenschr., 1889, No. 2. 536. FiJRBRiXGER. Untersuchungen und Vorschriften ilber die Desinfektion der Hande des Arztes, etc. Wiesbaden, 1888, 55 pages (Bergmann). 537. Entgegnung an Dr. Landsberg. Deutsche med. Wochenschr., 1889, No 2. 538. Zur Desinfektion der Hande des Arztes. Deutsche med. Wochenschr., 1888, No. 48. 539. SALOMONSON UND LEVISON. "Versuche mit verschiedenen Desinfektions-Appa- raten. Zeitschr. filr Hygiene, Bd. iv., 1883, p. 94. 540. SOYKA. Zur Theorie und Praxis der Desinfektion. Prager med. Wochenschr., 1888, Nos. 15 and 16. 541. BUDDE. Neue Konstruktion f ilr Dampf desinfektious-Apparate nebst Versuchen ilber ihre Funktionsfahigkeit. Zeitschr. filr Hygiene, Bd. vii., 1869, p. 269. 792 BIBLIOGRAPHY. 542. EDSON. Disinfection of dwellings by means of sulphur dioxide. New York Med. Record, vol. xxxvi., 1839, p. 533. 5*43. VON GERLOCZY. Versuche ilber die praktische Desinfektion von Abfall stoffen. Deutsche Viertel jahrsschr. fur offentl. Gcsundheitspflege, Band xxi. , 1889, p. 433. 544. NOCHT. Ueber die Verwendung von Kafbolseifenlosung zu Desinf ektions • zwecken. Zeitschr. filr Hygiene, Bd. vii., 1890, p. 521. 545. PFUHL. Ueber Desinfektion der Latrinen mit Kalk. Zeitschr. fur Hygiene, Bd. vii., 1890, p. 363. 546. ROHRBECK. Zur L5sung der Desinf ektionsf rage mit Wasserdampf. Cen- tralbl. filr Bakteriol , Bd. vi., 1689, p. 493. 547. BOLL. Zur Desinfektion der Hande. Deutsche med. Wochenschr., 1890. No. 17. 548. DK GIAXA. Sur I'actiondesinfectantedublanchimentdesmursau lait de chaux Ann. de Micrograph., 1890, p. 305. 549. KIRCHNER. Ueber dieNothwendigkeit und diebeste Art der Sputumsdesinfek- tion bei Lungentuberkulose. Centralbl. filr Bakteriol., Bd. ix., p. 41. 550. TEUSCHNER. Beitrage zur Desinfektion mit Wasserdampf. Zeitschr. filr Hygiene, Bd. ix., p. 492. 551. ALBRECHT. Die erste offentliche Desinf ektionsanstalt der Stadt Berlin. Anst. der Stadt Berlin filr die offentl. Gesundheitspflege, 1886, pp. 174-184. 552. LOFFLER. Welche Massregeln erscheinen gegen die Verbreitung der Diph- theric geboten ? Centralbl filr Bakteriol., Bd. viii., 1890, p. 663. 553. STERNBERG. The disinfection of excreta. Journ. of the Am. Med. Associa- tion, vol. xvii., 1891, p. 289. 554. WELCH. Conditions underlying the infection of wounds (disinfection of the hands). Am. Journ. Med. Sc., Philadelphia, Nov., 1891, p. 461. PAET THIRD. PATHOGENIC BACTERIA. I. MODES OF ACTION. 555. BOCKHART. Ueber secundiire Infektion bei Harnrohrentripper. Monatshefte fur prakt. Dermatol., 1887, No. 19. 556. BUMM. Ueber gonorrhoische Mischinfektionen beim Weibe. Deutsche med. Wochenschr., 1887, p. 1057. 557. STERN UND HIRSCHLER. Beitrag zur Lehre der Mischinfektion. Wiener med. Presse, 1888, p. 973. 558. BABES. Bakteriologische Untersuchungen iiber septische Processe des Kindes- alters. Leipzig, 1889. 559. ANTON UND FUTTERER. Untersuchungen ilber den Typhus abdominalis. Miinchener med. Wochenschr., 1888, p. 315. 560. WELCH. The histological lesions produced by the toxalbumiu of diphtheria. Bull, of the Johns Hopkins Hospital, vol. iii., 1892, p. 17. BIBLIOGRAPHY. 793 II. CHANNELS OF INFECTION. 561. SCHIMMELBUSCH. Infektion aus heiler Haut. Tagebl. der 61. Versammlung deutscher Naturforscher und Aerzte in Koln, 1888, p. 127. 562. ROTH. Ueber das Verhalten der Schleimhaute und der ausseren Haut in Bezug auf ihre Durchlassigkeit fur Bakterien. Zeitschr. filr Hygiene, Bd. iv., 1888, p. 151. 563. BRAUNSCHWEIG. Ueber Allgemeininf ektion von der unversehrten Augenbinde- haut aus. Fortschr. fur Med., 1889, No. 24. 564. KORKUNOFF. Beitrag zur Frage der Infektion durch Mikroorganismen von Seiten des Darmkanals. Abstract in Centralbl. fur Bakteriol., Bd. vi., 1889, p. 445. 565. BUCHNER. Untersuchungen uber den Durchtritt von Infektionserregern durch die intakte Lungenoberflache. Archiv filr Hygiene, Bd. viii., p. 145. 566. HILDEBRANDT. Experimentelle Untersuchuugen (iber das Eindringen patho- gener Mikroorganismen von den Luftwegen und der Lunge aus. Beitrage zurpathol. Anat. undPhysiol., Bd. ii.. 1888, p. 143. 567. KORKUNOFF. Zur Frage von der intestinalen Infektion. Archiv fur Hygiene, Bd. x., p. 485. III. SUSCEPTIBILITY AND IMMUNITY. 568. CHAUVEAU. De la predisposition et de 1'immunite pathologique. Compt. rend. Acad. des Sc., t. Ixxxix., 1879. 569. Du role de 1'oxygfine de 1'air dans 1'attenuation quasi instantanee des cultures virulentes par Faction de la chaleur. Ibid., xcvi., 1883. 570. De 1'attenuation des cultures virulentes par 1'oxygene comprime. Gaz. hebdom. de Med. et de Chir., 1884. 571. Sur la resistance des animaux de 1'esptice bovine au sang de rate et sur la preservation de ces animaux par les inoculations preventives. Compt. rend. Acad. des Sc., xci., 1880, p. 648. 572. Nouvelles experiences sur la resistance des moutons algeriens au sang de rate. Ibid., xc., p. 1396. 573. Des causes qui peuvent faire varier les resultats de 1'inoculation char- bonneuse sur les moutons algeriens ; influence de la quantite des agents in- fectants ; applications a la theorie de 1'immunite. Ibid., xc., p. 1526. 574. Nature de l'immunite des moutons algeriens centre le sang de rate; est-ce une aptitude de race ? Ibid , xci., p. 33. 575. De 1'attenuation des effets [des inoculations virulentes par Temploi de tres-petites quantites de virus. Ibid., xcii., 1881, p. 844. 576. Sur le mecanisme de 1'immunite. Ann. de Tlnstitut Pasteur, t. ii., 1888. 577. PASTEUR. Sur les maladies virulentes, et en particulier sur la maladie appelee vulgairement cholera des poules. Compt. rend. Acad. des Sc., xc., 1880, p. 239. 578. De 1'attenuation du virus du cholera des poules. Ibid., xci., p. 673. 579. Nou velles observations sur 1'etiologieet la prophylaxieducharbon. Ibid., xci., p. 697. 580. De 1'attenuation de virus et de leur retour a la virulence ; avec la collabo- ration de MM. Chamberlain et Roux. Ibid, xcii., 1881, p. 429. ;j81. Le rouget de pore ; avec la collaboration de MM. Chamberlain, Roux et Thuillier. Ibid., xcv., 1882, p. 1120. 582. Une statistique au sujet de la vaccination preventive centre le charbon, portant sur quatre vingt-cinq mille animaux. Ibid., xcv., p. 1250. 794 BIBLIOGRAPHY. 583. PASTEUR. Response au docteur Koch. Revue scient., Paris, xxxi., 1883, pp- 74-84. 584. STERNBERG. Explanation of acquired immunity in infectious diseases. Am. Journ. of the Med. Sciences, April, 1881. 585. Chapter on " Bacteria in Infectious Diseases." Bacteria, Magnin and Sternberg, 1884, pp. 241-252. ;586. Address in Medicine, delivered at Yale University, June 23d, 1892. Popular Science Monthly, Sept., 1892. 587. LOFFLER. Ueber Immunitatsfrage. Mitth. aus dem K. Gesundheitsamte, Bd. i., 1881. 588. KOCH. Ueber die Milzbrandimpfung. Cassel and Berlin, 1882. 589. MASSE. Des inoculations preventives dans les maladies virulentes. Paris, 1883, 590. GRAWITZ. Die Theorie der Schutzimpfung. Virchow's Archiv, Bd. Ixxxiv., 1881. 591. BUCHNER. Eine neue Theorie liber Erziehung von Immunitat gegen Infektions- krankheiten. Miinchen, 1883. 592. Ueber die bakterientodtende Wirkung des zellfreien Blutserums. Cen- tralbl. fur Bakteriol., Bd. v., 1889, No. 25, and Bd. vi., 1890, No. 1. 593. Ueber die mihere Natur der bakterientodtenden. Substanz im Blut- serum. Centralbl. fiir Bakteriol., Bd. vi., 1889. 594. Die chemische Reizbarkeit der Leucocyten und deren Beziehuug zur Entzundung und Eiterung. Berliner klin. Wochenschr. , 1890, No. 47. 595. Ueber Immunitat, deren natilrliches Vorkommen und kiinstliche Erzeu- gung. Munchener med. Wochenschr., 1891, Nos. 32 and 33. 596. STRAUSS ET CHAMBERLAND. Passage de la bacteridie charbonneuse de la mere au foetus. Compt. rend. Acad. des Sc., t. xcv., 1882. 597. MALVOZ. Sur la transmission intraplacentaire des microorganismes. Ann. de llnstitut Pasteur, t. ii., 1838, p. 121. 598. SALMON AND SMITH. Experiments on the production of immunity by hypo- dermic injection of sterilized cultures. Centralbl. fiir Bakteriol., Bd. ii., 1887, p. 543. 599. Roux. Immunite centre la septicemie conferee par les substances solubles. Ann. de 1'Institut Pasteur, t. i., 1888, p. 562. 600. Immunite contre le charbon symptomatique conferee par les substances solubles. Ibid., t. ii., p. 49. 601. Roux ET CHAMBERLAIN. Sur I'immunite contre le charbon conferee par les substances chimique*. Ibid., t. ii., p. 405. '602. WOOLRIDGE. Note on protection in anthrax. Proc. of the Roy. Soc., London, vol. xlii., 1887, p. 312. 603. Versuche liber Schutzimpfung auf chemischem Wege. Archiv fur Anat. und Physol., Physiol. Abthlg., Bd. iii., 1888, p. 527. 604. EMMERICH UND Di MATTEL Vernichtung von Milzbrandbacillen im Organis- mus. Fortschr. der Medicin, 1887, p. 653. 605. Untersuchungen iiber die Ursache der erworbenen Immunitat Ibid.» 1888, No. 19. 606. DE FREUDENKEICH. Antagonisms des bacteries. Ann. de Micrographie, 1889. -607. PFEFFER. Locomotorische Richtungsbewegungen durch chemische Reize. Arbeiten aus dem bot. Institut zu Tubingen, Bd. i., p. 363. -608. ALI-COHEN. Die Chemiotaxis als Hiilfsmittel der bakteriologischen Forschung. Centralbl. fiir Bakteriol., Bd. viii., 1890, p. 161. »609. GABRITCHEWSKY. Sur les proprietes chimiotaxiques des leucocytes. Ann. de 1'Institut Pasteur, t. iv., p. 346. BIBLIOGRAPHY. 795 '610. MASSART ET BOUDET. Recherches sur 1'irritabilite des leucocytes. Journ. de Med., de Chir. et de Pharra., 1890 (Feb. 20th). €11. Le chimiotaxisme des leucocytes et 1'infection microbienne. Ann. de 1'Institut Pasteur, t. v., 1891, p. 417. €12. CHARRIN ET ROGER. Influence de la fatigue sur 1'evolution des maladies micro- biennes. Compt. rend. Soc. Biol., Jan. 24th, 1890. €13. CANALIS ET MORPURGO. Influence du jefine sur la disposition aux maladies in- fectieuses. Resume in Ann. de 1'Institut Pasteur, t. iv., 1890. €14. LEO. Beitrag zur Immunitiltslehre. Zeitschr. filr Hygiene, Bd. vii., 1889, p. 505. €15. BOUCHARD. Essai d'une theorie de 1'infection. Verhandlungen des 10. Inter- nal. Med. Congress, Berlin, 1890. •€16. METSCHNIKOFF. Ueber eiue Sprosspilzkrankheit der Daphnien. Ein Beitrag zur Lehre liber den Kampf der Phagocyten gegen Krankheitserreger. Vir- chow's Archiv, Bd. xcvi., p. 177. >617. Ueber die Beziehung der Phagocyten zu den Milzbrandbacillen. Ibid., Bd. xcvii., p. 502. €18. Sur la lutte des cellules de 1'organisme centre 1'invasion des microbes. Ann. de 1'Institut Pasteur, t. i., 1887, p. 321. €19. Etudes sur I'immunite. Ann. de 1'Institut Pasteur, t. iii., 1889, p. 289 ; 2eme memoire, ibid., t. iv., p. 65; 3eme memoire, ibid., t. iv., p. 193; 4eme memoire, ibid., t. v., 1891, p. 465. 620. Deux travaux du laboratoirc de M. Baumgarten diriges contre la theorie des phagocytes. Ibid., t. iv., 1890, p. 85. Recherches sur 1'accoutumance aux produits microbiens. Ibid., t. v., 1891, p. 567. 622. FAHRENHOLZ. Beitrage zur Kritik der Metschnikoffschen Phagocytenlehre, etc. Inaug. Diss , Konigsberg, 1889. 623. CZAPELEWSKI. Uutersuchungen liber die Immunitiit der Tauben gegen Milz- brand. Beitrag zur allg. Pathol. undpath. Anat., Bd. vii., 1889, p. 47. 624. NUTTALL. Experimente iiber die bakterienf eindlichen Einfliisse des thierischen Korpers. Zeitschr. fur Hygiene, Bd. iv., 1888, p. 353. 625. FLUGGE. Studien iiber die Abschwiichung virulenter Bakterien und die erwor- bene Immunitat. Zeitschr. fur Hygiene, Bd. iv., 18S-8, p. 208. 626. HUEPPE. Historisch-Kritisches iiber den Impfschutz, welchen Stoffwechsel- produkte gegen die virulenten Parasiten verleihen. Fortschr. der Med., Bd. vi., 1888, No. 8. '627. PETRUSCHKY. Untersuchungen iiber die Immuuitat des Frosches gegen Milz- brand. Beitrage zur path. Anat., etc., v. Ziegler, Bd. iii., 1888, p. 357. -628. SIROTININ. Ueber die entwicklungshemmenden Stoffwechselprodukte der Bakterien und die sogenannte Retentionshypothese. Zeitschr. fur Hygiene, Bd. iv., 1888, p. 262. 629. WOLFHEIM. Ein weiterer Beitrag zur Phagocytenlehre. Beitrage zur pathol. Anat., etc., v. Ziegler, Bd. iii., 1888, p. 405. 630. WYSSOKOWITSCH. Ueber die Ursachen der Immunitat. Wratch., 1888, p. 428. Abstract in Centralbl. fur Bakteriol., Bd. v., 1889, p. 103. 631. BITTER. Kritische Bemerkungen zu E. Metschnikoff's Phagocytenlehre. Zeitschr. fur Hygiene, Bd. iv., 1888, p. 299. •632. BOUCHARD. L'influence qu'excerce sur la maladie charbonneuse 1'inoculation du bacille pyocyanique. Compt. rend. Acad. des Sc. , t. cviii., 1889, p. 713. '633. Essai d'une theorie de 1'infection. Transactions of Tenth Intern. Med. Congress, Berlin, 1890. 796 BIBLIOGRAPHY. 634. CHARRIN ET GUIQUARD. Action du bacille pyocyanique sur la bacteridie charbonneuse. Compt. rend. Acad. des Sc., t. cviii., 1889, p, 764. 635. HANKIN. Immunity produced by an albumose isolated from anthrax cultures. Brit. Med. Journ., 1889, p. 810. 636 A bacteria killing globulin. Proc. of the Roy. Soc., London, 1890, May 22d. 637. Report on the conflict between the organism and the microbe. Brit. Med. Journ., 1890, p. 65. 638. Ueber die schiitzenden Eiweisskb" rper der Ratte. Centralbl. filr Bakte- riol., Bd. ix., pp. 336, 372. 639. - — On immunity. The Lancet, 1891, p. 339. 640. BAUMGARTEN. Beitrasje zur pathologischen Mykologie. Experimentelle Ar- beiten tlber die Bedeutung der " Phagocyten " fiir Immunitat und Heilung. Centralbl. fiir klin. Med., 1888, No. 26. 641. Ueber das " Experimentum crucis" der Phagocytenlehre. Beitrage zur patholog. Anat., etc., v. Ziegler, Bd. vii., 1889. 642. LUBARSCH. Ueber die Bedeutung der Metschnikoff'schen Phago<;yten filr die Vernichtung der Bacillen im Froschkorper. Tagebl. der 61. Versammlung deutscher Naturforscher und Aerzte in Koln, 1888, p. 84. 643. Ueber die bakterienvernichtenden Eigenschaften des Blutes und ihre Be- ziehungen zur Immunitat. Centralbl. fiir Bakteriol., Bd. vi., 1889. 644. Ueber die Ursachen der Immunitat. Fortschr. der Med., Bd. viii., 1890V No. 17. 645. • Untersuchungen iiber die Ursachen der angeborenen und erworbenea Immunitat. Zeitschr. fur klin. Med., 1891 (Sep. Abdr., 163 pages). 646. NISSEN. Zur Kenntniss der bakterienvernichtenden Eigenschaft des Blutes.. Zeitschr. fur Hygiene, Bd. vi., 1889, p. 487. 647. TCHISTOVITSCII. Des phenomenes de phagocytose dans les poumons. Ann. de 1'Institut Pasteur, 1889, p. 337. 648. BRIEGER UND FRANKEL. Untersuchungen liber Bakteriengifte. Berliner klin. Wochenschr., 1890, Nos.ll and 12. 649. Ueber Immunisirungsversuche bei Diphtheric. Ibid., 1890, No. 49. 650. BEHRING UND KITASATO. Ueber das Zustandekommen der Diphtheric Im- munitat und der Tetanus-Immunitat bei Thieren. Deutsche med. Wochen- schr., 1890, No. 49. 651. BEHRING. Untersuchungen tiber das Zustandekommen der Diphtherie-Im- munitatbei Thieren. Deutsche med. Wochenschr., 1890, No. 50. 652. KITASATO. Experimentelle Untersuchungen liber das Tetanusgift. Zeitschr. fiir Hygiene, Bd. x., p. 2tt7. 653. G-AMELEIA. Sur le pouvoir antitoxique de 1'organisme animal. La Semaine med., 1890, No 56. 654. OGATA. Ueber die bakterienfeindliche Substanz des Blutes. Centralbl. fiir Bakteriol., Bd. ix., 1891, p. 597. 655. PETRUSCHKY. Der Verlauf der Phagocyten-Contro verse. Fortschr. der Med., Bd. viii., 1890, No. 12. 656. Entgegnung auf F. Hueppe's " Bemerkungen " u. s. w. Fortschr. der Med., Bd. viii., 1890, No. 15. 657. PHISALIX. Etude experimentale sur le role attribue aux cellules lymphatiques,. etc. La Semaine med., t. x., 1890, No. 49. 658. ROGER. Proprietes bactericides du serum pour le streptocoque de 1'erysipele-. Le Bulletin med., 1890, No. 87, p. 966. BIBLIOGRAPHY. 797 659. SAWTSCHENKO. Zur Frage ilber die Immunitat gegen Milzbrand. Centralbl. fiir Bakteriol., Bd. ix., pp. 473, 493, 528. 660. STERN. Ueber die Wirkung des menschlichen Blutes und anderer Korperflils- sigkeiten auf pathogene Mikroorganismen. Zeitschr. fur klin. Med., Bd. xviii., 1890. 661. TIZZONI UND CATTANI. Ueber die Eigenschaften des Tetanus- Antitoxins. Centralbl. fur Bakteriol., Bd. ix., 1891, p. 685. 662. FernereUntersuclmngen liber das Tetanus- Antitoxin. Ibid., Bd. x., 1891, p. 33. 663. KLEMPERER, G. UND F. Versuche ilber Immunisirung und Heilung bei der Pneu- mokokken-Infektion. Berliner klin. Wocheiischr. , 1891, Nos. 34 and 35. 664. EMMERICH UND FOWITZKY. Milnchener med. Wochenschr., 1891, No. 32. 665. EHRLICH. Experimented Untersuchungen ilber Immunitiit. Deutsche med. Wochenschr., 1891, No. 32. 666. Centralbl. fur Bakteriol., Bd. x., 1891, p. 763. 667. Deutsche med. Wochenschr., 1891, No. 44. 668. SANARELLI. Weitere Mittheilungen uber Gifttheorie und Phagocytose. Cen- tralbl. fiir Bakteriol., Bd. x., 1891, p. 514. 669. EMMERICH. Die Ursache der Immunitat, etc. Milnchener med. Wochenschr. , 1891, Nos. 19 and 20. 670. CHARRIN. Compt. rend, de la Societe de Biologic, Paris, 1890, No. 17. 671. CHARRIN ET GAMELEIA. Ibid., No. 19. 672. SEWELL. Journal of Physiology, vol. viii., 1887, p. 203. IV. PYOGENIC BACTERIA. 673. OGSTON. Report upon microorganisms in surgical diseases. British Medical Journal, London, 1881, pp. 369-375, 1 pi. 674. ROSEXBACH. Mikroorganismeu bei den Wundinfektionskrankheiten des Men- schen. Wiesbaden, 1884. 675. PASSET. Ueber Mikroorganismen der eitrigen Zellgewebsentzilndung des Men- schen. Fortschr. der Med., 1885, No. 2. 676. GRAWITZ. Ueber die Ursachen der subkutanen Entzilndung und Eiterung. Virchow's Archiv, Bd. cviii., p. 67. l'>?7. STEINHAUS. Die .Etiologie der acuten Eiterungen. Leipzig, 1889. 678. SCHEURLEN. Weitere Untersuchungen ilber die Entstehung der Eiterung, etc. Fortschr. der Med., 1887, p. 762. 679. WYSSOKOWITSCH. Ueber den Ursprung der Eiterung. Wratsch., 1887, p. 667. U'O. BUCHNER. Ueber eiterungserregende Stoffe in der Bakterienzelle. Centralbl. fiir Bakteriol., Bd. viii., 1890, p. 311. 681. HOPPA. Bakteriologische Mittheilungen aus dem Laboratorium der chirurg. Klinik des Prof. Mass in Wilrzburg. Fortschr. der Med., 1886, p. 75. 082. BUMM. Zur ^Etiologie der puerperalen Mastitis. Archiv fiir Gymikologie, Bd. xxvii., 1886. 683. ULLMANN. Die Fundorte der Staphylokokken. Zeitschr. fiir Hygiene, Bd. iv., 1888, p. 55. 634. BOCKHART. Ueber die JStiologie und Therapie der Impetigo, des Furunkels und der Sykosis. Monatshef te f ilr prakt. Dermatol., Bd. iv., 1887, No. 10. 685. BIONDI. Die pathogenen Mikroorganismen des Speichels. Zeitschr. fiir Hygiene, Bd. ii., 1887, p. 489. 686. VIGNAL. Recherches sur les micro-organismes de la bouche. Archives de Physiol. norm, et path., 1886, pp. 325-414. 67 798 BIBLIOGRAPHY. 687. VON BESSER. Ueber die Bakterien der normalen Luftwege. Beitrage zur pathol. Anat., etc. (Ziegler's), Bd. iv., 1889, p. 331. 688. WRIGHT. Nasal bacteria in health. New York Med. Jouru., 1889, July 27th. 639. ESCHERICH. Die Darmbakterien des Siiuglings. Stuttgart, 1886. 690. LUBBERT. Der Staphylococcus pyogenes aureus und der Osteomyelitiskokkus. Wlirzburg, 1886, 102 pp., 2 pi. 691. HADELICH. Ueber die Form und Grossen-Verhiiltnisse des Staphylococcus pyo- genes aureus. Inaug. Diss., Wlirzburg, 1887. 692. ERNST. A consideration of the bacteria of surgical diseases. Phila. Med. Times, 1886, October 16th and 30th. 693. BIUEGER. Untersuchuugen liber Ptomaine. Berliner klin. Wochenschrift, 1886, p. 281. 694. GARTNER UND PLAGGE. Ueber die desinflcireude Wirkung wasserigen Car- bolsaurelosungen. Archiv fur klin. Chir., Berlin, 1885, xxxii., p. 403. 695. BEHRING. Ueber Quecksilbersublimat in eiweisshaltigen Flilssigkeiten. Cen- tralbl. fur Bakteriol., Bd. iii., 1888, pp. 27, 65. 696. TARNIER ET VIGNAI,. Recherches experimentales relatives a 1'action de quelques antiseptiques sur le streptocoque et le staphylocoque pyogenes. Arch, de Med. exper. et d'Anat. pathol., 1890, No. 4. 697. ABBOTT. Corrosive sublimate as a disinfectant against the Staphylococcus pyo- genes aureus. Bull. Johns Hopkins Hospital, Baltimore, 1891, vol. ii.,"p. 50. 698. WYSSOKOWITSCH. Ueber die Schicksale der in's Blut injicirten Mikroorgan- ismen in Korper der Warmbluter. Zeitschr. fur Hygiene, Bd. i., 1886, p. 3. 699. GARRE. Zur ^Etiologie akut. eitriger Entzumluugen. Fortschr. der Med., 1885, No. 6. 700. BUMM. Ueber die Einwirkung pyogener Mikroorganismeu auf's Bindegewebe, etc. Wilrzb. phys.-med. Gesellsch., 10. Sitzg., May 12th, 1888. 701. BECKER. Vorl. Mitth. fiber den die akute infektiose Osteomyelitis erzeugenden Mikroorganismus. Deutsche med. Wochenschrift, 1883. 702. KRAUSE. Ueber einen bei der akuten infektiosen Osteomyelitis vorkommenden Mikrokokkus. Fortschr. der Med., Bd. ii., 1884. 703. RODET. Etude experimental sur I'osteomyelie infectieuse. Compt. rend. Acad. des Sc., xcix., p. 569. 704. KRASKE. Zur ^Etiologie und Pathogenese der akuten Osteomyelitis. Berliner klin. Wochenschr., 1886, p. 262. 705. WEICHSELBAUM. Zur ^Etiologie der akuten Endocarditis. Centralbl. fur Bakteriol., Bd. ii., 1887. 706. Ueber Endocarditis pneumonica. Wiener med. Wochenschr., 1888, Nos. 35 and 36. 707. Beitrage zur ^Etiologie und path. Anat. der Endocarditis. Ziegler's Beitrage, Bd. iv., 188*. pp. 127-222. 708. FRANKEL, E., UND SA'NGER. Untersuchungen liber die ^Etiologie der Endo- carditis. Virchow's Archiv, Bd. cviii., 1887, p. 286. 709. PRUDDEN. An experimental study of mycotic ulcerative endocarditis. Am. Journ. of the Med. Sc., 1887. 710. On the etiology of diphtheria. Atner. Jouru. of the Med. Sc. , 1889. 711. RIBBERT. Ueber experimentelle Myo- und Endocarditis. Fortschr. der Med., 1886, p. 1. 712. VON EISELSBERG. Nachweis von Erysipel-Kokkea in der Luft chirurgischer Krankenzimmer. Von Langenbeck's Archiv, Bd. xxxv., 1887. 713. FORTCNATI. Azione degli stafilococchi piogeni nelle ferite della cornea. Boll. d'Ocul., t. x., p. 109. BIBLIOGRAPHY. 799 714. GALLEXGA. Del nesso f ra la blefarite cigliare e la cherato-conguintivite eczema- tosa. Annal. di Ottalmol., t. xvi., p. 492. 715. FEHLEISEN. Die ^Etiologie des Erysipels. Berlin, 1883. 716. MANFREDI E TRAVERSA. Sull' azione fisiologica e tossica det prodotti di col- tura dello streptococcodell'erisipela. Giornale Internaz. delle Scienze med., annox.,^1888. 717. CLIVIO E MONTI. Sull' eziologia della peritoaite puerperale. Estratto degli letti del XII. Congresso medico. 718. CZERNIEWSKI. Zur Frage von den puerperalen Erkrankungen. Archiv fiir Gyniikologie, Bd. xxxiii., 1888. 719. WIDAL. Etude sur 1'infection puerperale, la phlegmasia alba et 1'erysipele. Paris, 1889. 720. BUMM. Ueber die Aufgaben weiterer Forschungen auf dem Gebiete der puer- peralen Wuudinfektion. Archiv fur Gyniikologie, Bd. xxxiv., 1889. 721. Zur /Etiologie des septischen Peritonitis. Miluchener med. Wochenschr., 1889, No. 42. 722. FRANKEL, E. Zur Lehre von der Identitilt des Streptococcus pyogenes und erysipelatis. Centralbl. fiir Bakteriol., Bd. vi., p. 691. 723. VON EISELSEKRG. Contribuzione allo studio dello streptococco dell' erisipela. Archiv per le scienze mediche, vol. xi., 1887, p. 159. 724. EMMERICH. Nachweis von Erysipelkokken in einen Sektionssaal. Tageblatt der 59. Versamml. Deutsch. Naturforscher und Aerzte zu Berlin, 1886, p. 433. 725. FLUGGE. Die Mikroorganismen. 2d ed., Leipzig, 1886, p. 153. 726. GOTTSTEIN. Beitrage zur Lehre von der Seplikamie. Deutsch. med. Wochen- schrift, 1890, No. 24. 727. LEVY TJND SCHRADER. Bakteriologisches ilber Otitis media. Archiv fur ex- per. Pathol. und Pharmakol., Bd. xxvi., p 223. 728. NETTER. Recherches bacteriologiques sur les otitesmoyennesaigues. Annales des Maladies de 1'Oreille et de Larynx, 1888. 729. SCHEIBE. Mikroorganismen der akuten Mittelohrerkrankungen. Zeitschr. fur Ohrenheilk., Bd. xix., 1889. 730. HAIJERMANN. Zur Pathogenese der eitrigen Mittelohrentzundung. Archiv fiir Ohrenheilk., 1889, p. 119. 731. BORDOXI-UFFREDUZZI. Centralbl. fur Bakteriol., Bd. ix., 1891, p. 390. 732. PAULSEX. Mikroorganismen in der gesunden Nasenhohle und beim akuten Schnupfen. Centralbl. fiir Bakteriol., Bd. viii., p. 344. 733. VON BESSER. Die Mikroorganismeu der Luftwege. Abstract in Centralbl. fur Bakteriol., Bd. v., 1889, p. 714. 734. • Ueber die Bakterien der normalen Luftwege. Zeigler's Beitrage zur pathol. Anat., Bd. iv., 1889, p. 331. 735. WIDMARK. Nagra bakteriologisk-oftalmiatriska studier. Nordisk Opthal. Tidisskrift, 1888. 736. SHOUGOLOWICZ. Zur Frage von dem Mikroorganismus des Trachoms. St. Petersburger med. Wochenschr., 1890, Nos. 28-30. 737. FICK. Ueber Mikroorganismen im Conjunktivalsack. Wiesbaden, 1887. 738. GIFFORD. Beitrage zur Lehre vender sympathischen Ophthalmic. Archiv fiir Augenheilkunde von Knapp und Schweigger, Bd. xvii., 1886, p. 14. 739. Ueber das Vorkommen von Mikroorganismen bei Conjunctivitis eczema- tosa, etc. Ibid., Bd. xvi., p. 197. "740. BOSSOWSKI. Ueber das Vorkommeu von Mikroorganismen in Operations- 800 BIBLIOGRAPHY. wunden unter dem antiseptischen Verbande. Wiener med. Wochenschr., 1887, Nos. 8 and 9. 741. WELCH. Conditions underlying the infection of wounds. Am. Journ. of the Med. Sc., Phila., 1891, p. 439 (November). 742. LANNELONGUE ET ACHARD. Etude experimentale des osteomyelites ii staphy- locoques et SL streptocoques. Ann. del'Institut Pasteur, t. v., 1891, p. 209. " GONOCOCCUS." 743. NEISSER. Ueber eine der Gonorrhoea eigenthumliche Mikrokokkus-Form vorlaufige Mittheilung. Centralbl. fur die med. Wissensch., 1879, No. 28. 744. Die Mikrokokken der Gonorrhoea. Deutsche med. Wochenschr. , 1882, p. 279. 745. BUMM. Der Mikroorganismus der gonorrhoischen Schleimhaut-Erkrankungen. Wiesbaden, 1885, 146 pp., 4 pi. 746. LESTIKOW. Ueber Bakterien bei den venerischen Krankheiten. Charite-An- nalen, vol. vii., p. 750. 747. Berliner klin. Wochenschr., 1882, p. 500. 748. BOCKHART. Beitrag zur ^Etiologie und Pathologic des Harnrohrentrippers. Vierteljahresschrift f ilr Dermatol. und Syph. , 1883, p. 3. 749. Ueber die Bedeutuug der Gonokokken fiir Diagnose und Therapie. Ver- handl. der deutschen dermatolog. Gesellschaft, i., p. 133. Wien, 1889. 750. STEINSCHNEIDER. Ueber seine in Verbindung mit Dr. Galewsky vorgenom- menen Untersuchungen ilber Gonokokken und Diplokokken in der Harn- rohre. Verhandl. der deutschen dermatolog. Gesellschaft, i., p. 159. Wien, 1889. 751. Ueber Vulvo-vaginitis gonorrhoica kleiner Madchen. Ibid., p. 170. 752. Zur Differenzirung der Gonokokken. Berliner klin. Wochenschr., 1890, No. 24. 753. SCHROTTER UND WiNKLER. Ueber Reinkulturen der Gonokokken. Abstract in Centralbl. fur Bakteriol., Bd. ix., p. 679. 754. STERNBERG. Thermal death-point of the " gonococcus." Report of Com. on Disinfectants, A. P. H. A., Concord, 1883, p. 146. 755. AUFUSO. II gonococco di Neisser. LI& Riforma med., 1891, p. 328. V. BACTERIA IN CROUPOUS PNEUMONIA. 756. FRIEDLANDER. Die Schizomyceten bei der akuten fibrinosen Pneumonie. Virchow's Archiv, Bd. Ixxxvii. , 1882. 757. Die Mikrokokken der Pneumonie. Fortschr. der Med., Bd. i., 1883. 758. Weitere Arbeiten liber die Schizomyceten der Pneumonie und der Meningitis. Fortschr. der Med., 1886, p. 702. 759. TALAMON. Communication a la Societe anatom. de Paris, November 30th, 1883. 760. AFANASSIEW. Societe de Biologic, seance du 21 mai, 1884. 761. SALVIOLI. Arch, pour les Sc. med., t. viii., 1884. 762. ZIEHL. Ueber das Vorkommen der Pneumoniekokken im pneumonischen Sputum. Centralbl. fur die med. Wissensch., 1883. 763. FRIEDLANDER UND FROBENIUS. Berliner klin. Wochensch., 1883. 764. PLATANOW. Ueber die diagnostische Bedeutung der Pneumoniekokkeu. In- aug. Diss., Wilrzburg, 1884. 765. MATRUY. Ueber Pneumoniekokken. Wiener med. Presse, June, 1883. BIBLIOGRAPHY. 801 766. GERMAIN-SEE. Des maladies specifiques du poumons. Paris, 1885. 767. KLEIN. Etiology of acute croupous pneumonia, etc. Fourteenth Annual Re- port of the Local Government Board, London, 1885. 768. STERNBERG. A fatal form of septicaemia in the rabbit produced by the subcu- taneous injection of human saliva. National Board of Health Bulletin, vol. ii., Washington, 1881; also Johns Hopkins University, Stud. Biol. Lab., Baltimore, vol. ii., 1882, pp. 183-200, 1 plate. 769. Experiments with disinfectants. Johns Hopkins University, Stud. Biol. Lab., Baltimore, ii., 1882, pp. 201-212; also in National Board of Health Bulletin, Washington, vol. iii., 1881, No. 4. 770. Induced septicaemia in the rabbit. American Journal of the Medical Sciences, Philadelphia, Ixxxiv., 1882, pp. 69-76. 771. Virulence of normal human saliva. Medical Times, Philadelphia, Nov. 4th, 1882. 772. Germicide value of therapeutic agents. American Journal of the Medi- cal Sciences, 1883, pp. 321-343. 773. Paper in the American Journal of the Medical Sciences, July, 1885. Ibid. , July, 1886. 774. The etiology of croupous pneumonia. The Medical Record, New York, vol. xxxv., 1889, p. 281. 775. Micrococcus Pasteuri. Journal of the Royal Microscopical Society, Lon- don, 1886. 776. Micrococcus pneumoniae crouposse. Centralblatt filr Bakteriol., Bd. xii., 1892, p. 53 ; also, The Medical News, Phil., vol. lx., 1892, p. 153. 777. PASTEUR. Sur une maladie nouvelle provoquee par la salive d'un enfant mort de la rage. Compt. rend. Acad. des Sc., Paris, xcii., 1881, pp. 159-165. 778. CLAXTON. Virulence of normal human saliva. Medical Times, Philadelphia, 1882, p. 627. 779. FRANKEL, A. Bakteriologische Mittheilungen. Verhandl. des Vereins f iir in- nere Med., July 13th, 1885; Deutsche med. Wochenschrift, 1885, No. 31, p. 546. 780. Zeitschrift fur klin. Med., Bd. x. 1886, Heft 5 und 6. 781. Ueber einen Bakterienbefund bei Meningitis cerebrospinalis, nebst Be- merkungen uber die Pneumonie-Mikrokokken. Deutsche med. Wochen- schr., 1886, No. 13. 782. Weitere Beitrage zur Lehre von den Mikrokokken der genuinen fibrino- sen Pneumonie. Zeitschrift filr klin. Med., Bd. xi., 1886, Heft 5 und 6. 783. Ueber die bakterioscopische Untersuchung eitriger pleuritischer Er- giisse, etc. Charite-Annalen, xiii., p. 147. 784. WEICHSELBAUM. Ueber die ^Etiologie der akuten Lungen- und Rippenfell- Entzilndungen. Wiener med. Jahrbiicher, 1886, p. 483. 785. Ueber die JEtiologie der akuten Meningitis cerebrospinalis. Fortschritte der Med., Bd. v., 1887, Nos. 18 and 19. 786. Ueber Endocarditis pneumonica. Wiener med. Wochenschr., 1888, Nos. 35 and 36. 787. Ueber seltenerere Localisationen des pneumonischen Virus. Wiener klin. Wochenschr., 1888, Nos. 28-32. 788. Der Diplococcus pneumoniae als Ursache der primareu, akuten Peritoni- tis. Centralbl. fur Bakteriol., Bd. v., 1889, p. 33. 789. Bakteriologische und pathologisch-anatomische Untersuchungen uber Influenza und ihre Komplikationen. Wiener klin. Wochenschr., 1890, 802 BIBLIOGRAPHY. 790. WOLF. Der Nachweis der Pneumonie-Bakterien in Sputum. Wiener med. Blatter, 1887, Nos. 10-14. 791. NETTER. Du microbe de la pneumonic dans la salive. Compt. rend, hebdom. des seances de la Soc. de Biol., 1887, No. 34. 792. Du microbe de Friedlander dans la salive, etc. Ibid., December 24th, . 1887. 793. Recherches sur les meningites suppurees. France Med., 1889, No. 64. 794. De la pleuresie metapneumonique et de la pleuresie purulente pneumo- coccique primitive. Bull, et memoires de la Societe Med. des Hopitaux de Paris, 1889. 795. Recherches bacteriologiques sur les otites moyennes aigugs. Annales des Maladies de 1'Oreille, etc., 1888, p. 493. 796. GAMELEIA. Etiologie de la pneumonic fibrineuse. Ann. de 1'Institut Pasteur^ vol. ii., p. 440. 797. MONTI. Contributio allo studio della meningite cerebrospinale. Riforma medica, 1889, Nos. 58 and 59. 708. Sull' eziologia del reumatismo articolare acuto. Ibid. , 1889, No. 54. 799. FOA. Weitere Untersuchungen liber die JEtiologie der Pneumonie. Deutsche med. Wochenschrift, 1889, p. 21. 800. Sulla biologia del dipplococco lanceolata. Associazione medica italiana, Padova, 1889. Abstract in Riforma medica, 1889, No. 233. 801. THUE. Untersuchungen liber Pleuritis und Pericarditis bei der crouposen Pneumonie. Centralbl. flir Bakteriol., Bd. v., 1889, p. 38. 802. ZAUFAL. Neue Falle von genuiner akuter Mittelohrentziindung veranlasst durch den Diplococcus pneumonias. Prager med. Wochenschrift, 1889, Nos. 6-12. Ibid., No. 15. Ibid., No. 36. 803. GABBI. Studio sull' artrite sperimentale da virus pneumonico. Lo Sperimen- tale, 1889. 804. JAKOWSKI. Zur ^Etiologie der akuten crouposen Pneumonie. Zeitschrift fiir Hygiene, Bd. vii., 1889, p. 237. 805. TESTI. Di una rarissima eomplicazione della pneumonite fibrinosa. Riforma medica, 1889, Nos. 281 and 282. 806. BORDONI-UFFREDUZZI UND GRADENioo. Ueber die ^Eti 'logic der Otitis media. Centralbl. fur Bakteriol., Bd. vii., 1890, pp. 529, 556, 695. 807. BORDONI-UFFREDUZZI. Neuer Streptococcus oder Diplococcus lanceolatus ? Centralbl. fiir Bakteriol., Bd. vi., p. 670. 808. Ueber die Widerstandsfilhigkeit des pneumonischeu Virus in den Aus- wiirfen. Ibid., Bd. x., p. 305. 809. FOA E BORDONI-UFFREDUZZI. Sulla eziologia della meningite cerebro- spinale epidemica. Archivio per le Sci. med.,xi., 1887, p. 385. 810. GUARNIERI. Studi sull' eziologia della polmonite. Atti della R. Accad. med. di Roma, 1888-89, p. 447. 811. BONOMB. Ueber die Unterscheidungsmerkmale zwischen clem Streptococcus der epidemischen Cerebrospinal-Meningitis und dem Diplococcus pneu- moniae. Centralbl. flir Bakteriol., Bd. vii., 1890, p. 402. 812. NEUMANN. 1st der Mikrococcus pyogenes tenuis (Rosenbach) mit dem Pneu- moniekokkus (Frankel-Weichselbaum) identisch? Centralbl. fiir Bakteriol., Bd. vii., p. 177. 813. SCHEIBE. Bakteriologiscb.es zur Otitis media bei Influenza. » Centralbl. fiir Bakteriol., Bd. viii., 1890, p. 225. 814. GABBI TJND PURITZ. Beitrag zur Lehre der seltenen Lokalisatiouen dos Virus BIBLIOGRAPHY. 803 pneumoniaa (Periarthritis, Endocarditis, und Meningitis). Centralbl. fur Bakteriol.,Bd. viii., 1890, p. 137. 815. ORTMANN UND SAMTER. Beitrag zur Lokalisation des Diplococcus pneu- moniaa. Virchow's Archiv, Bd. cxx., Heft 1. 816. DUPLAY. Parotide a pneumocoques. La Semaine med., 1891, No. 2. 817. KOPLIK. The etiology of empyema in children. Archives of Pediatrics, 1890 (October). 818. BANTI. Sull' eziologica delle pneumoniti acute. La Sperimentale xliv., 1890, pp. 349, 461, 573. 819. EMMERICH. Paper read at Internal. Med. Congress, London, 1891. Abstract in Journ. Am. Med. Assoc., September 12th, 1891, p. 418. 820. KLEMPERER, G. UND F. Versuche liber Immunisirung und Heilung bei der Pneumokokken-Infektion. Berliner klin. Wochenschrift, 1891, Nos. 34 and 35. 821. KRUSE TJND PANSINI. Untersuchungen liber den Diplococcus pneumonias und verwandte Streptokokken. Zeitschrift flir Hygiene, Bd. xi., 1892, p. 279. VI. PATHOGENIC MICROCOCCI NOT DESCRIBED IN SECTIONS IV. AND V. DIPLOCOCCUS INTERCELLULARIS MENINGITIDIS. 822. WEICHSELBAUM. Ueber die ^Etiologie der akuten Meningitis cerebrospinalis. Fortschr. der Med., Bd. v., 1887, Nos. 18 and 19. STAPHYLOCOCCU8 SALIVARIUS PYOGENES. 823. BIONDI. Die pathogenen Mikroorganismen des Speichels. Zeitschrift fur Hygiene, Bd. ii., 1887, p. 194. MICROCOCCUS OF PROGRESSIVE TISSUE NECROSIS IN MICE. 824. KOCH. Wundinfektionskrankheiten. Leipzig, 1878. MICROCOCCUS OP PROGRESSIVE ABSCESS FORMATION IN RABBITS. Op. tit. (No. 824). MICROCOCCUS OF PY/EMIA IN RABBITS. Op. tit. (No. 824). MICROCOCCUS OF SEPTICAEMIA IN RABBITS. Op. tit. (No. 824). MICROCOCCUS SALIVARIUS SEPTICUS. Op. tit. (No. 823). MICROCOCCUS 8UBFLAVUS. 825. BUMM. Der Mikroorganismus der gonorrhoischen Schleimhauterkrankungen. Wiesbaden, 1885, p. 20. MICROCOCCUS OF TRACHOMA (?). 826. SATTLER. Zehender's klin. Monatsblatter, 1881. 827. MICHEL. Ueber den Mikroorganismus bei der sog. agyptischen Augenentziln- dung (Trachom). Sitzungsber. der Wlirzburger phys.-med. Gesellschaft, 23. Januar, 1886. Ibid., Archiv filr Augenheilkunde, Bd. xvi., 1886, p. 367. 828. KARTULIS. Zur .^Etiologie der ilgyptischen katarrhalischen Conjunctivitis. Centralbl. fur Bakteriol., Bd. i., 1887. 804 BIBLIOGRAPHY. MICROCOCCUS TETRAGENUS. 829. KOCH. Mitth. aus dem K. Gesundheitsamte, Bd. ii., p. 42. 830. GAFPKY. Von Langenbach's Archiv fur Chir., Bd. xxviii., Heft 3. MICROCOCCUS BOTRYOGENUS. 831. JOHNE. Beitrage zur yEtiologie der Infektionsgeschwulste. Bericht viber die Veterinarwesen im Kgrch. Sachsen filr das Jahr 1884, p. 46. 832. Ueber mykotische Bindegewebswucherung bei Pferden. Deutsche Zeitschrift filr Thiermed. und Path., Bd. xii., 1886, p. 137. MICROCOCCUS OF MANFREDI. 833. MANFREDI. Ueber eineu neuen Mikrokokkus als pathogenes Agens bei infek- tiosen Tumoren. Fortschr. der Med., 1886. MICROCOCCUS OF BOVINE MASTITIS. 834. KITT. Untersuchungen uber die verschiedenen Formeu der Euterentzundtmg. Deutsche Zeitschrift fiir Thiermed. und Path., Bd. xvii., 1885, p. 1. MICROCOCCUS OF BOVINE PNEUMONIA (?). 835. POELS UND NOLENS. Das Contagium der Lungenseuche. Fortschr. der Med., 1886, No. 7. STREPTOCOCCUS SEPTICUS. 836. FLUGGE. Die Mikroorgauismen. 2d ed., 1886, p. 154. STREPTOCOCCUS BOMBYCIS. NOSEMA BOMBYCIS. 837. BECHAMP. Micrococcus bombycis. Compt. rend. Acad. des Sc., Ixiv., 1867. 838. PASTEUR. Etudes sur les maladies des vers a soie. Paris, 1870. MICROCOCCUS OF HEYDENREICH. 839. HEYDENREICH. Volume of 116 pages, published in St. Petersburg, 1888 (Rus- sian). Abstract in Ceutralbl fur Bakteriol., Bd. v., 1888, p. 163. MICROCOCCUS OF DEMME. 840. DEMME. Beitrage zur Kentniss des Pemphigus acutus. Verhandl. des V. Cong, fiir innere Med. in Wiesbaden, 1886. STREPTOCOCCUS OF MANNEBERG. 841. MANNEBERG. Zur ^Etiologie des Morbus Brightii acutus. Centralbl. fiir klin. Med., 1888, No. 30. MICROCOCCUS ENDOCARDITIDIS RUGATUS. 842. WEICHSELBAUM. Beitrage zur ^Etiologie und path. Anat. der Endocarditis. Ziegler's Beitrage, Bd. iv., 1888, p. 127. MICROCOCCUS OF GANGRENOUS MASTITIS IN SHEEP. 843. NOCARD. Note sur la mammite gangreneuse des brebis latieres. Ann. de 1'Institut Pasteur, t. i., 1887, p. 417. STREPTOCOCCUS OF MASTITIS IN COWS. 844. NOCARD ET MOLLERAU. Sur une mammite contagieuse .des vaches latieres. Ann. de 1'Institut Pasteur, t. i., 1887, p. 109. BIBLIOGRAPHY. 805 STREPTOCOCCUS CORYZ.E CONTAGIOS.E EQUORUM. 845. SCHUTZ. Der Streptokokkus der Druse der Pferde. Archiv f ttr wissensch. undprakt. Thierheilk. , 1888, p. 172. ELEMATOCOCCUS BOVI8. 846. BABES. Die ^Etiologie der seuchenhaften Hiimoglobiaurie des Rindes. Vir- chow's Archiv, Bd. cxv., 1889. MICROCOCCUS OINGIV^i PYOGENES. 847. MILLER. Die Mikroorganismen der Mundhohle. Leipzig, 1889, p. 216. PSEUDODIPLOCOCCUS PNEUMONI/E. 848. BONOME. Pleuro-pericarditis und Cerebrospinal-Meningitis sero-fibrinosa durch einen dem Diplococcus pneumonicus sehr ahnlichen Mikroorganismus er- zeugt. Centralblatt fi\r Bakteriol., Bd. iv., 1888, p. 321. STREPTOCOCCUS SEPTICUS LIQUEFACIENS. 849. BABES. Bakteriologische Untersuchungen liber septische Prozesse des Kindes- alters. Leipzig, 1889. MICROCOCCUS OF KIRCHNER. 850. KIRCHNER. Bakteriologische Untersuchungen ilber Influenza. Zeitschr. fur Hygiene, Bd. ix., 1891, p. 528. MICROCOCCUS NO. II. OF FISCHEL. 851. FISCHEL. Eine bakteriologische-experimentelle Studie liber Influenza. Zeit- schr. fur Heilkunde, Bd. xii., 1891. STREPTOCOCCUS OF BONOME. 852. BONOME. Sull' eziologia della meningite cerebro-spiuale epidemica. Archive per le Scienze mediche, vol. xiii., 1890. MICROCOCCUS OF ALMQUIST. 853. ALMQUIST. Pemphigus neonatorum, bakteriologisch und epidemiologisch be- Ituchtet. Zeitschr. fur Hygiene, Bd. x., 1891, p. 253. STREPTOCOCCUS PYOSEPTICUS. 854. HERICOURT ET RICHET. Compt. rend. Acad. des Sc., cvii., 1888, p. 690. STREPTOCOCCUS PERNICIOSUS PSITTACORUM. 855. EBERT. Virchow's Archiv, ,Bd. Ixxx. 856. WOLFF. Virchow's Archiv, Bd. xcii. MICROCOCCUS OF FORBES. 857. FORBES. Studies of the contagious diseases of insects. Bull. 111. State Lab. of Nat. Hist. vol. ii., 1886, p. 257. VII. THE BACILLUS OF ANTHRAX. 858. POLLENDER. Viertcljahrsschr. f ilr ger. Med., Bd. viii., 1855. 859. DAVAINE. Recherches sur les maladies charbonneuses. Compt. rend. Acad. des Sc., Ivii., pp. 220, 351, 386, et lix., 1863, p. 393. 806 BIBLIOGRAPHY. 860. DAVAINE. Recherches sur la nature et la const, anat. de la pustule maligne. Ibid., lx., 1865, p. 1296. 861. Sur la presence constante des Bacteridies dans les auimaux infectes de maladies charbonneuses. Ibid., Ixi., 1886, p. 368. 862. Action de la chaleur sur le virus charbonneux. Ibid., Ixxvii., 1873. 863. Rec. de Med. vet., vol. iv., 1877. 864. PASTEUR, Etiologie du charbon. Bull. Acad. de Med., Paris, 1879, viii., pp. 1222-1234. 865. Recherches sur 1'etiologie et la prophylaxie de la maladie charbonneuse dans le departement d'Eure-et-Loire. Rec. de Med. vet., Paris, 1879, pp. 193^198. 866. Sur 1'etiologie du charbon. Compt. rend. Acad. des Sc., 1880, xci., pp. 86-94. 867. Sur 1'etiologie des affections charbonneuses. Ibid., xci., pp. 455-459. 868. Sur la non-recidive de 1'affection charbonueuse. Ibid., xci., p. 531. 869. Nouvelles observations sur 1'etiologie et la prophylaxie du charbon. Ibid., xci., p. 697. 870. Sur la longue duree de la vie des germes charbonneux et sur leur con- servation dans les terres cultivees. Ibid., xcii., 1881, p. 209. 871. - — Resultats des vaccinations charbonneuses pratiquees pendant les mois de juillet, aout, et septembre, 1881. Arch, vet., Paris, vii., 1882, p. 177. De 1'attenuation de virus et de leur retour a virulence; avec la collabora- tion de MM. Chamberlain et Roux. Ibid., xcii., pp. 429-435. 873. De la possibilite de rendre les moutons refractaires au charbon par la methode des inoculations preventives; avec la collaboration de MM. Cham" berlain et Roux. Ibid., xcii., 1881, pp. 662, 666. 874. Une statistique au sujet de la vaccination preventive contre le charbou, portant sur quatre-vingt-cinq mille animaux. Ibid., xcv., 1882, p. 1250. 875. Reponse au docteur Koch. Rev. scient., Paris, xxxi., 1883, pp. 74-84. 876. TOTJSSAINT. Recherches experimentales sur la maladie charbonneuse. Paris, 1879. 877. De I'immuuite pour le charbon, acquise i la suite d'inoculations preven- tives. Compt. rend. Acad. des Sc., xci., 1880, p. 135. 878. Sur quelques points relatifs & 1'immunite charbonneuse. Ibid., xciii., p. 163. 879. CHATJVEAU. Etudes sur le sang de rate en Algerie. Journ. de Med. vet., Lyon, 1880, pp. 449 and 505. 880. Etude experimentale de 1'actiou exercee sur 1'agent infectueux par 1'or- ganisme des moutons plus ou moins refractaires au sang de rate. Compt. rend. Acad. des Sc., xci., 1880, p. 880. 881. Sur la resistance des animaux de 1'espece bovine au sang de rate, etc. Ibid., xci., 1880, p. 648. 882. - - — Nouvelles experiences sur la resistance des moutons algeriens au sang de rate. Ibid., xc., 1880, p. 1396. 883. - — Des causes qui peuvent faire varier les resultats de 1'inoculation char- bonneuse sur les moutoas algeriens; influence de la quantite des agents in- fectants; applications a la theorie de rimmunite. Ibid., xc., 1880, p. 1526. 884. Nature de 1'immunite des moutous algeriens contre le sang de rate; est- ce une aptitude de race ? Ibid., xci., p. 33. 885. De 1'attenuation des effets des inoculations virulentes par 1'emploi de tres- petites quantites de virus. Ibid., xcii., 1881, p. 844. 886. Etude experimentale des conditions qui permettent de rendre iisuel BIBLIOGRAPHY. 807 1'emploide la methode de M. Toussuint pour attenuer le virus charbonneux, etc. Ibid., xciv., 1882, p. 1694. 887. De 1'attenuation directe et rapide des cultures virulentes par 1'action de la chaleur. Ibid., xcvi., 1883, p. 553. 888. De la faculte prolifique des agents virulents attenues par la chaleur, etc. Ibid., p. 612. 889. Du role de 1'oxygSne de 1'air dans 1'attenuation, etc. Ibid., p. 678. 890. Sur le transformation en microbiologie pathogfene: Des limites, des con- ditions et des consequences de la variabilite du Bacillus anthracis. Ibid., cix., 1889, p. 597. 891. KOCH. Untersuchungen iiber Bakterien. Beitrage zur Biologic der Pflanzen, Bd. ii., Heft 2, 1876. 892. Zur /Etiologie des Milzbrandes. Mittb. aus dem K. Gesundheitsamte, Berlin, Bd. i , 1831. 893. Ucbcr die Milzbrandimpfung. Eine Entgegnung auf den von Pasteur in Genf gehaltenen Vortrag. Kassel und Berlin, 1882, 37 pages. 894. SEMMEU. Der Milzbraud und das Milzbrandkontagium. Jena, 1882. 895. ROLOPF. Der Milzbrand. Berlin, 1883. 896. BOLLINGER. Zur ^Etiologie des Milzbrandes. Sitzungsber. der Ges. fiir Morphol. und Physiol. zu Milnchen, 1885. 897. Ueber die Rcgenwiirmer als Zwischentrager des Milzbrandgiftes. Ar- beiten aus dem path. Institut zu Milnchen. Stuttgart, Itr86, p. 209. 898. BUCHNER. Die Umwandlung der Milzbrandbakterien in unschadliche Bakte- rieu. Virchow's Archiv, Bd. xci., 1883. 899. — Neue Versuche iiber Einathmung von Milzbrandsporen. Miinchener med. Wochenschr., 1887, No. 52. 900. - Ueber die Ursache der Sporenbildung beim Milzbrandbacillus. Cen- tralbl. fiir Bakteriol., Bd. viii., p. 1. 901. KOCH, GAFFKY UND LOFFLER. Experimeutelle Studien iiber Abschwilchung der Milzbrandbacillen durch Filtterung. Mitth. aus dem K. Gesundheits- amte, Bd. ii., 1884, p. 147. 902. FALK. Ueber das Verhalten von Infektiousstoffen im Verdauuugskanale. Virchow's Archiv, Bd. xcii., 1883. 903. ARLOING. Influence de la lumiere blanche et des ses rayons constituants sur le developpement et les proprietes du Bacillus anthracis. Arch, de physiol. norm, et pathol., 1886, p. 209. 904. FRANCK. Ueber Milzbrand. Ein Beitrag zur Lehre von der ortlichen und zeitlichen Disposition. Zeitschr. fiir Hygiene, Bd. i., 1886, p. 369. 905. Roux. De 1'action de la lumiere et de 1'air sur les spores de la bacteridie du charbon. Ann. de 1'Institut Pasteur, 1887, p. 445. 906. STRAUSS. Le charbon des animaux et de riiomme. Paris, 1887, 220 pp. 907. REINHOLD. Zur ^Etiologie des Milzbrandes. Zeitschr. fiir Hygiene, Bd. iv., 1888, p. 498. 908. - - Weiterer Beitrag zur Milzbrandatiologie. Ibid., Bd. v., 1889, p. 506. 909. BEHKING. Ueber die Ursache der Immunitat von weissen Ratten gegen Milz- brand. Centralbl. fur klin. Med., 1888, No. 38. 910. Beitrage zur ^Etiologie des Milzbrandes. Zeitschrift fiir Hygiene, Bd. vi., 1889, p. 117. Ibid., Bd. vii., 1889, p. 171. 911. HANKIN. Immunity produced by an albumose isolated from anthrax cultures. British Med. Journ., 1889, p. 810. 912. KARLINSKY. Zur Kentniss der Verbreitungswege des Milzbrandes. Centralbl. fiir Bakteriol., Bd. v., 1889, p. 5. 808 BIBLIOGRAPHY. 913. WYSSOKOWITSCH. Ueber Schutzimpfungen gegen Milzbrand in Russland. Fortschr. der Med., 1889, p. 1. 914. PERRONCITO. Studien iiber Immunitat gegen Milzbrand. Centralbl. fur Bak- teriol., Bd. v.,1889, p. 503. 915. CZAPLEWSKI. Untersuchungen tiber die Immunitat der Tauben gegeu Milz- brand. Beitr. zur allg. Path., etc. (Ziegler's), Bd. vii., 1889, p. 47. 916. FRANK. Ueber den Untergang von Milzbrandbacillen im Thierkorper. Cen- tralbl. fiir Bakteriol., Bd. iv., 1888, pp. 710, 737. 917. GAMELEIA. Etude sur la vaccination charbonneuse. Ann. de 1'Institut Pasteur, 1888, p. 517. 918. ROSENBLATH. Beitriige zur Pathologic des Milzbrandes. Virchow's Archiv, Bd. cxv., 1889, p. 371. 919. PHILIPOWICZ. Ueber das Auftreten pathogener Mikroorganismen im Harne. Wiener med. Blatter, 1885, p. 22. 920. TRAMBUSTI E MAFFUCCI. Sull' eliminazione dei virus dall' organismo ani- male. Rivista internaz. di Med. e Chir., 1886, Nos. 9 and 10. 921. STRAUSS ET CHAMBERLAIN. Archiv de Physiol., 1883, p. 436. 922. STRAUSS. Sur le passage de la bacteridie charbonneuse de la mere au foetus. Compt. rend. Soc. de Biol., 1889, pp. 409 and 498. 923. WOLFF. Ueber erbliche Uebertragung pathogener Mikroorganismen. Virchow's Archiv, Bd. cv., 1886, p. 192. 924. HOFFA. Ueber die Natur des Milzbrandgiftes. Wiesbaden, 1886. 925. WOLFFHUGEL UND RiEDEL. Die Vermehrung der Bakterien im Wasser. Ar- beiten aus dem K. Gesundheitsamte, Bd. ii., 1836, p. 455. 926. MARTIN. The chemical products of the growth of Bacillus anthracis, and their physiological action. Proc. of the Royal Soc., London, 1890 (May 22d). 927. OSBORNE. Die Sporenbildung des Milzbrandbacillus auf Nahrboden von ver- schiedenen Gehalt an Nahrstoffen. Archiv fur Hygiene, Bd. xi., p. 51. 928. PETERMANN. Recherches sur I'immunite centre le charbon. Ann. de 1'In- stitut Pasteur, vol. vi., 1892, p. 32. VIII. THE BACILLUS OF TYPHOID FEVER. 929. EBERTH. Der Bacillus des Abdominaltyphus. Virchow's Archiv, Bd. Ixxxi., 1880. Ibid., Bd. Ixxxiii., 1881. 930. BRQWICZ. Handbuch der path. Anat., Birch-Hirschfeld, 1875. 931. FISCHEL. Pragermed. Wochenschr., 1878, p. 33. 932. GAFFKY. Zur ^Etiologie des Abdominaltyphus. Mitth. aus dem K. Gesund- heitsamte, Bd. ii., 1884. 933. KOCH. Mitth. aus dem K. Gesundheitsamte, Bd. i., 1881, p. 46. 934. MEYER. Uutersuchungen iiber den Bacillus des Abdominaltyphus. Inaug. Diss., Berlin, 1881. 935. KLEBS. Archiv fiir exper. Path, und Pharmakol., Bd. xii., xiii., xv., 1880-81. 936. Archiv fiir exper. Path, und Pharm., Bd. xiii. 937. HEIN. Centralbl. fur die med. Wiss., October 4th, 1884. 938. LETZERICH. Virchow's Archiv, Bd. Ixviii., 1876. 939. ALMQUIST. Nord.med. Ark , Stockholm, xiv., 1882, No. 10. 940. MARAGLIANO. Centralbl. fiir die med. Wiss., 1882, No. 41. 941. TAYON. Gaz. med. de Montpellier, May, 1885. 912. Sur le microbe de la tievre typhoide de rhomme ; culture et inocula- tion. Compt. rend. Acad. des Sc., t. c. et ci., 1886. BIBLIOGRAPHY. 809 943. PFEIFFER. Ueber den Nachweis der Typhusbacillen im Darminhalt und Stuhl- gang. Deutsche med. Wocbenschr., 1885, p. 500. 944. KLEIN. Rep. Local Govt. Board, London, 1875. 945. BAHRDT. Arcbiv der Heilkunde, 1876, p. 156. 946. VON MOTSCHUKOFFSKY. Centralbl. fur die med. Wiss., 1876, No. 11. 947. WALDER. Inaug. Diss., Zurich, 1879. 948. CORNIL ET BABES. Les Bacteries. Paris, 1885, p. 432. 949. BRIEGER. Weitere TJntersuchungen uber Ptomaine. Berlin, 1885. 950. SEITZ. Bakteriologische Studien zur Typhusatiologie. Munchen, 1886. 951. FRANKEL, A. Zur Lehre von den pathogenen Eigenschaften des Typhusbacil- lus. Centralbl. fur klin. Med., 1886, No. 10. 952. MICHAEL. Typhusbacillen im Trinkwasser. Fortschr. der Med., 1886, No. 11. 953. WOLFFHUGEL UND RiEDEL. Die Vermehrung der Bakterien im Wasser. Ar- beiten aus dem K. Gesundheitsamte, Bd. i., 1886, p. 455. 954. BEUMER UND PEIPER. Bakteriologische Studien uber die atiologische Be- deutung der Typhusbacillen. Zeitschrift fur Hygiene, Bd. i., 1886, p. 489. Ibid., Bd. ii., 1887, p. 110 ; ibid., p. 382. 955. FRANKEL, E., UNO SIMMONDS. Zur atiologischen Bedeutung des Typhusbacil- lus. Centralbl. fur klin. Med., 1886, No. 39. 956. Weitere Untersuchungen ilber die JEtiologie des Abdominaltyphus. Zeitschrift filr Hygiene, Bd. ii., 1887, p. 138. 957. PHILIPOWICZ. Ueber die diagnostische Verwerthung der Milzfunktion bei Typhus abdominalis. Wiener med. Blatter, 1886, No. 6 and 7. 958. SIROTININ. Die Uebertragung von Typhusbacillen auf Versuchthiere. Zeit- schrift filr Hygiene, Bd. i., 1886, p. 465. 959. BiRCH-HiRSCHFELD. Ueber die Zilchtung von Typhusbacillen in gefarbten Nahrlosungen. Archiv filr Hygiene, 1887, p. 341. 960. CHANTEMESSE ET WIDAL. Recherches sur le bacille typhique et 1'etiologie de la fiSvre typhoide. Arch, de Phys. norm, et path., 1887, p. 217. 961. De 1'immunite contre le virus de la fievre typhoide conferee par les sub- stances solubles. Ann. de 1'Institut Pasteur, t. ii., 1888, p. 54. 962. STERNBERG. The bacillus of typhoid fever. The Med. News, Phila., April 30th, 1887. 963. The thermal death-point of the typhoid bacillus. Rep. of Com. on Dis- infectants in volume published by the American Public Health Assoc. in 1888, p. 140. 964. BUCHNER. Ueber die vermeintlichen Sporen der Typhusbacillen. Centralbl. filr Bakteriol., Bd. iv., 1S88, p. 353. 965. KITASATO. Ueber das Verhalteu der Typhus- und Cholerabacillen zu saure- oder alkalihaltigen Nahrboden. Zeitschrift filr Hygiene, Bd. iii., 1888, p. 408. 966. PFUHL. Zur Sporenbildung der Typhusbacillen. Centralbl. filr Bakteriol., Bd. iv., 1888, p. 769. 967. THOINOT. Sur la presence du bacille de la fievre typhoide dans 1'eau de la Seine 3, Ivry. La Semaine med., 1887, p. 135. 968. MACE. Sur la presence du bacille typhique dans le sol. Compt. rend. Acad. des Sc., cvi., p. 1564. 969. SEMMER. Zur Frage uber das Vorkommen des Typhus bei Thieren. Virchow's Archiv, Bd. cxii., 1888, p. 203. 970. VAUGHAN AND NOVY. Experimental studies on the causation of typhoid fever, with special reference to the outbreak at Iron Mountain, Mich. The Medical News, 1888, p. 92. 810 BIBLIOGRAPHY. 971. DE GIAXA. Ueber das Verhalten einiger pathogenen Mikroorganismen im Meerwasser. Zeitschrift fiir Hygiene, Bd. vi., p. 162. 972. GRANCHER ET DESCHAMPS. Rechcrches sur le bacille typhique dans le sol. Arch, de Med. exp. et d'Anat. path., vol. i., 1889, p. 5. 973. HEIM. Ueber das Verhalten der Krankheitserreger der Cholera, des Unterleibs. typhus, und der Tuberculose in Milch, Butter, Molken and Kase. Arbeiteu aus dem K. Gesundheitsamte, Bd. v., 1889, p. 294. 974. HESSE. Unsere Nahrungsmittel als Nilhrboden fur Typhus und Cliolera. Zeitschrift fur Hygiene, Bd. v. , p. 527. 975. KARLINSKY. Untersuchungen fiber das Verhalten der Typhusbacillen in typhosen Dejektionen. Centralbl. fiir Bakteriol., Bd. vi., 1889, p. 65. 976. - — Ueber das Verhalten einiger pathogenen Bakterien im Trinkwasser. Archiv fiir Hygiene, Bd. ix., p. 432 Ibid., Bd. x., p. 464. 977. Untersuchungen ilber das Vorkommen der Typhusbacillen im Harn. Prager raed. Wochenschr., 1890, Nos. 35 and 36. 978. NEUMANN. Ueber Typhusbacillen im Urin. Berliner klin. Wochenschr., 1890, No. 6. 979. SCHILLEK. Zum Verhalten der Erreger der Cholera und des Unterleibstyphus in dem Inhalt der Abtrittsgruben und Abwasser. Arbeiten aus dem K. Ge- sundheitsamte, Bd. vi., 1890. 980. Beitrag zum Wachsthum der Typhusbacillen auf Kartoffeln. Arbeiten aus dem K. Gesundheitsamte, Bd. v., 1889, p. 312. 981. KITASATO. Die negative Indolreaktion der Typhusbacillen im Gegensatz zu anderen ahnlichen Bacillenarten. Zeitschr. fiir Hygiene, Bd. vii., 1889, p. 515. 982. MARTINOTTI E BARBACCI. Presenza dei bacilli del tifo nell' acqua potabile. Gioruale della R. Accad. di Med. di Torino, 1889, No. 8. 983. PETRUSCHKY. Die Anwendung der Lackmusreaktion zur Differenzirung der Typhusbacillen von ahnlichen Bakterienarten. Centralbl. fiir Bakteriol., Bd. vi., 1889, p. 660. 984. STRAUS ET DUBARRY. Recherches sur la duree de la vie des microbes patlio- genes dans 1'eau. Arch, de Med. exper. et d'Anat. pathol., t. i., 1889, p. 5. 985. UFFELMANN. Die Dauer derLebensfahigkeit von Typhus- und Cholerabacillen in Fakalmassen. Centralbl. fiir Bakteriol., Bd. v , 1889, p. 497. 986. GASSER. Sur un nouveau precede de diagnostique differentiel du bacille d'Eberth. La Semaine med.. 1890, No. 31. 987. Culture du bacille typhique sur milieux nutritifs colores. Arch, de Med. exper. et d'Anat. path., 1890. No. 6. 988. JANOWSKY. Zur Biologic der Typhusbacillen. Centralbl. fiir Bakteriol., Bd. viii., 1^90, pp. 167, 193, 230, 262, 417, 449. 989. SMITH. Einige Bemerkungen fiber Saure- und Alkalibildung bei Bakterien. Centralbl fttr Bakteriol., Bd viii., p. 389. 990. VINCENT. Sur un uouveau procede d'isolement du bacille typhique clans 1'eau. Compt. rend Soc. de Biol.. 1890, No. 5. 991. CASSEDEBAT. Sur un bacille pseudo typhique trouve dans les eaux de riviere. Compt. rend. Acad. des Sc., t. ex., 1889. 992. Le bacille d'Eberth-Gaffky et les bacilles pseudo-typhiques dans les eaux de riviere. Ann. de 1'Institut Pasteur, 1890, p. 625. 993. FINKELNBURG. Ueber einen Befund von Typhusbacillen im Brunnenwasser, nebst Bemerkungen ilber die Sedimentirmethode der Untersuchung auf pathogene Bakterien in Flilssigkeiten. Centralbl. fiir Bakteriol., Bd. ix., 1891, p. 301. BIBLIOGRAPHY. 811 994. HOLZ. Experimentale Untersuchungen liber dea Naclnveis der Typhusbacil- len. Zeitschr. fiir Hygiene, Bd. viii., 1890, p. 143. 995. VINCENT. Presence du bacille typhique dansl'eau de Seine pendant le mois de juillet, 1890. Ann. de 1'Institut Pasteur, 1890, p. 772. 996. JAEGER. Zur Kenntniss der Verbreitung des Typhus durch Kontagion und Nutzwasser. Zeitschr. flir Hygiene. Bd. x., 1891, p. 197. 997. LASER. Ueber das Verhalten von Typhusbacillen, Cholerabakterien und Tu- berkelbacillen in der Butter. Zeitschr. fiir Hygiene, Bd. x , 1891, p. 513. 998. BABES. Ueber Variabilitat und Varietaten des Typhusbacillus. Zeitschr. fiir Hygiene, Bd. ix., 1890, p. 323. 999. FOOTE. The detection of the Bacillus typhosus in water. Med. Record, New York, 1891, p. 506. 1000. PARIETTI. Metodo di ricerca del Bacillo del tifo Jnelle acque potabili. Ri- vista d'igiene e sanita pubblica. 1890. 1001. KAMEN. Zum Nachweise der Typhusbacillen im Trinkwasser. Centralbl. fiir Bakteriol., Bd. xi., 1892, p. 33. IX. BACTERIA IN DIPHTHERIA. 1002. KLEBS. Arch, f ilr exper. Pathol., Bd. iv., 1875. 1003. — — Medical Congress at Wiesbaden, 1883. 1004. LOFFLER. Uutersuchungen ilber die Bedeutung der Mikroorganismen fiir die Entstehung der Diphtheritis bei Menschen, etc. Mitth. aus dem K. Ge- sundheitsamte, Bd. ii., 1884, p. 421. 1005. Die Ergebuisse weiterer Untersuchungen ilber die Diphtheriebacillen. Centralbl. fiir Bakteriol., Bd. ii., 1837, p. 105. 1006. Der gegenwiirtige Stand der Frage nach der Entstehung der Diph- theric. Deutsche med. Wochenschr., 1890, Nos. 5 and 6. 1007. - Bemerkungen zu der Arbeit von Prof. E. Klein, " Zur ^Etiologie der Diphtheric." Centralbl. fur Bakteriol., Bd. vii., 1890, p. 528. 1008. Welche Massregeln erscheinen gegen die Verbreitung der Diphtheric geboten ? Centralbl. fiir Bakteriol., Bd. viii., 1890, p. 663. 1009. VON HOFFMANN. Untersuchungen iiber den LSffler'schen Bacillus der Diph- therie und seine pathogene Bedeutung. Wiener med. Wochenschr., 1888, Nos. 3 and 4. 1010. PUTZ. Ueber croupos-diphtheritische Erkrankungen unserer Hausthiere und deren Beziehungen zur Diphtheric des Menschen. Oesterr. Zeitschr. fiir Veterinar-Wissenschaften, Bd. i., 1887. 1011. ESSER. 1st Diphtheritis des Menschen auf Kalber ilbertragbar ? Thiermed- Rundschau, 1888, No. 9. 1012. Roux ET YERSIN. Contribution a 1'etude de la diphtheric. Ann. de 1'Institut Pasteur, t. ii., 1838, p. 629. 1013. Ibid., t. iii., 1889, p. 273. 1014. Ibid., t. iv., 1890, p. 385. 1015. PKUDDEN. On the etiology of diphtheria. Am. Journ. of the Med. Sciences, 1889. 1016. Studies on the etiology of diphtheria, 2d series. Med. Record, New York, vol. xxxix., 1891, p. 445. 1017. BABES. Croup und Diphtheric. Wiener klin. Wochenschr., 1889, No. 14. 1018. Die Gewebsveranderungen bei experimenteller Diphtheric. Centralbl. fur Bakteriol., Bd. viii., 1890, p. 741. 812 BIBLIOGRAPHY. 1019. BABES. Untersuchungen liber den Diphtheriebacillus und die experiinentelle Diphtheric. Virchow's Archiv, Bd. cxix.. 1890, p. 460. 1020. HOLZINGER. Zur Frage der Scharlachdiphtherie. Inaug. Diss., Munchen, 1889. 1021. KOLISKO UND PALTAUF. Zum Wesen des Croups und der Diphtheric. Wiener klin. Wochenschr., 1889, No. 8. 1022. ORTMANN. Berliner klin. Wochenschr., 1889. No. 10. 1023. ZARNIKO. Beitrag zur Kenntniss des Diphtheriebacillus. (Inaug. Diss.) Cen- tralbl. ftlrBakteriol., Bd. vi., 1889. 1024. WURZ ET BOURSES. Arch, de Med. experimeutale, t. ii., 1890, p. 341. 1025. ESCHERICH. Zur .Etiologie der Diphtheric. Centralbl. fur Bakteriol. , Bd. vii., 1890, p. 8. 1026. KLEIN. Zur ^Etiologie der Diphtheric. Centralbl. fiir Bakteriol., Bd. vii., 1890, pp. 489, 521. 1027. Nachtrag zum ';Weiteren Beitrag zur ./Etiologie der Diphtheric." Centralbl. fur Bakteriol., Bd. viii., 1890, p. 7. 1028. D'EspiNE. Rev. med. de la Suisse romande, 1888, p. 49. 1029. BEHRING. Untersuchungen iiber das Zustandekommen der Diphtheric- Immu- nitat bei Thieren. Deutsche med. Wochenschr., 1890, No. 50. 1030. BEHRING UND KITASATO. Ueber das Zustandekommen der Diphtherie-Im- munitat und der Tetanus-Immunitat bei Thieren. Deutsche med. Wochen- schr., 18'JO, No. 49. 1031. BRIEGER UND FRANKEL. Untersuchungen liber Bakteriengifte. Berliner klin. Wochenschr., 1890, Nos. 11 and 12. 1032. Ueber Immunisirungsversuche bei Diphtheric. Berliner klin. Wochen- schr., 1890, No. 49. 1033. WELCH AND ABBOTT. The etiology of diphtheria. Bull. Johns Hopkins Hos- pital, vol. ii., 1891, p. 25. 1034. ABBOTT. The relation of the pseudo-diphtheritic bacillus to the diphtheritic bacillus. Ibid., vol. ii., 1891, p. 110. 1035. Further studies upon the relation of the pseudo-diphtheritic bacillus to the diphtheritic bacillus. Ibid., vol. ii., 1891, p. 143. 1036. (No. 48.) Roux ET YERSIN. Ann. del'Institut Pasteur, t. iv., 1890, p. 409. 1037. (No. 49 and No. 50.) LOFFLER. Mitth. aus dem K. Gesundheitsamte, Bd. ii., 1884. 1038. (No. 51.) RIBBERT. Ueber einen bei Kaninchen gefundenen pathogenen Spaltpilz. Deutsche med. Wochenschr., 1887, No. 8. X. BACTERIA IN INFLUENZA. 1039. BABES. Vorlaufige Mittheilungen liber eiuige bei Influenza gefundene Bak- terien. Centralbl. fur Bakteriol., Bd. vii., 1890, pp. 233, 460, 496, 533, 561, 598. 1040. Ueber die bei Influenza gefundeue f einen Bakterien. Deutsche med. Wochenschr., 1892, No. 6. 1041. BOUCHARD. Recherches bacteriologiques sur la grippe et ses complications. La Semaine med., 1890, No. 5. 1042. FISCHEL. Beobachtungen wahrend der lufluenzaepidemie. Prager med. Wochenschr., 1890, No. 9. 1043. Eine bakteriologisch-experimentelle Studie liber Influenza. Zeitschrift flir Heilkunde, Bd. xii., 1891. 1044. JOLLES. Zur JStiologie der Influenza. Wiener med. Blatter, 1890, No. 4. BIBLIOGRAPHY. 813 1045. KIRCHNER. Untersuchungen liber Influenza. Centralbl. fur Bakteriol , Bd. vii., 1890, p. 361. 1046. — Bakteriologische Untersuchungen ilber Influenza. Zeitschrift fur Hygiene, Bd. ix., 1890, p. 528. 1047. KLEBS. Ein Blutbefund bei Influenza. Centralbl. fur Bakteriol., Bd. vii., 1890, p. 145. 1048. Deutsche med. Wochenschr., 1890, No. 14. 1049. KOWALSKI. Bakteriologische Untersuchungen ilber die Influenza. Wiener klin. Wochensch., 1890, Nos. 13 and 14. 1050. KRUSE, PANSINI UND PASQUALE. Influenzastudien. Centralbl. filr Bakteriol., Bd. vii., 1890, p. 657. 1051. LEVY. Bakteriologische Befunde bei Influenza. Berliner klin. Wochenschr., 1890, No. 7. 1052. PRIOR. Bakteriologische Untersuchungen liber die Influenza und ihre Kompli- kationen. Miinchener med. Wochenschr., 1890, Nos. 13-15. 1053. RIBBERT. Anatomische und bakteriologische Beobachtungen liber Influenza. Deutsche med. Wochenschr., 1890, No. 5. 1054. Weitere bakteriologische Mittheilungen liber Influenza. Ibid., No. 15. 1055. VAILLARD. Le streptocoque et la grippe. La Semaine rned., 1890, No. 7. 1056. WEICHSELBAUM. Bakteriologische und pathologisch-anatomische Untersuch- ungen tiber Influenza und ihreKomplikationeu. Wiener klin. Wochenschr., 1890, Nos. 6-10. 1057. ZAUFAL. Bakteriologisches zur Mittelohrentziindung bei Influenza. Prager med. Wochenschr., 1890, No. 9. 1058. FRIEDRICH. Untersuchungen liber Influenza. Arbeiten aus dein K. Gesund- heitsamte, Bd. vi., 1890, Heft 2. 1059. PRUDDEN. Bacterial studies on the influenza and its complicating pneumonia. Medical Record, New York, 1890, p. 169. 1060. SCHEIBE. Bakteriologisches zur Otitis media bei Influenza. Centralbl. fiir Bakteriol., Bd. viii., 1890, p. 225. 1061. BEIN. Bakteriologische Untersuchungen liber Influenza. Zeitschrift fiir klin. Med., xvii., 1890, Heft 6. 1062. PFEIFPER. Vorlaufige Mittheilungen liber den Erreger der Influenza. Deutsche med. Wochenschr., 1892, No. 2. 1063. CANON. Ueber einen Mikroorganismus im Blute von Influenzakranken. Deutsche med. Wochenschr., 1892, No. 2. 1064. Ueber Ziichtung des Influenzabacillus aus dem Blute von Influenza- kranken. Ibid., 1892, No. 3. 1065. KITASATO. Ueber den Influenzabacillus und sein Kulturverfahren. Deutsche med. Wochenschr., 1892, No. 2. XI. BACILLI IN CHRONIC INFECTIOUS DISEASES. TUBERCULOSIS. 1066. VILLEMAN. Etude sur la tuberculose. Paris, 1868. 1067. COHNHEIM. Uebertragbarkeit der Tuberkulose. Berlin, 1877. 1068. KOCH. Die ^Etiologie der Tuberkulose. Berliner klin. Wochenschr., 1882. 1069. Mittheilungen aus demK. Gesundheitsamte, Bd. ii., 1884. 1070. - Kritische Besprechung der gegen die Bedeutung der Tuberkelbacillen gerichteten Publicationen. Deutsche med. Wochenschr., 1883, No. 10. 1071. Weitere Mittheilung liber das Tuberkulin. Deutsche med. Wochen- schr., 1891, No. 43. 814 BIBLIOGRAPHY. 1072. DOUTRELPONT. Die ^Etiologie des Lupus vulgaris. Yierteljahresschr. fiir Dermatol. und Syph., 1884. 1073. CORNIL ET LELOIR. Recherches, etc., sur la natur du lupus. Arch, de Physiol. norm, et path. , 1884. 1074. GAFFKY. Verhalten der Tuberkelbacillen im Sputum. Mitth. aus dem K. Gesundheitsamte, Bd. ii., 1884. 1075. ZIEHL. Bedeutung der Tuberkelbacillen filr Di'agnose und Prognose. Deutsche med. Wochenschr., 1883, No. 5. 1076. MALASSEY ET VIGNAL. Sur le microorganisme de la tuberculose zoogloeique. Compt. rend. Acad. desSc., t. xcix.,p. 200. 1077. EHRLICH. Deutsche med. Wochenschr., Bd. viii., 1882. Ibid., Bd. ix., 1883. 1078. GIBBS. Lancet, London, 1883. 1079. BABES. Der erste Nachweis des Tuberkelbacillus im Harn. Centralbl. fiir die med. Wissensch., 1883. 1080. LUSTIG. Ueber Tuberkelbacillen im Blut bei an allg. akuter Miliartub. Erkrankten. Wiener med. Wochenschr., 1884, No. 48. 1081. WEIGERT. Deutsche med. Wochenschr., 1883, No. 24. 1082. Zur Theorie der tuberkulosen Riesenzellen. Deutsche med. Wochen- schr. , 1885, p. 599. 1083. MULLER. Ueber den Befund von Tuberkelbacillen bei f unaijt Knochen und Gelenkaffektionen. Centralbl. fur Chir., 1884. 1084. BROUILLY. Note sur la presence de bacilles dans les lesion^^R'urgicales tuber culeuses. Rev. de Chir., vol. iii., 1883. 1085. SCHILL UND FISCHER. Mitth. aus dem K. Gesundheitsan^WBd. ii., 1884. 1086. JOHNE. Zur ^Etiologie der Htihnertuberkulose. DeutscJ^Eeitschr. filr Thier- med. und vergl. Pathol., Bd. x., p. 155. 1087. Zur Pathogenese der Tuberkulose beim Pfc Jl. Bericht ilber das Veterinarwesen im Kgrch. Sachsen fur das Jahr 18m, p. 52. 1088. RIBBERT. Ueber die Verbreitungsweise der Tube*elbacillen bei Hiihnern. Deutsche med. Wochenschr., 1883, No. 28. M 1089. WEICHSELBAUM. Tuberkelbacillen im Blut. WJffner med. Jahrb., 1883. 1090. kZusammenfassender Bericht iiber die^Etiologie der Tuberkulose. Centralbl. fur Bakteriol., Bd. iii., 1888, No^e. 1091. BANG. Ueber Eutertuberkulose der Milchkiihe. Deutsche Zeitschr. fiir Thier- med. und vergl. Pathol., Bd. xi. 1092. Tuberkelbacillen in der Milch tuberkuloser Kilhe. Abstract in Cen- tralbl. fur klin. Med., 1888, p. 898. 1093. 1st die Milch tuberkuloser Kilhe virulent, wenn das Enter nicht ergrif- fen ist ? Internal. Med. Congress in Berlin, 1890. Centralbl. filr Bakteriol., Bd. ix., p. 144. 1094. STERNBERG. Injection of finely powdered inorganic material into the abdo- minal cavity of rabbits does n0t induce tuberculosis; an experimental re- search, with pathological notes by Wm. T. Councilman. Amer. Journ. of the Med. Sci., Jan., 1885. 1095. CORNIL ET MEGOIN. Tuberculose et diphtheric des .^allinaces. Journ. de 1'Anat., 1885, p. 268. 1096. KRASKE. Ueber tuberkulose Erkrankung von Wunde. Centralbl. fiir Chi- rurgie, 1885, No. 4. 1097. NATHAN. Ueber das Vorkommen von Tuberkelbacillen bei Otorrhoen. Deut- sches Archiv fiir klin. Med., Bd xxv., 1835, p. 491. 1098. NEELSEN. Methode zum Nachweis von Tuberkelbacillen. Fortschr. der Med., 1885, p. 200. BIBLIOGRAPHY. 815 1099. ORTMANX. Ueber Tuberkulose der weiblichen Brustdrilse, mit besonderer Berilcksichtigkeit der Riesenzellenbildung. Virchow's Archiv, Bd. c., 1885, p. 365. 1100. RUTIMEYER. Ueber das Vorkommen von Tuberkelbacillen im Blut und Milz- saft bei allgemeiner Miliartuberkulose. Centralbl. fiir klin. Med., 1885, p. 353. r 0 1101. SIRENA E PERNICE. Sulla transmissibilita della tuberculosi per mezzo degli sputi dei tisici. Gaz. degli Ospitali, 1885, No. 25. 1 102. STICKER. Ueber das Vorkommen von Tuberkelbacillen im Blute bei der akuten allgemeinen Miliartuberkulose. Centralbl. fiir klin. Med., 1885, p. 441. 1103. TSCHERNING. Inoculationstuberkulose beim Menschen. Fortschr. der Med., 1885, p. 65. 1104. ULRICH. Nachweis der Tuberkelbacillen bei Conjunktivaltuberkulose. Cen- tralbl. filrprakt. Augenheilk., 1885, Heft 12. 1105. VOLTOLINI. Ueber ein besonderes Erkennungszeichen der Tuberkelbacillen. Breslauer arztl. Zeitschr., 1885, No. 15. 1106. WESENER. Kritische und experimentelle Beitrage zur Lehre von der Ftitter- ungstuberkulose. Freiburger akadeniische Habilitationsschrift, 1885. 1107. BENDER. Ueber die Beziehungen des Lupus vulgaris zur Tuberkulose. Deuljgche med. Wochenschr., 1886, p. 396. 1108. BERGKAMMER. Kasuistischer Beitrag zur Verbreitung der Miliartuberkulose und Etnwanderung der Tuberkelbacillen in die Blutbahn. Virchow's Ar- chiv, Bfflkii., 1886, p. 397. 1109. BIEDERT. Win Verfahren, den Nachweis vereinzelter Tuberkelbacillen zu sichern, e»k Berliner klin. Wochenschr., 1886, No. 42. Ibid., 1887, No. 2. 1110. BOLLINGER. WJeber intestinale Tuberkulose bei Huhnern durch Genuss tuber- kuloser SpulJL Deutsche med. Zeitung, 1885, No. 78. 1111. VON BRUNN. E^itrag zur Lehre von der Uebertragbarkeit der Tuberkel- bacillen. Deufltehe med. Wochenschr., 1886, p. 178. 1112. CAVAGNIS. ContreVil virus tubercolare e contre la tuberculosi : Tentativi sperimentali. Atfl^del R. Institute Veneto di Scienze, Lett, et Arti, t. iii., iv., v., serie vi., 18$5-86. 1113. EHRLICH. Beitrage zurVTheorie der Bacillenfarbung. Charite-Annalen, 1886. 1114. FRANKE. Zur Fiirbung der Tuberkelbacillen in Geweben (Schnitten). Deutsche med. Wochenschr., 1886, p. 397. 1115. GARRE. Zur ^Etiologie der kalten Abscesse, etc. Deutsche med. Wochenschr., 1886, p. 581. 1116. HANOT. Contribution a 1'etudede la tuberculose cutanee. Archiv. de Physiol. norm, et pathol., 1886, p. 25. 1117. KIRSTEIN. Ueber den Nachweis der Tuberkelbacillen im Urin. Deutsche med. Wochenschr., 1886, p. 249. 1118. MULLER. Experimentelle Erzeugung typischer Knochentuberkulose. Cen- tralbl. fiir Chirurgie, 1886, No. 14. 1119. Deutsche Zeitschrift fur Chirurgie, Bd. xxiv., 1886, p. 37. 1120. NASSE. Beitriige zur Kenntniss der Arterientuberkulose. Virchow's Archiv, Bd. cv., 1886, p. 173. 1121. NEESE. Ein Beitrag zur Tuberkulose des Auges. Archiv filr Augeuheil- kunde, Bd. xvi., 1886, Heft 3 and 4. 1122. NOCARD ET Roux. Sur la culture du microbe de la tuberculose. Soc. de Biol., seance du 11 decembre, 1886. 1123. Sur la culture du bacille de la tuberculose. Ann. de 1'Institut Pasteur, 1887, p. 19. 816 BIBLIOGRAPHY. 1124. ADAM. Haufigkeit der Tuberkulose bei den geschlachteten Rindern auf dem Schlachthofe zu Augsburg. Adam's Wochenschr. f iir Thierheilkunde, 1886, No. 17. 1125. CADEAC ET MALET. Etude experimentale de la transmission de la tuberculose par 1'air expire et par 1'atmosphere. Revue de Med., 1887, No. 7. 1126. Compt. rend. Acad. des Sc.. t. cv., 1887, p. 1190. 1127. Recherches experimentales sur la virulence des matieres tuberculeuses dessechees, putriflees ou congelees. Lyon Med., 1888, p. 229. 1128. VON EISELBERG. Beitrage zur Impftuberkulose beim Menschen. Wiener med. Wochenschr., 1887, No. 53. 1129. ERNST. Gabbet's Farbung der Tuberkelbacillen. Correspondenzbl. fur Schweitzer Aerzte, Jahrg. xvii., 1887. 1130. ERNST. How far may a cow be tuberculous before her milk becomes danger- ous as an article of food? Am. Journ. of the Med. Sc., 1889 (November). 1131. FINGER. Lupus und Tuberkulose. Zusammenfassende Darstellung des jetzi- gen Standes dieser Frage. Centralbl. filr BakterioL, Bd. ii., 1887, p. 348. 1132. Ueber die sogenannte Lichenwarze und ihre Stellung zu Lupus und zu Tuberkulose. Deutsche med. Wochenschr., 1880, No. 5. 1133. LELOIR. Neue Untersuchungen tiber die Beziehung zwischen Lupus und Tuberkulose. Ann. de Dermatol. et de Syph., vii., 18B6, p. 328. 1 134. PFEIPFER. Ein neuer Fall von Uebertragung der Tuberkulose des Rindes auf den Menschen. Zeitschrift fiir Hygiene, Bd. iii., 1887, p. 189. 1135. SOUZA. Procede rapide de coloration a froid des bacilles tuberculeux dans les crachats. Compt. rend. Soc. de Biol., 1887, No. 25. 1136. SPILMANN ET HAUSHALTER. Dissemination du bacille de la tuberculose par les mouches. Compt. rend. Acad. des Sc., t. cv., 1887, p. 352. 1137. VOLSCH. Beitrag zur Frage nach der Tenacitat der Tuberkelbacillen. Zieg- ler's Beitrage zur path. Anat. und Physiol., Bd. ii., Heft 11. 1138. DOR. Methode de coloration rapide des bacilles de la tuberculose et de la lepre. Lyon Med., 1888, No. 18. 1139. HEYDENREICH. Die Struktur des Tuberkelbacillus. Wratch., 1887, No. 33. 1140. HOFFMANN. Ueber die Verbreitung der Tuberkulose durch unsere Stuben- fliege. Correspondenzbl. der arztlichen Kreis- und Bezirksvereine im Ko- nigreich Sachsen, 1888, No. 12. 1141. LUBIMOFF. Zur Technik der Farbung von Tuberkel- und Leprabacillen. Cen- tralbl. filr BakterioL, Bd. iii., 1888, p. 540. 1142. PALOWSKI. Culture des bacilles de la tuberculose sur la pomme de terre. Ann. de 1'Institut Pasteur, 1888, p. 303. 1143. PEUCH. Note sur la contagion de la tuberculose par le lait non-bouilli et la viande crue. Revue veterin., \8^S, pp. 649-653. 1144. STRAUS UND WURTZ. Unempfanglichkeit der Hlihner f tir Filtterungstuber- kulose. (Tuberculosis Congress in Paris, 1888.) Referat Wiener med. Presse, 1888, p. 1278. 1145. Einfluss des Magensaftes auf die Tuberkelbacillen. Ibid. 1146. TRUDEAU. An environment experiment repeated. Med. News, Philadelphia, 1888, No. 17. 1147. Hydrofluoric acid as a destructive agent to the tubercle bacillus. Ibid., No. 18. 1148. - — Sulphuretted hydrogen vers"s the tubercle bacillus. Ibid., 1887, p. 570. 1149. UTZ. Die Fiitterungstuberkulose der Sch weine. Bad. hierarztl. Mitth. , 1889, p. 7. BIBLIOGRAPHY. 817 1150. YILLEMIN. Etudes experimentales de 1'action de quelques agents chimiques sur le developpement du bacille de la tuberculose. Bull, general de therapeut., 1888, p. 550. 1151. YERSIN. De 1'action de quelques antiseptiques et de la chaleur sur le bacille de la tuberculose. Ann. de 1'Institut Pasteur, t. ii., 1888, p. 60. 1152. Etude sur le developpement du tubercule experimental. Ibid., p. 245. 1153. HAMMERSCHLAG. Bakteriologisch-chemische Untersuchungen der Tuberkel- bacillen. Sitzungsber. der K. Akad. der Wissensch. in Wien, Dec. 13th, 1888. 1154. HERMAN. Precede rapide de coloration du bacille tuberculeux. Ann. de 1 Institut Pasteur, t. iii., 1889, p. 160. 1155. HIRSCHBEKGER. Experimentelle Beitrage zur Infektiositat der Milch tuberku- lOser Kilhe. Inaug. Diss., Miinchen, 1889. 1156. KASTNER. Experimentelle Beitrage zur Infektiositat des Fleisches tuberku- loser Kinder. Milnchener med. Wochenschr., 1889, Nos. 34, 35. 1157. KITT. Eine vereinfachte Tuberkelbacillenfarbung. Monatsschr. fiir prakt. Thierheilkunde, i , p. 123. 1158. KRUGER. Einige Untersuchungen des Staubniederschlages der Luft in Bezug auf seinen Gehalt an Tuberkelbacillen. Inaug. Diss., Bonn, 1889. 1159. MAFFCCCI. Richerche sperimentali sull'azione dei bacilli della cuberculosi dei gallinacci e dei mammiferi nella vita embrionale ed adulta del polio. Riforuaa medica, 1889, Nos. 209 and 213, 1160. Sulla infezione tubercolare degli embrione di polio. Giornale di Anat., Fisiol. e Pathol. degli Animali, 1889, fasc. ii. 1161. Ueber die Wirkung der reinen, sterilen Kulturendes Tuberkelbacillus. » Centralbl. fur allg. Path, und path. Anat., 1890, No. 26. 1162. - Die Hiihnertuberkulose. Zeitschr. fur Hygiene, Bd. xi., 1892, p. 445. 1163. Di MATTEL Delia presenza del bacillo tubercolare sulla superficie del corpo dei tisici. Bull, della R. Accad. di Roma, 1888-89, fasc. i. 1164. MARTIN. Note sur la culture du bacille de la tuberculose. Archiv de Med. exper. et d'Anat. path., 1889, p. 77. 116-j. NEISSER. Ueber die Struktur der Lepra- und Tuberkelbacillen, etc. 1. Con- gress der Deutsch. Dertnatol. Gesellsch. in Prag, 1889. 1166. SCHILL. Tuberkelbacillenfarbung auf dem Objekttrager. Centralbl. fiir Bakteriol. Bd v., 1889, p. 340. 1167. SCHMIDT MUHLHEIM. Ueber den Nachweis und das Verhalten von Tuberkel- keimen in der Kuhmilch. Archiv fiir anim. Nahrungsmittelk., Jahrg. v., 1889, Nos. 1 and 3. 1168. STEINHEIL. Ueber die Infektiositat des Fleisches bei Tuberkulose. Inaug Diss., Miinchen, 1889. 1169. GEBHARDT. Experimentelle Untersuchungen fiber den Einfluss der Ver- dilnnung auf die Wirksamkeit des tuberkulosen Giftes. Virchow's Archiv, Bd. cxix., p. 127. 1170. CADIOT, GILBERT ET ROGER. Tuberculose des volailles. La Semaine med., x., 1890, No. 45. 1171. CZAPLEWSKI. Zum Nachweis der Tuberkelbacillen im Sputum. Centralbl. fiir Bakteriol., Bd. viii., p. 685. 1172. FORSTER. Ueber den Einfluss des Raucherns auf die Infektiositat des Fleisches perlsiichtiger Riuder. Mi'mchener med. Wochenschr., 1890, No. 16. 1173. KUHNE. Die Untersuchung von Sputum auf Tuberkelbacillen. Centralbl. fur Bakteriol , Bd. viii., p. 293. 818 BIBLIOGRAPHY. 1174. BOLLINGER. Ueber die Infektionswege des tuberkul5sen Giftes. Internal. Med. Congress of Berlin, 1890. Centralbl. fur Bakteriol., Bd. ix., p. 140. 1175. COURMONT ET DOR. De la production, chez le lapin, des tumeurs blanches experimentales, par inoculation intraveineuse de culture du bacille de Koch attenue. La Province Med., 1890, p. 529. 1176. JOLLES UNO AD. Zur Kenntniss der chemischen Natur des Kochins. Inter- nal, klin. Rundschau, Bd. v., 1891, p. 10. 1177. KOSTJURIN UND KRAiNSKi. Ueber die Wirkung von Faulniss- und Tuberkel- toxinen auf Thiere, etc. Wratsch., 1891. Abstract in Centralbl. filr Bak- teriol., Bd. ix., 1891, p. 445. 1178. NICKEL. Zur Biochemie der Bakterien. Centralbl. filr Bakteriol., Bd. ix., 1891, p. 333. 1179. Roux. Quelques remarques a propos de la colorabilite du bacille de la tuber- culose. La Province Med., 1891, p. 37. 1180. SCHNIRER. Zur Frage nach der Verb-reitung der Tuberkelbacillen ausserhalb des Korpers. Wiener med. Presse, 1891, p. 3. 1181. TIZZONI UND CATTANI. Ueber das Vorhandensein eines gegen Tuberkulose immunisirenden Princips im Blute von Thieren, welche nach der Methode von Koch behandelt worden sind. Centralbl. filr Bakteriol., Bd. xi., 1892, p. 82. 1182. NOCARD. Application des injections de tuberculine au diagnostique de la tuberculose bovine. Ann. de 1'Institut Pasteur, vol. vi., 1892, p. 44. 1183. SAWIZKY. Zur Frage tlber die Dauer der infektiosen Eigenschaften des ge- trockneten tuberkulosen Sputunis. Inaug. Diss., St. Petersburg, 1891. Ab- stract in Centralbl. filr Bakteriol., Bd. xi., 1892, p. 153. 1184. PASTOR. Eine Methode zur Gewinnung von Reinkulturen der Tuberkelbacil- len aus dem Sputum. Centralbl. filr Bakteriol., Bd. xi., 1892, p. 233. 1185. KITASATO. Gewinnung von Reinkulturen der Tuberkelbacillen und andere pathogener Bakterien aus Sputum. Zeitschrift filr Hygiene, Bd. xi., 1892, p. 441. BACILLUS LEPR^E. 1186. NEISSER. Breslauer arztl. Zeitschrift, 1879. 1187. Weitere Beitrage zur ^Etiologie der Lepra. Archiv. filr path. Anat., etc., Berlin, Ixxxiv., 1881, p. 514. Ibid., Bd. ciii., 1886, p. 355. 1188. HANSEN, A. Bacillus leprae. Nord. med. Ark., Stockholm, xii., 1880, p. 1. 1189. Virchow's Archiv, Bd. Ixxix. 1190. Studien tiber Bacillus leprae. Virchow's Archiv, Bd. xc., 1882, p. 542. 1191. Die Lage der Leprabacillen. Virchow's Archiv, Bd. ciii., 1886. p. 388. 1192. BABES. Etude comparative des bacteries de la lepre et de la tuberculose, Compt. rend. Acad. des Sc., t. xcvi., 1883. 1193. ARNING. Ueber das Vorkommen der Bacillus leprse bei Lepra anaesthetica s. nervorum. Virchow's Archiv, Bd. xcvii. 1194. DAMSCH. Uebertragungsversuche von Lepra auf Thiere. Virchow's Archiv, Bd. xcii., 1883. 1195. KOBER. Uebertragungsversuche von Lepra auf Thiere. Virchow's Archiv, 1882. 1196. Vossius. Uebertragungsversuche von Lepra auf Kaninchen. Ber. ilber den Ophthalmologencongress in Heidelberg, 1881. 1197. UNNA. Ueber Leprabacillen. Deutsche med. Wochenschr., 1885, No. 32. 1198. Zur Farbung der Leprabacillen. Monatshefte filr prakt. Dermatologie, redig. v. Uuna in Hamburg ; Erganzungsheft, 1885, p. 47. BIBLIOGRAPHY. 819 1199. UNNA. Wo liegeu die Leprabacillen ? Deutsche med. Wochenschr., 1886, p. 123. 1200. Die Bacillenklumpen inder Haut sind keine Zellen. Virchow's Archiv, Bd. ciii., 1886, p. 553. 1201. Die Leprabacillen in ihrem Verhaltniss zum Hautgewebe. Dermatolog. Studien, Heft 1, Hamburg, 1886. 1202. GUTTMANN. Ueber Leprabacillen. Berliner klin. Wochenschr., 1885, p. 81. 1203. VIRCHOW. Demonstration von Lepra laryngis. Berliner klin. Wochenschr., 1885, p. 189. 1204. MELCHER UND ORTMANN. Uebertragung von Lepra auf Kaninchen. Berliner klin. Wochenschr., 1885, p. 193. 1205. • Experimentelle Darm- und Lymphdrilsen-Lepra bei Kaninchen. Ber- liner klin. Wochenschr., 1886, No. 9. 1206. CAMPANA. Ancora della trapiantazione della lepra negli animal! bruti. Boll. della Accad. med. de Genova, 1886, No. 7. 1207. Nochmals die Uebertragung der Lepra auf Thiere. Vierteljahresschr. filr Dermatol. und Syph., 1887, p. 435. 1208. Tentativi ripetuti ma senza risultato positivo nella cultura del bacillo leproso. Riforma med., 1889, Nos. 243 and 244. 1209. Un bacillo simile al bacillo leproso aviluppatosi in tentativi di cultura di tessuti con lepra tubercolare. La Riforma med., 1891, p. 159. 1210. BAUMGARTEN. Ueber die Farbungsunterscheide zwischen Lepra- und Tuber- kelbacillen. Centralbl. fur Bakteriol., Bd. i., 1887, p. 573. 1211. Tuberkel- und Leprabacillen. Jbid., Bd. ii., 1887, p. 291. 1212. - — Replik. (to Bordoni-Uffreduzzi ; see No. 1214). Berliner kliu. AVochen- schr., 1889, p. 217. 1213. BORDONI-UPFREDCZZI. Ueber die Cultur der Leprabacillen. Zeischr. filr Hygiene, Bd. iii., 1887, p. 178. 1214. Zur Frage der Leprabacillen. Berliner klin. Wochenschr., 1888, p. 216. 1215. LELOIR. Essais d'inoculation de la lepre aux auimaux. Ann. de Dermatol. etdeSyph., 1887, p. 625. 1216. WESENER. Zur Farbung der Lepra- und Tukerkelbacillen. Centralbl. flir Bakteriol., Bd. ii., 1887, p. 131. Ibid., p. 450. 1217. Uebertragungsversuche von Lepra auf Kaninchen. Mi'mchener med. Wochenschr., 1887, No. 18. 1218. CORNIL. La contagion de la lepre. Bull, de 1'Acad. de Med., seance du 19 juin, 1888. 1219. LUBIMOFF. Zur Technik der Farbung von Tuberkel- und Leprabacillen. Centralbl. fiir Bakteriol., Bd. ii., 1888, p. 540. 1220. BEAVEN RAKE. Trans. Path. Soc. Lond., vol. xxxviii., 1887, p. 439. 1221. British Med. Journal, 1888, p. 215. 1222. Vossius. Ueber die Uebertragbarkeit der Lepra auf Kaninchen. Zeitschr. fiir vergl. Augenheilkunde, Bd. iv. 1223. DAUBLER. Ueber Lepra und deren Contagiositiit. Monatsschr. filr prakt. Dermatol., Bd. viii., 1889, p. 123. 1224. NEISSER. Ueber die Struktur der Lepra- und Tuberkelbacillen, etc. Archiv fiir Dermatol. und Syph., 1889, p. 29. Ibid., p. 42. 1225. STALLARD. The bacillus of leprosy. Brit. Med. Journ., 1889 (Dec. 21st). 1226. BABES ET KALINDERO. Sur la reaction produite par le remede de Koch chez les lopreux. La Semaine med., 1891, No. 3. 820 BIBLIOGRAPHY. BACILLUS MALLEI. 1227. LOFFLER UND ScHUTZ. Ueber den Rotzpilz. Deutsche med. Wockenschr. , 1882, No. 52. 1228. LOFFLER. Die ^tiologie der Rotzkrankheit. Arbeiten aus dem K. Gesund- heitsamte, Bd. i., 1886, p. 141. 1229. ISRAEL. Berliner klin. Wochenschr. , 1883, No. 11. 1230. BOUCHARD, CAPITAN ET CHARRIN. Bull, de 1'Acad. d. Sc., 1882, No. 51. 1231. WEICHSELBAUM. Zur jEtiologie der Rotzkrankheit des Menschen. "Wiener med. Wochenschr., 1885, p. 21. 1232. KITT. Versuche ilber die Zuchtung des Rotzpilzes. Jahresber. der Miinchen. Thierarzneisch., 1883-84. 1233. Impfrotz bei Waldmausen. Centralbl. fiir Bakteriol., Bd. ii., 1887, p. 241. 1234. CADEAC ET MALET. Transmission de la morve de mire au foetus. Progres med., 1886, No. 4. 1235. La resistance du virus morveux ft 1'action destructive des agents at- mospheriques et de la chaleur. Ibid., 1886, No. 34. 1236. • La transmission de la morve sur le pore et de mere du foetus. Rec. de med. veterin., 1886, No. 5. 1237. Etude experimental de la transmission de la morve par contagion mediate ou par infection. Revue de Med., 1887, No. 5. 1238. Ueber Impfrotz bei Wiihlratten. Oesterr. Monatsschr. fur Thierheilk., 1888, No. 1. 1239. KRANZFELD. Zur Kenntniss dea Rotzbacillus. Centralbl. fur Bakteriol., Bd. ii., 1887, p. 273. 1240. BABES. Eindringen der Rotzbacillen durch die unverletzte aussere Haut. Acad. de Med., Paris, 1888. 1241. BAUMGARTEN. Zur Frage der Sporenbildung bei den Rotzbacillen. Cen- tralbl. ftlr Bakteriol., Bd. iii., 1888, p. 397. 1242. KUHNE. Ueber Furbung der Bacillen in Malleusknoten. Fortschr. der Med. , Bd. vi., 1888, p. 860. 1243. STRAUS. Sur la vaccination contre la morve. Compt. rend. Acad. des. Sc., t. cviii., p. 530. 1244. FINGER. Zur Frage der Immunitat und Phagocytose beim Rotz. Ziegler's Beitrage zur pathol. Anat., Bd. vi., 1889, Heft 4. 1245. SALMON. Glanders. Reports of the Bureau of Animal Industry for the years 1887 and 1888 (Washington). 1246. SMITH. On the influence of slight modifications of culture media on the growth of bacteria as illustrated by the glanders bacillus. Journ. of Comp.. Med. and Vet. Archives, March, 1890. 1247. CORNIL. Sur la penetration des bacilles de la morve a travers la peau intact. La Semaine med.. 1890, No. 22. 1248. PREUSSE. Versuche mit Rotzlymphe — Mallei'n. Berliner thierarztliche Woch- enschr., 1891, No. 29. 1249. EBER. Ueber Rotzlymphe— Malleiin. Centralbl. fiir Bakteriol. , Bd. xi., 1892, p. 20. BACILLUS OF LUSTGARTEN. 1250. LUSTGARTEN. Wiener med. Wochenschr., 1884, No. 47. 1251. Die Syphilisbacillen. Wien, 1885. 1252. DE GIACOMI. Neue Farbungsmethode der Syphilisbacilleu. Correspondenzbl. fiir Schweitzer Aerzte, Bd. xv. BIBLIOGRAPHY. 821 1253. ALVAREZ ET TAVEL. Bull, de 1'Acad. de Med., 1885 (August). 1254. DOUTRELPONT UND ScHUTZ. Deutsche med. Wochenschr., 1885, No 19. 1255. KLEMPERER. Ueber Syphilis- und Smegmabacillen. Deutsche med. Wochen schr., 1885, p. 809. 1256. BITTER. Ueber Syphilis- und Smegmabacillen. Virchow's Archiv, Bd. cvi., 1886, p. 209. 1257. DOUTRELPONT. Ueber die Bacillen bei Syphilis. Vierteljahresschr. f l\r Der- matol. und Syph., Bd. xiv., 1887, p. 101. 1258. MATTERSTOCK. Ueber Bacillen bei Syphilis. Mitt, aus der med. Klin, der Univ. Wilrzburg, 1886. 1259. VON ZEISSEL. Untersuchungen ilber den Lustgarten'schen Bacillus in Syphi- lisprodukten und Sekreten derselben. Wiener med. Presse, 1885, No. 48. 1260. Die Wesenheit des Syphiliscontagium. Allg. Wiener med. Zeitung, 1887, Nos. 32-34. 1261. BENDER. Die Bacillen bei Syphilis. Centralbl. fur Bakteriol., Bd. ii., 1887, Nos. 11 and 12. 1262. TAVEL. Zur Geschichte der Smegmabacillen. Centralbl. fur Bakteriol., Bd. ii., 1887, p. 673. 1263. MARCUS. Nouvelles recherches sur le microbe de la syphilis. These de Paris, 1888. 1264. MARKUSE. Ueber den jetzigen Stand der Syphilis und Smegmabacillenfrage. Vierteljahresschr. filr Dermatol. und Syphilis, 1888, p. 343. 1265. LEWY. Ueber Syphilis- und Smegmabacillen. Inaug. Diss., Bonn, 1889. BACILLUS OF EVE AND LINGARD. 1266. EVE AND LINGARD. On a bacillus cultivated from the blood and tissues in syphilis. The Lancet, London, 1886 (April 10th). MICROCOCCI OF DI8SE AND TAGUCHI. 1267. DISSE UND TAGUCHI. Deutsche med. Wochenschr., 1886, p. 235. BACILLUS OF RHINOSCLEROMA (?). 1268. CORNIL ET ALVAREZ. Sur les microOrganismes du rhinosclerome. Bull. Acad. de Med., Paris, 1885. 1269. PALTAUF UND VON EISELBERG. Zur ^Etiologie des Rhinoskleroms. Fort- schr. der Med., 1886, Nos. 19 and 20. 1270. WOLKOWITSCH. Zur Histologie und parasitilren Natur des Rhinoskleroms. Centralbl. filr die Wissensch., 1886, No. 47. 1271. — : Das Rhinosklerom. Langenbeck's Archiv, Bd. xxxviii., Heft 2 and 3. 1272. DITTRICH. Ueber das Rhinosklerom. Zeitschrift f ilr Heilkunde, Bd. viii., 1887, p. 251 . 1273. Entgegnung auf die kritischen Bemerkungen des Herrn Babes. Centralbl. filr Bakteriol., Bd. iii., 1888, p. 146. 1274. Zur ^Etiologie des Rhinoskleroms. Centralbl. filr Bakteriol., Bd. v., 1889, No. 5. 1275. BABES. Antwort auf Herrn Dittrich's Entgegnung, dessen Artikel ilber Rhinosklerom. Centralbl. filr Bakteriol. , Bd. ii., 1887, p. 617. 1276. MELLK. Les bacilles du rhiuosclerome. Abstract in Baumgarten's Jahresbe- richt, Bd. iv., 1888, p. 228. 1277. Neue Fiirbemethode filr Rhinosklerombacillen. Ibid. 822 BIBLIOGRAPHY. 1278. STEPANOW. Ueber die Impfungen des Rhinoskleroms. Centralbl. fur Bak- teriol., Bd. v., 1889, p. 549 (abstract). 1279. ZAGARI. Richerche etiologiche sull' rinoscleroma. Giorn. intern, delle Sci. mediche, 1889, No. 4. 1280. PAWLOWSKI. Ueber die JEtiologie und Pathologic des Rhinoskleroms, etc. Centralbl. filr Bakteriol., Bd. ix., 1891, p. 742. BACILLUS OP KOUBASOFF. 1281. KOUBASOFF. Die Mikroorganismen der krebsartigen Neubildungen. Abstract in Centralbl. fur Bakteriol., Bd. vii., 1890, p. 317. BACILLUS OF NOCARD. 1282. NOCARD. Maladie des boaufs de la Guadeloupe. Ann. de 1'Institut Pasteur, vol. ii., 1888, p. 293. XII. BACILLI WHICH PRODUCE SEPTICAEMIA IN SUSCEPTIBLE ANIMALS. BACILLUS SEPTICAEMIA H/EMORRHAGIC^E. BACILLUS OF CHOLERA IN DUCKS. BACILLUS OF HOG CHOLERA. BACILLUS OF BELFANTI AND PASCAROLA. BACILLUS OF SWINE PLAGUE (MARSEILLES). BACILLUS SEPTICUS AGRIGENUS. 1283. DAVAINE. Recherches, etc., de la septicemie. Bull, de fl'Acad. de Med., t. viii., 1879, p. 121. 1284. PASTEUR. Sur les maladies virulentes, et en particulier sur la maladie appelee vulgairement cholera des poules. Compt. rend. Acad. des Sc., xc., 1880, p. 239. 1285. De 1'attenuation du virus du cholera des poules. Ibid., xci., p. 673. 1286. PERRONCITO. Ueber das episootische Typhoid der Hiihner. Arcbiv fur wiss. und prakt. Thierheilk., 1879. 1287. KITT. Mittheilungen ilber die Typhoidseuche des Geflilgels. Allg. deutsche Gefliigelzeitung, 1885 (Feb. 15th). 1288. — ^— Beitrage zur l£enntniss der Geflilgelcholera und deren Schutzimpfung. Deutsche Zeitschr. filr Thiermedicin und vergl. Pathol., Bd. xiii., 1886. 1289. KOCH. ^Etiologie der Wundinfektionskrankheiten. Leipzig, 1878, p. 59. 1290. GAFFKY. Experimentelle erzeugte Septikamie. Mitth. aus dem K. Gesund- heitsamte, Bd. i., 1881. 1291. Die Geflugelcholera. Zusammenfassender Bericht. Centralbl. filr Bakteriol., Bd. i., 1887, p. 305. 1292. KLEIN. Report on infectious pneumo-euteritis of the pig. Rep. Med. Off- Local Govt. Board, London, 1878, pp. 169-280, 26 pi. 1293. Bemerkungen ilber die ./Etiologie der Schweineseuche. Fortschr. der Med., Bd. vi., 1888, p. 929. 1294. BUCH. Zur Kenntniss der Schweineseuche. Archiv filr wiss. und prakt. Thierheilk., Bd. xiii., 1887, p. 332. 1295. CORNIL ET CHANTEMESSE. Etiologie de la pneumouie contagieuse des pores- Le Bulletin medical, 1887, p. 1363. BIBLIOGRAPHY. 823 1296. ORESTE AND ARMANNI. Studii e richerche intorno al barbone dei buffali. Atti del R. Institute d'Incoraggiamento alle Sci. nat., econom., et technol., 1887. 1297. CORNIL ET TOUPET. Sur une maladie nouvelle des canards. Bull, de la Soc. nat. d' Acclimation, 1888 (June 20th). 1298. HUEPPE. Ueber die Wildseuche und ihre Bedeutung fiir Nationalokonomie und Hygiene. Berliner klin. "Wochenschr. , 1886, p. 753. 1299. GAMELEIA. Zur ^Etiologie der Hilhnercholera. Centralbl. fiir Bakteriol., Bd. iv., 1888, p. 161. 1300. GRAFFUNDER. Zur Kenntniss der Schweineseuche. Deutsche Zeitschrift fiir Thiermed., 1888, p. 391. 1301. SALMON AND SMITH. The bacterium of swine plague. Am. Monthly Mic. Journ., 1886, p. 204. 1302. SALMON. On swine plague. Second An. Rep. of the Bureau of Animal In- dustry, Washington, 1886 (for the year 1885). Ibid.. 1887, p. 603. 1303. Further investigations on the nature and prevention of hog cholera. Rep. of the Com. of Agriculture for 1887, p. 481 (Washington, 1888). 1304. : — Hog cholera : its history, nature, and treatment, etc. Washington, 1889. 1305. Hog cholera in other countries. Rep. of Bureau of Animal Industry, 1888, p. 159. 1306. Investigations of fowl cholera. Rep. Com. of Agriculture for 1881 and 1882. 1307. SMITH. Contribution to the study of the microbe of rabbit septictemia. Journ. of Comp. Med. and Surg., vol. viii., 1887, p. 24. 1308. Zur Kenntniss des Hogcholerabacillus. Centralbl. fiir Bakteriol., Bd. ix., 1891, pp. 253, 307, 339. 1309. Zur Kenctniss der americanischen Schweineseuche. Zeitschrift fiir Hygiene, Bd. x., 1891, p. 480. 1310. • Special report on the cause and prevention of swine plague. U. S. Dept. of Agriculture, Bureau of Animal Industry, Washington, 1891. 1311. SCHUTZ. Ueber die Schweineseuche. Arbeiten aus dem K. Gesundheitsamte, 1886, Bd. i., p. 376. 1312. Die Schweinepest in Danemark. Archiv fiir wissensch. und prakt. Thierheilk., 1888, p. 376. 1313. EBERTH UND SCHIMMELBUSCH. Der Bacillus der Frettenseuche. Virchow's Archiv, Bd. cxv., 1889, p. 282. Ibid., cxvi., 1889, p. 327. 1314. FROHLING. Ueber amerikanische Schweineseuche, hog cholera, swine plague. Schweiz. Arch, fiir Thierheilk., xxx., p. 116. 1315. BILLINGS. Dr. Salmon's latest : Hog cholera and swine plague two distinct diseases. The Nebraska Farmer, 1887, p. 365. 1316. Swine plague, with especial reference to the porcine pests of the world. Lincoln, Neb., 1888. 1317. The Southern cattle plague (Texas fever) of the United States. Lin- coln, Neb., 1888. 1318. Dr. E. Salmon's swine plague and hog cholera critically considered. Lincoln, Neb., 1889. 1319. Are the German "Schweineseuche" and the "swine plague "of the Government of the United States identical diseases ? American Naturalist, 1895 (March 12th). 1320. Evidence showing that the report of the " board of inquiry concerning swine diseases" was fixed. Lincoln, Neb., 1890. 824 BIBLIOGRAPHY. 1321. RIETSCH ET JOBERT. L'epidemie des pores & Marseille en 1887. Compt. rend. Acad. des Sc., t. cvi., 1888 (No. 15). 1322. SELANDER. Ueber die Bakterien der Schweinepest. Centralbl. fur Bakteriol., Bd. iii., 1888, No. 12. 1323. BLEISCH UND FIEDELER. Beitrag zur Kenntniss der Schweineseuche. Zeit- schrift filr Hygiene, Bd. vi., 1889, p. 401. 1324. REPORT OP THE U. S. BOARD OF INQUIRY concerning epizootic diseases among swine. U. S. Department of Agriculture, 1889. 1325. RIECK. Eine infektiose Erkrankung der Kanarienv5gel. Deutsche Zeitschrift filr Thiermed., Bd. xv., 1889, p. 68. 1326. SEMMER UND NONIEWICZ. Die Schweineseuche. Oesterr. Monatsschr. filr Thierheilk., 1889, No. 4. 1327. WERTHEIM. Bakteriologische Untersuchungen ilber die Cholera gallinarum. Archiv f&r exp. Pathol. und Pharm., Bd. xxvi., 1889, p. 61. 1328. RACCUGLIA. Ueber die Bakterien der amerikanischen Swine-Plague (Hog Cholera) und der deutschen Schweineseuche. Centralbl. f ilr Bakteriol. , Bd. viii., 1890, p. 289. 1329. BUNZL-FEDERN. Bemerkungen liber Wild- und Schweineseuche. Centralbl. fur Bakteriol., Bd. ix., 1891, p. 787. 1330. Untersuchungen iiber einige seuchenartige Erkrankungen der Schweine. Archiv fiir Hygiene, 1891. 1331. SCHWEINITZ. A preliminary study of the ptomaines from the culture liquids of the hog-cholera germ. Phila. Med. News, 1890, p. 237. 1332. NOVY. The toxic products of the bacillus of hog cholera. Phila. Med. News, 1890, p. 231. 1333. CANEVA. Ueber die Bakterien der hamorrhagischen Septikamie (Hueppe), Hog-Cholera (Salmon), Swineplague (Billings), Swinepest (Selander), Rin- derseuche (Billings), Biiffelseuche(Oreste-Armanni), Marseille'sche Schweine- seuche (Jobert, Rietsch), Frettenseuche (Eberth). Centralbl. fiir Bakteriol., Bd. ix., 1891, p. 557. 1334. FROSCH. Ein Beitrag zur Kenntniss der Ursache der amerikanischen Schweine- seuche, etc. Zeitschrift fiir Hygiene, Bd. ix., 1890, p. 235. Ibid., Bd. x., 1891, p. 509. 1335. WELCH. Johns Hopkins Hospital Bulletin, December, 1889. BACILLUS ERYSIPELATOS SUIS. 1336. KOCH. Wundinfektionskrankheiten. Leipzig, 1878. 1337. PASTEUR. Le rouget du pore; avec le collaboration du MM. Chamberlain, Roux et Thuillier. Compt. rend. Acad. des Sc., xcv., 1882, p. 1120. 1338. PASTEUR ET THUILLIER. Bull, de 1'Acad. de Med., Paris, xcvii., 1883. 1339. LOFFLER. Experimentelle Untersuchungen iiber Schweinerothlauf . Arbeiten aus dem K. Gesundheitsamte, Bd. i., 1885. 1340. SCHUTZ. Ueber den Rothlauf der Schweine und die Impfung desselben. Ar- beiten aus dem K. Gesundheitsamte, Bd. i., 1885, p. 56. 1341. Archiv fiir wissensch. und prakt. Thierheilk., Bd. xii., 1886, Heft 1. 1342. LYDTIX UND SCHOTTELIUS. Der Rothlauf der Schweine. Wiesbaden, 1885. 1343. LYDTIN. Schutzimpfungen gegen den Rothlauf [der Schweine. Bad. thier- arztl. Mitth., 1886, p. 9. 1344. HESS UND GUILLEBEAU. Zur Schutzimpfung gegen Schweineseuche. Schwei- zer Archiv fiir Thierheilk., Bd. xxviii., 1886, Heft 8. BIBLIOGRAPHY. 825 1345. KITT. Beitrage zur Kenntniss des Stabschenrothlauf der Schweine und dessen Schutzimpfung. Revue fur Heilk. und Thierzucht, 1886. 1346. Untersuchungen ilber dea Stabschenrothlauf und dessen Schutz- impfung. Centralbl. fur Bakteriol., Bd. ii., 1887, p. 693. 1347. PAMPOUKIS. Les bacilles du rouget. Archiv de Physiol. norm, et pathol., 1886, p. 89. 1348. HAFNER. Die Schutzimpfung gegen den Rothlauf der Schweine. Bad. thierarztl. Mitth , 1889, p. 17. 1349. HESS. Der Stabschenrothlauf und die Schweineseuche. Thiermed. Vortrilge, herausgegeben voii Schneidemuhl, Bd. i., Heft 1. 1350. JAKOBI. Beitrag zur Schutzimpfung gegen den Rothlauf der Schweine. Ber- liner thierarztl. Wochenschr., 1888, No. 50. 1351. PETRI. Ueber die Widerstandsfahigkeit der Bakterien des Schweinerothlaufs in Reinkulturen und in Fleisch rothlaufkranker Schweine gegen Kochen, Schmoren, Braten, Salzen, Einpftkeln und Rauchern. Arbeiten aus dem K. Gesundheitsamte, Bd. vi., 1890, Heft 2. BACILLUS COPROGENES PARVUS. 1352. BIENSTOCK. Zeitschr. furklin. Med., Bd. viii., Heft 1. BACILLUS CAVICIDA. 1353. BRIEGER. Berliner klin. Wochenschr., 1884, No. 14. 1354. ESCHERICH. Die Darmbakterien des Sauglings. Stuttgart, 1886, p. 74. BACILLUS CAVICIDA HAVANIENSIS. 1355. STERNBERG. Report on the etiology and prevention of yellow fever. Wash- ington, 1891, p. 202. BACILLUS CRASSUS 8PUTIGENUS. 1356. FLUGGE. Die Mikroorganismen. 2d ed., 1886, p. 260. BACILLUS PYOGENES FCETIDUS. 1357. PASSET. Untersuchungen ilber die eitrigen Phlegmone des Menschen. Ber- lin, 1885. PROTEUS HOMINIS CAPSULATUS. PROTEUS CAPSULATUS 8EPTICUS. 1358. BORDONI-UFFREDUZZI. Ueber den Proteus hominis capsulatus, etc. Zeit- schr. for Hygiene, Bd. iii., 1888, p. 333. 1359. BANTI. Sopra quatro nuove specie di protei o bacilli capsulati. Dal. Giornale medico : Lo Sperimentali, 1888 (August). BACILLUS ENTERITIDIS. 1360. GARTNER. Correspondenzbl. des allg. arztl. Vereins von Thuringen, 1888. 1361. KAKLINSKY. Zur Kenntniss des Bacillus enteritidis, Gartner. Centralbl. f iir Bakteriol., Bd. vi., 1889, No. 14. BACILLUS OF GROUSE DISEASE. 1362. KLEIN. Ueber eine akute infektiose Krankheit des schottischen Moorhuhnes (Lagopus Scoticus). Centralbl. fur Bakteriol., Bd. vi., 1889, p. 36. Ibid., p. 259. 826 BIBLIOGRAPHY. BACILLUS GALLINARtTM. 1363. KLEIX. Ueber eine epidemische Krankheit der Hiihner, verursacbt durch einen Bacillus. Centralbl. fur Bakteriol., Bd. v., 1889, p. 689. Ibid., Bd. vi., p. 259. BACILLUS SMARAGDINUS FCETIDUS. 1364. REIMANN. Inaug. Diss., Wilrzburg, 1887. BACILLUS PNEUMOSEPTICUS. 1365. BABES. Progres medical roumain, 1889 (April 6th). BACILLUS CAPSULATUS. 1366. PFEIFFER. Ueber einen neuen Kapselbacillus. Zeitschr. fiir Hygiene, Bd. vi., 1889, p. 145. BACILLUS HYDROPHILUS PUSCUS. 1367. SANARELLI. Ueber einen neuen Mikroorganismus des Wasser, welcher flir Thiere mit veranderlicher und konstanter Temperatur pathogen ist. Cen- tralbl. fur Bakteriol., Bd. ix., pp. 193, 222. BACILLUS TENUIS SPUTIGENUS. 1368. PANSINI. Bakteriologische Studien liber den Auswurf. Virchow's Archiv, Bd. cxxii., 1890. BACILLUS OF LASER. 1369. LASER. Ein neuer, f ilr Versuchsthiere pathogener Bacillus, aus der Gruppe der Fretten-Schweineseuche. Centralbl. fur Eakteriol., Bd. xi., 1892, p. 184. BACILLUS TYPHI MURIUM. 1370. LOFFLER. Ueber Epidemieen unter den in hygienischen Institute zu Greifs- wald gehaltenen Miiusen, und liber die Bekilmpfung der Feldmausplage. Centralbl. fttr Bakteriol., Bd. xi., 1892, p. 13). BACILLUS OF CAZAL AND VAILLARD. 1371. CAZAL ET VAILLARD. Sur une maladie parasitaire de I'homme transmissible au lapin. Ann. de 1'Institut Pasteur, vol. v., 1891, p. 353. BACILLUS OP BABES AND OPRESCU. 1372. BABES ET OPRESCU. Sur un bacille trouve dans un cas de septicemie hemor- rhagique presentant certains caracteres du typhus exanthematique. Ann. de Tlnstitut Pasteur, vol. v., 1891, p. 274. BACILLUS OF LUCET. 1373. LUCET. Dysenteric epizootique des poules et des dindes. Ann. de 1'Institut Pasteur, vol. v., 1891, p. 312. CAPSULE BACILLUS OF LOEB. 1374. LOEB. Ueber einen bei Keratomalacia infantum beobachteten Kapselbacillus. Centralbl. fur Bakteriol., Bd. x., 1891, p. 369. BIBLIOGRAPHY. 827 XIII. PATHOGENIC AEROBIC BACILLI NOT DESCRIBED IN PREVIOUS SECTIONS. BACILLUS COLI COMMUNIS. 1375. EMMERICH. Die Cholera in Neapel. Deutsche med. Wochenschr., 1884, No. 50. 1376. WEISSER. Ueber die Emmerich'schen sogenannten Neapler Cholerabakterien. Zeitschr. fur Hygiene, Bd. i., 1886, p. 315. 1377. ESCHERICH. Die Darmbakterien des Sauglings. Stuttgart, 1886. 1378. BAGINSKY. Ueber GahrungsvorgangeimkindlichenDarmkanal, etc. Deutsche med. Wochenschr., 1888. 1379. BOOKER. A study of some of the bacteria found in the dejecta of infants af- flicted with summer diarrhoea. Trans, of the Ninth Internat. Med. Con- gress, vol. iii. 1380. Second communication. Trans, of the Am. Pediatric Society, 1889, p. 198. 1381. STERNBERG. Recent researches relating to the etiology of yellow fever. Trans. Assn. Am. Physicians, vol. iii., 1888, p. 321. 1382. FRANKEL, A. Ueber peritoneal Infektion. Wiener klin. Wochenschr., 1891, Nos. 13-15. BACILLUS LACTIS AEROGENES. ESCHERICH. Op. cit. (No. 1377). BAGINSKY. Op. cit. (No. 1378). BOOKER. Op. cit. (No. 1379). 1383. JEFFRIES. A contribution to the study of the summer diarrhoeas of infancy. Trans. Am. Pediatric Society, vol. i., 1889, p. 249. BACILLUS ACIDIFORMANS. 1384. STERNBERG. Report on the etiology and prevention of yellow fever. Wash- ington, 1891, p. 200. BACILLUS CUNICULICIDA HAVANIENSIS. STERNBERG. Op. cit. (No. 1384), p. 187. BACILLUS LEPORIS LETHALIS. STERNBERG. Op. cit. (No. 1384), p. 167. BACILLUS PYOCYANUS. 1385. GESSARD. De l-a, pyocyanine et de son microbe. These de Paris, 1882. 1386. Nouvelles recherches sur le microbe pyocyanique. Ann. de 1'Institut Pasteur, vol. iv., 1890, p. 89. 1387. FRICK. Bakteriologische Mittheilungen liber das grilne Sputum und ilber die grilnen Farbstoff producirenden Bacillen. Virchow's Archiv, Bd. cxvi., 1889. 1388. WASSEUZUG. Sur la formation de la matiere colorante chez le Bacillus pyocy- anus. Ann. de 1'Institut Pasteur, vol. i., 1887, p 581. 1389. ERNST. Ueber einen neuen Bacillus des blauen Eiters (Bacillus pyocyanus ft). Zeitschrift fur Hygiene, Bd. ii., 1887, p. 369. 1390. LEDDERHOSE. Ueber den blauen Eiter. Tagebl. der 60. Versamml. Deutscher Naturf. und Aerzte in Wiesbaden, 1887, p. 295. 828 BIBLIOGRAPHY. 1391. LEDDERHOSE. Deutsche Zeitschrift fur Chirurg., 1888. 1392. CHAUUIN. La maladie pyocyanique. Paris, 1889. 1393. BOUCHARD. Influence qu'exerce sur la maladie charbonneuse 1'inoculation du bacille pyocyanique. Compt. rend. Acad. des Sc., t. cviii., 1889, p. 713. 1394. BABES. Note sur quelques matieres colorantes et aromatiques produites par le bacille pyocyanique. Compt. rend. Soc. de Biol. , Paris, 1889, p. 438. 1395. WOODHEAD ET WOOD. De 1'action antidotique exercee par les liquides pyo- cyaniques sur le cours de la maladie charbonneuse. Compt. rend. Acad. des Sc.,t. cix., 1889, No. 26. 1396. ROGER. Des modifications qu'on peut provoquer dans les fonctions d'un microbe chromogene. Compt. rend. Soc. de Biol., 1887. PROTEUS VULGARIS. 1397. HAUSER. Ueber Faulnissbakterien. Leipzig, 1885, 94 pp., 15 pi. 1398. CHEYNE. Report on a study of certain of the conditions of infection. British Med. Journ., 1886 (July 31st;. 1399. FOA ET BONOME. Sur les maladies causees par les microorganismes du genre Proteus (Hauser). Archives ital. de Biologic, t. viii., 1887. 1400. - Ueber Schutzimpfungen. Zeitschrift fur Hygiene, Bd. v., 1888, p. 415. 1401. BOOKER. Trans. Am. Pediatric Soc., vol. i., 1889, p. 206. PROTEUS OP KARLINSKY. 1402. KARLINSKY. Ein neuer pathogener Spaltpilz (Bacillus murisepticus pleomor- phus). Centralbl. fur Bakteriol., Bd. v., 1889, p. 193. PROTEUS MIRABILIS. HAUSER. Op. cit. (No. 1397). PROTEUS ZENKERI. HAUSER. Op. cit. (No. 1397). PROTEUS SEPTICU8. 1403. BABES. Bakteriologische Untersuchungen Tiber septische Prozesse des Kindes- alters. Leipzig, 1889. PROTEUS LETHALIS. 1404. BABES. Progres med. roumain, 1889 (April 6th). BACILLUS A OP BOOKER. BOOKER. Op. cit. (No. 1379). BACILLUS ENDOCARDITIDIS GRISEUS. 1405. WEICHSELBAUM. Beitrage zur JEtiologie und pathol. Anatomic der Endocar- ditis. Ziegler's Beitrage, Bd. iv., 1888, p. 119. BACILLUS ENDOCARDITIDIS CAPSULATUS. WEICHSELBAUM. Op. cit. (No. 1405), p. 127. BACILLUS OP LESAGE. 1406. LESAGE. De la diarrhee verte des enf ants du premier age. Bull, med., 1887 (Oct. 26th). BIBLIOGRAPHY. 829 BACILLUS OP DEMME. 1407. DEXIME. Zur Kenntniss der schweren Erytheme. Fortschr. der Med., 1888, No. 7. BACILLUS (EDEMATIS AEROBICUS. 1408. KLEIN. Centralbl. fiir Bakteriol., Bd. x., p. 186. BACILLUS OF LETZERICH. 1409. LETZERICH. Untersuchungen und Beobachtungen uber Nephritis bacillosa interstitialis primaria ; eine neue Mykose. Zeitschr. fur klin. Med., Bd. xiii., 1887, p. 33. BACILLUS OP SCHIMMELBUSCH. 1410. SCHIMMELBUSCH. Ein Fall von Noma. Deutsche med. Wochenschr., 1889> No. 26. BACILLUS FOZTIDUS 1411. HAJEK. Die Bakterien bei der akuten und chronischen Coryza, etc. Berliner klin. Wochenschr., 1888, No. 33. BACILLUS OP LUMNITZER. 1412. LUMNITZER. Centralbl. fiir Bakteriol., Bd. iii., p. 621. BACILLUS OP TOMMASOLI. 1413. TOMMASOLI. Di una nuova forma di sicosi. Giornale italiano delle mallattie veneree e delle pelle, 1889, No. 3. BACILLUS OP SCHOU. 1414. SCHOU. Untersuchungen liber Vaguspneumonie. Fortschr. der Med., 1885, No. 15. BACILLUS NECROPHORUS. 1415. LOFPLER. Mitth. aus dem K. Gesundheitsamte, Bd. ii., p. 493. BACILLUS COPROGENES FO3TIDUS. 1416. SCHOTTELIUS. Der Rothlauf der Schweine. Wiesbaden, 1885. BACILLUS OXYTOCUS PERNICIOSUS. 1417. FLUGGE. Die Mikroorganismen. 2d ed., Leipzig, 1886, p. 268. BACILLUS SAPROGENES II. ROSENBACH. Op. cit. (No. 674). BACILLUS OP AFANASSIEW. 1418. AFANASSIEW. St. Petersburger med. Wochenschr., 1887, Nos. 39^12. PNEUMOBACILLUS LIQUEFACIBN8 BOVIS. 1419. ARLOING. Compt. rend. Acad. des Sc., t. cvi. BACILLUS PSEUDOTUBERCULOSIS. 1420. PFEIPPER. Ueber die bacillure Pseudotuberculose bei Nagethiere. Leipzig, 1889. BACILLUS GINGIV.* FYOGENES. 1421. MILLER. Die Mikroorganismen der Mundhohle. Leipzig, 1889, p. 216. 69 830 BIBLIOGRAPHY. BACILLUS DENTALIS VIRIDANS. MILLER. Op. cit. (No. 1421), p. 218. BACILLUS PULP^E PYOGENES. MILLER. Op. cit. (No. 1421), p. 219. BACILLUS SEPTICUS KERATOMALACLiE. BABES. Op. cit. (No. 1403). BACILLUS SEPTICUS ACUMINATUS. BABES. Op. cit. (No. 1403). BACILLUS SEPTICUS ULCERIS GANGR.ENOSI. BABES. Op. cit. (No. 1403). BACILLUS OP TRICOMI. 1422. TRICOMI. II micro- parassita della gangrena senile. Atti della Soc. italiana di Chirurgia, 1887 (April 20th). BACILLUS ALBUS CADAVERIS. 1423. STRASSMANN UND STRECKER. Bakterien bei der Leichenfaulniss. Zeitsclir. fiir Medicinalbeamte, 1888, No. 3. BACILLUS VARICOSITS CONJUNCTIVE. 142 1. GOMBERT. Reciierches experiment, sur les microbes des conjunctives. Mont- pellier; Paris, 1889. BACILLUS MENINGITIDIS PURULENT^E. 1425. NEUMANN UWD SCHAEFFER. Zur ^Etiologie der eitrigen Meningitis. Vir- chow's Archiv, Bd. cix., 1887, p. 477. BACILLUS SEPTICUS VESIC.E. 1426. CALDO. Deux nouveaux bacilles dans les urines pathologiques. Bull, de la Soc. anatom. de Paris, 1887, p. 339. BACILLUS OF GESSNER. 1427. GESSNER. Archiv filr Hygiene, Bd. ix., p. 129. BACILLUS CHROMO-AROMATICUS. 1428. GALTIER. Sur un microbe pathogene chromo-aromatique. Compt. rend. Acad. des Sc., t. cvi., 1888, p. 1368. BACILLUS CANALIS CAPSULATUS. 1429. MORI. Ueber die pathogenen Bakterien des Kanalisationswassers. Zeitschr. ftlr Hygiene, Bd. iv.( 1888, p. 97. BACILLUS CANALIS PARVUS. MORI. Op. cit. (No. 1429). BACILLUS INDIGOGENUS. 1430. ALVAREZ. Compt. rend. Acad. des Sc., t. cv., 1887, p. 286. BIBLIOGRAPHY. 831 BACILLUS OF KARTULIS. 1431. KARTULIS. Zur JEtiologie der agyptischen katarrhalischen Conjunctivitis. Centralbl. filr Bakteriol., Bd. i., 1887, p. 289. 1432. WEEKS. Der Bacillus des akuten Bindehautkatarrhs. Arch, filr Augenheilk., Bd. xvii., 1887, p. 318. BACILLUS OF UTPADEL. 1433. UTPADEL. Ueber eiuen pathogenen Bacillus aus Zwischendeckf illlung. Ar- chiv fur Hygiene, Bd. vi., 1887. 1434. GESSNER. Ueber die Bakterien in Duodenum des Menschen. Archiv fiir Hy- giene, Bd. ix., 1889, p. 128. BACILLUS ALVEI. 1435. CHESHIRE AND CHEYNE. The pathogenic history, and history under cultiva- tion, of a new bacillus, the cause of a disease of the hive bee hitherto known as foul brood. Journ. of the Royal Mic. Soc. of London, 1885 (March). 1436. KLAMANN. Ueber die Faulbrut der Bienen. Bienenwirthschaftl. Centralbl. (Hannover), 1888, Nos. 18 and 19. BACILLUS OF ACNE CONTAGIOSA OF HORSES. 1437. DIECKERHOFF UND GRAWiTZ. Die Acne contagiosa des Pferdes und ihre yEtiologie. Virchow:s Archiv, Bd. cii., 1885, p. 148. BACILLUS NO. I. OF ROTH. 1438. ROTH. Ueber pathogene Mikroorganismen in den Harden. Zeitschr. filr Hygiene, Bd. viii., 1890, p. 296. BACILLUS NO. II. OF ROTH. ROTH. Op. cit. (No. 1438). BACILLUS OF OKADA. 1439. OKADA. Ueber einen neuen pathogenen Bacillus aus Fussbodenstaub. Cen- tralbl. filr Bakteriol., Bd. ix., 1891, p. 442. BACILLUS OF PURPURA H^EMORRHAGICA OF TIZZONI AND GIOVANNINI. 1440. TIZZONI E GIOVANNINI. Recherche batteriologiclre e sperimentali sulla genesi dell' infezione emorragica. Atti della R. Accad. delle Sci. di Bologna, 1889. 1441. Ziegler's Beitrage, Bd. vi., 1889, p. 300. BACILLUS OF PURPURA H^ESfORRHAGICA OF BABES. 1442. BABES. Ueber Bacillen des hiimorrhagischen Infektion des Menschen. Cen- tralbl. filr Bakteriol., Bd. ix., 1891, p. 719. BACILLUS OF PURPURA H^EMORRHAGICA OF KOLB. 1443. KOLB. Arbeiten aus dem K. Gesundsheitsamte, Bd. viii., 1891. BABES. Op. cit. (No. 1442). BACILLUS HEMINECROBIOPHILUS. 1444. ARLOING. Compt. rend. Acad. des Sc., cvi., p. 1365. Ibid., cvii., p. 1167. 832 BIBLIOGRAPHY. XIV. PATHOGENIC ANAEROBIC BACILLI. BACILLUS TETANI. 1445. NICOLAIEK. Deutsche med. Wochenschr. , 1884, No. 42. 1446. Beitrage zur ^Etiologie des Wundstarrkrampfes. Inaug. Diss., Got- tingen, 1885. 1447. CARL E RATTONE. Studio sperimentale sull' etiologia del tetano. Gior. della R. Accad. di Med. di Torino, 1884 (March). 1448. ROSENBACH. Zur JEtiologie des Wundstarrkrampfes beim Menschen. Archiv fur klin. Chirurgie, Bd. xxxiv., 1886, p. 306. 1449. BEUMER. Zur atiologischen Bedeutung der Tetanusbacillen. Berliner kliu. Wochenschr., 1887, No. 30. 1450. Zur ^tiologie des Trismus sive Tetanus neonatorum. Zeitschrift fur Hygiene, Bd. iii., 1887, p. 242. 1451. BONOME. Sull' eziologia del tetano. Giorn. del R. Accad. di Med. di Torino, 1886. 1452. Fortschr. der Med., Bd. v., 1887, p. 690. 1453. BRIEGEU. Zur Kenntniss der ^Etiologie des Wundstarrkrampfes. Berliner klin. Wochenschr., 1887, p. 311. 1454. Ueber das Vorkommen von Tetanin bei einem an Wundstarrkrampf erkrankten Individuum. Berliner klin. Wochenschr., 1888, No. 17. 1455. FERRARI. Le microbe du tetanus. La Semaine med., 1887, No. 15. 1456. GIORDANO. Contribute all' eziologia de tetano. Giorn. della Accad. di Med. di Torino, 1887, Nos. 3 and 4. 1457. HOCHSINGER. Zur .^Etiologie des menschlichen Wundstarrkrampfes. Cen- tralbl. fur Bakteriol., Bd. ii., 1887, Nos. 6 and 7. 1458. MORISANI. Richerche sperimentali sulla eziologia del tetano traumatico. Na- poli, 1887. 1459. OHLMULLER TJND GOLDSCHMIDT. Ueber einen Bakterienbef und bei mensch- lichen Tetanus. Centralbl. filr klin. Med., 1887, No. 31. 1460. PEIPER. Zur yEtiologie des Trismus sive Tetanus neonatorum. Centralbl. f iir klin. Med., 1887, No. 42. 1461. SHAKESPEARE. Preliminary report of experimental researches concerning the infectious nature of traumatic tetanus. Trans. IX. Internal. Med. Congress. 1462. BOSSANO. Attenuation du virus tetanique par le passage sur le cobaye. Compt. rend. Acad. des Sc., t. cvii., 1888, p. 1172. 1463. VON EISELBERG. Experimented Beitrage zur ^Etiologie des Wundstarr- krampfes. Wiener klin. Wochenschr., 18S8, Nos. 10-13. 1464. FRIEDBERGER. Starrkrampf beim Pferde, Ueberimpfung auf weisse Mause. Jahresbericht der K. Central-Thierarztneisch. in Miinchen, 1887-88, p. 53. 1465. LAMPIASI. Richerche sull' etiologia del tetano. Giorn. intern, delle Scienze med., 1888. 1466. RAUM. Zur ^Etiologie des Tetanus. Zeitschrift flir Hygiene, Bd. v., 1889, p. 509. 1467. RIETSCH. Sur le tetanos experimentale. Compt. rend. Acad. des Sc., t. cvii., 1888, p. 400. 1468. WIDENMANN. Beitrag zur ^Etiologie des Wundstarrkrampfes. Zeitschrifl f iir Hygiene, Bd. v., 1889, p. 522. 1469. BAERT ET VERHOOGEN. Sur le bacille de Nioolaier et son role dans la patho- genic du tetanos. Bull, de la Soc. Beige de Microscopic, t. xv., 1889. BIBLIOGRAPHY. 833 1470. BELFAXTI E PASCAROLA. Nuovo contribute allo studio batteriologico del tetano. Riforma medica, 1889, No. 71. Ibid., 1890, No. 94. Ibid., 1890, No. 155. 1471. BOSSANO ET STEULLET. Resistance des germes tetaniques a Faction de cer- tains antiseptiques. Compt. rend. Soc. de Biol., 1889, p. 614. 1472. DALL' ACQUA E PARIETTI. Contributo sperimentale all' eziologia del tetano traumatico. Riforma medica, 1890 (March). 1473. KITASATO. Ueber den Tetanuserreger. Deutsche med. Wochenschr., 18b9, No. 31. 1474. Ueber den Tetanusbacillus. Zeitschrift fur Hygiene, Bd. vii., 1889, p. 225. 1475. Experimentelle Untersuchungen ilber das Tetanusgift. Zeitschrift filr Hygiene, Bd. x., 1891, p. 267. 1476. BEHRING. Ueber Immunisirung und Heilung von Versuchsthieren beim Tetanus. Zeitschrift fur Hygiene, Bd. xii., 1X92, p. 45. 1477. SORMANI. Ancora sui neutralizzanti del virus tetanigens, etc. Rendiconti del R. Instituto lombardo, 1889 (November 21st). 1478. Ueber ^Etiologie, Pathogenese und Prophylaxie des Tetanus. Trans. X. Internat. Med. Congress. Abstract in Centralbl. filr Bakteriol., Bd. x., 1891, pp. 421, 580. 1479. TIZZONI E CATTANI. Recherche batteriologiche sul tetano. Riforma me- dica,. 1889, No. 86. 1480. Sui caratteri morfologica del bacillo di Rosenbach e Nicolaier. Ibid., 1889, No. 126. 1481. Richerche sull' eziologia del tetano. Ibid., 1889, No. 142. 1482. - - Ulteriori richerche sul' tetano. Ibid., 1889, No. 148. 1483. - — Sulla diffusione del virus tetanico nell' organismo. Ibid., 1889, No. 162. 1484. - — - Ulteriori richerche sui caratteri delle colture del bacillo del tetano. Ibid., 1889, No. 293. 1485. - - Ueber das Tetanusgift. Centralbl. filr Bakteriol., Bd. viii., 1890, p. 69. 1486. Sulla resistenza del virus tetanico agli agenti chimici e fisici. Riforma med., 1890, No. 83. 1487. Ueber die Art einem Thiere die Immunitat gegen Tetanus zu iiber- tragen. Centralbl. filr Bakteriol.. Bd. ix., 1891, p. 189. 1488. Ueber die Eigenschaften des Tetanus- Antitoxins. Ibid., p. 685. 1489. Fernere Untersuchungen ilber das Antitoxin des Tetanus. Ibid., Bd. x., 1891, p. 33. 1490. - - Sull' attenuazione del bacillo del tetano. La Riforma med., 1891, p. 157. 1491. WIDENMANN. Beitrag zur ^Etiologie des Wundstarrkrampfes. Zeitschrift fur Hygiene, Bd. v., 1889. 1492. TIZZONI, CATTANI UND BAGNIS. Bakteriologische Untersuchungen liber den Tetanus. Zicgler's Beitrage, Bd. vii , Heft 4. 1493. BABES UND PUSCARIN. Versuche ilber Tetanus. Centralbl. fur Bakteriol., Bd. viii., 1890, p. 74. 1494. BEHRING UND KITASATO. Ueber das Zustandekommen der Diphtherie-Im- munitat und der Tetanus-Immiinitat bei Thieren. Deutsche med. Wochen- schr., 1890, No. 49. 1495. SANCHEZ-TOLEDO ET VEILLON. De la presence du bacille du tetanos dans les excrements du chevalet du boeuf i\ 1'etat sain. La Semaine med., 1890, No. 45. 1496. Recherches microbiologiques et experimentales sur le tetanos. Arch. de Med. experiment, et d'Anat. path., 1890, p. 11. 834 BIBLIOGRAPHY. 1497. PEYRAUD. Etiologie du tetanos ; sa vaccination chimique par la strychnine. La Semaiue med., 1890, No. 44. 1498. RENVERS. Zur ^Etiologie des Wundstarrkrampfes. Deutsche med. "Wochen- schr., 1890, No. 32. 1499. VAILLARD ET VINCENT. Recherches experimentales sur le tetanos. La Semaine med., 1891, No. 5. 1500. r- Contribution 3,1'etude du tetanos. Ann. de 1'Institut Pasteur, 1891, No. 1. 1501. TURCO. Alcune richerche sperimentali sulla diffusione del virus tetanico e sulla sua resistenza agli agenti esterni. La Riforma med., 1891, No. 236. 1502. VAILLARD. Sur 1'immunite contre le tetanos. Compt. rend, de la Soc. de Biol., 1891, No. 7. BACILLUS (EDEMATIS MALIGNI. 1503. PASTEUR. Sur le vibrion septique. Bull. Acad. de Med., 1887 and 1881. 1504. KOCH. Mitth. aus dem K. Gesundheitsamte, Bd. i., 1881, p. 54. 1505. GAFFKY. Experimented erzeugte Septikamie, etc. Mitth. aus dem K. Ge- suudheitsamte, Bd. i., 1881, p. 12. 1506. TRIFAUD. De la gangrene gazeuse foudroyante. Rev. de Chir., t. iii. 1507. LUSTIG. Zur Kenntniss bakteriamischer Erkrankuugen bei Pferden (malignes Oedem). Jahresber. der K. Thierarzneischule zu Hannover, 1883-84. 1508. KITT. Untersuchungen liber malignes Oedem und Rauschbrand bei Haus- thieren. Jahresber. der K. Thierarzneischule in Miinchen, 1883-84. 1509. HESSE, W. UND R. Ueber Zuchtung der Bacillen des malignen Oedems. Deutsche med. Wocheoschr., 1885, No. 14. 1510. CHAUVEAU ET ARLOING. Etudeexperimentalesur la septicemie gangreneuse. Arch, veter., 1884, pp. 366, 817. 1511. Bull. Acad. de Med., 1884 (May 6th and Aug. 19th). 1512. Roux ET CHAMBERLAXD. Immunite contre la septicemie conferee par les substances solubles. Ann. de 1'Institut Pasteur, vol. i., 1887, p. 562. 1513. ROGER. Quelques effets des associations microbiennes. Compt. rend. Soc. deBiol., 1889, p. 35. 1514. KITASATO UND WEYL. Zur Kenntniss der AnaGroben. Zeitschr. fur Hygiene, Bd. viii., 1890, p. 41. 1515. VAN COTT. Untersuchungen liber das VorkomYnen der Bacillen des malignen Oedems in der Moschustinktur. Centralbl. flir Bakteriol., Bd. ix., 1891, p. 303. 1516. VERNEUIL. Note sur les rapports de la septicemie gangreneuse et du tetanos, etc. La Semaine med., vol. x., 1890, No. 48. BACILLUS CADAVERIS. STERNBERG. Op. cit. (No. 1384), p. 212. BACILLUS OF SYMPTOMATIC ANTHRAX. 1517. FESER. Studium tlber den sog. Rauschbrand des Rindes. Zeitschrift fur prakt. Veterinarwissensch., Bern, 1876. 1518. BOLLINGER UND FESER. Wochenschr. f lir Thierheilkunde, 1878. 1519. ARLOING, CORNEVIN ET THOMAS. Sur I'inoculabilite du charbon symptoma- tique et les caracteres qui le differencient du sang de rate. Compt. rend. Acad. des Sc., Paris, xc., 1880, pp. 1302-1305. 1520. De 1'inoculation du charbon symptomatique par injection intraveineuse. BIBLIOGRAPHY. 835 et de 1'immunite conferee au veau, au mouton et a la chevre par ce proc6de. Compt. rend Acad. des Sc., Paris, xci., 1880, pp. 734-736. 1521. ARLOING, CORNEVIN ET THOMAS. Sur la cause de I'immunite des adultes et de 1'espece bovine contre le charbon symptomatique ou bacterien, dans les locaiites ou cette maladie est frequente. Compt. rend. Acad. des Sc., Paris, xciii., 1881, pp. 605-609. 1522. Mecanisme de 1'infection dans les differents modes d'inoculation du charbon symptomatique ; application a 1'interpretation des faits cliniques et & la metkode des inoculations preventives. Compt. rend. Acad. des Sc. , Paris, xcii., 1881 pp. 1246-1248. 1 528. Nouvelles reckerches experimentales sur la maladie inf ectieuse appelee charbon symptomatique. Journ. de Med. vet. et de ZoOtech., Lyon, 1881, 3s., vi.,pp. 290-300. 1524. Experiences publiques sur la vaccination du charbon symptomatique. Arch, vet., Paris, vi., 1881, pp. 721-727. 1525. Note relative a la conservation et a la destruction de la virulence du microbe du charbon symptomatique. Rec. de Med. vet., Paris, 1882, 6 s., ix., pp. 467-472. 1526. Moyen de conf erer artificiellement 1'iinmunite contre le charbon symp- tomatique ou bacterien avec du virus attenue. Compt. rend. Acad. des Sc., Paris, xcv., 1882, pp. 189-191. 1527. Le charbon symptomatique ; troisieme rapport a M. le Ministre de 1'Agriculture sur le resultat des inoculations preventives. Arch, vet., Paris, vii., 1882, pp. 767-771. 1528. Modifications que subit le virus du charbon symptomatique ou bac- terieu sous 1'influence dequelques causes de destruction. Compt. rend. Soc. de Biol., Paris, 7 s., iv., 1883, pp. 121-128. 1529. Le charbon symptomatique du bceuf . 2eme ed., Paris, 1887. 1530. EHLERS. Untersuchungen liber den Rauschbraudpilz. Inaug. Diss., Rostock, 1884. 1531. KITT. Untersuchungen liber malignes Oedem und Rauschbrand bei Haus- thieren. Jahresber. der K. Thierarzneischule in Miinchen, 1883-84. 1532. YersucheubereinmaligeRauschbrand-Schutzimpfung. Ibid., 1886-87, p. 91. 1533. Ueber Abschwachung des Rauschbrandvirus durch stromende Wasser- dampfe. Centralbl. filr Bakteriol., Bd. iii., 1888, pp. 572, 605. 1534. HESS. Bericht liber die Schutzimpf ungen gegen Rauschbrand, etc. , im Kanton Bern wiihrend der Jahre 1886-88. Bern, 1889. 1535. HAFNER. Die Rauschbrandimpfungen in Baden. Bad. thierarztl. Mitth., 1887, p. 33. Ibid., 1889, No. 2. 1536. ROGOWITSCH. Zur Kenntniss der Wirkung des Rauschbrandbacillus auf den thierischen Organismus. Ziegler's Beitrage, Bd. iv., 1888, Heft 4. 1537. Roux. Immunite contre le charbon symptomatique, confere par les sub- stances solubles. Ann. de 1'Iustitut Pasteur,' vol. ii., 1888, p. 49. 1538. STREBEL. Resultat der Rauschbrand-Schutzimpfung im Kanton Freiburg. Schweizer Archiv fur Thierheilk., Bd. xxx., p. 87. 1539. SUCHANKA. Resultate der Rauschbrandimpfungen des Jahres 1887 im Her- zogthume Salzburg- Oesterreich. Monatsschr. filr Thierheilk., 1888, p. 161. Ibid., 1889, No. 3. 1540. WOLFF. Schutzimpfungen' gegen Rauschbraud. Berliner Archiv filr wis- sensch. und prakt. Thierheilk., 1883, p. 191. 1541. KITASATO. Ueber den Rauschbrandbacillus und sein Kulturverfahren. Zeit- schr. fur Hygiene, Bd. vi., 1889, p. 105. Ibid., Bd. viii., p. 55. 836 BIBLIOGRAPHY. 1542. ROGER. Inoculation du charbon symptomatique au lapin. Corapt. rend. Soc. de Biol., 1889, pp. 77 and 242. 1543. De quelques causes qui modifient 1'immunite naturelle. Ibid., p. 476. 1544. WILDNER. Die Resultate der im Jahre 1888 in Niederosterreich vorgenom- menen Rauschbrand-Schutzimpfungen. Oesterr. Monatsschr. fiir Thier- heilk., 1889, No. 12. 1545. BOVET. Des gaz produits par la fermentation anaerobienne. Ann. de Micro- graphic, t. ii., 1890, No. 7. XV. PATHOGENIC SPIRILLA SPIRILLUM OBERMEIERI. 1546. OBERMEIER. Vorkommen feinster, eigene Bewegung zeigender Faden im Blutevon Recurrenskranken. Centralbl. filr die med. Wissensch., 1873, No. 10. 1547. Weitere Mittheilungen ilber Febris recurrens. Berliner klin. Wochen- schr., 1873, No. 35. 1548. ENGEL. UeberdieObermeier'schenRecurrensspirillen. Berliner klin. Wochen- schr., 1873, No. 35. 1549. MOCZUTOWSKY. Experimeutaluntersuchung tlber die Inoculationsflihigkeit der Typhen. Deutsches Archiv fiir klin. Med., Bd. xxiv., 1876. 1550. HEYDENREICH. Der Parasit des Rilckfallstyphus. Berlin, 1877. 1551. Kocn. Deutsche med. Wochenschr., 1879. 1552. WEIGERT Zur Technik der mikroskopischen Bakterienuntersuchungen. Vir- chow's Archiv, Bd. Ixxxiv., 1881, p. 292. 1553. CARTER. Contribution to the experimental pathology of spirillum fever ; its communicability by inoculation to the monkey. Med. Chir. Trans. , Lon- don, 1880, 2d series, xlv., pp. 7, 148. 1554. - - Aspects of the blood spirillum in relapsing fever. Trans. Internal. Med. Congress, London, 1881, p. 334. 1555. METSCHNIKOPP. Ueber den Phagocytenkampf beim Ruckfallstyphus. Vir- chow's Archiv, Bd. cix., 1887, p. 176. 1556. SOUDAKEWITCH. Recherches sur la fievre recurrente. Ann. de 1'Institut Pasteur, t. v., 1891, p. 545. SPIRILLUM ANSERUM. 1557. SAKHAROFP. Spirochseta anserina et la septicemie des oies. Ann. de 1'In- stitut Pasteur, t. v., 1891, p. 564. SPIRILLUM CHOLER^E ASIATIC/E. 1558. KOCH. Ueber die Cholerabakterien. Deutsche med. Wochenschr., 1884. Ibid., 1885, Nos. 19, 20, 37a, 38, 39. 1559. VAN ERMENGEM. Recherches sur le microbe du cholera asiatique. Paris and Brussels, 1885. 1560. Note sur 1'inoculation des produits de culture du bacille-virgule. Bull. de 1'Acad. Roy. de Med. de Belgique, 3eme serie, xviii. 1561. GIBIER ET VAN ERMENGEM. Recherches .exper. sur le cholera. Compt. rend. Acad. des Sc., t. ci., 1885. 1562. PPEIPPER. Ueber die Cholera in Paris. Deutsche med. Wochenschr., 1885, No. 2. BIBLIOGRAPHY. 837 1568. PFEIFFER. Choleraspirillen in der Darmwand. Deutsche med. Wochen- schr., 1887, Nos. 11 and 12. 1564. Entgegnung auf den Artikel : Ueber Thierversuche bei Cholera asiatica des Herrn Hueppe. Ibid., No. 23. 1565. Schlusswort zu Herrn Hueppe's " Bemerkungen, etc." Ibid., No. 31. 1566. Untersuchungen fiber das Choleragift. Zeitschrift fur Hygiene, Bd. ii., 1892, p. 393. 1567. STRAUSS, Roux, THUILHER ET NOCARD. Compt. rend. Soc. de Biol. , Paris. 1883. 1568. BUCHNER. Ueber Cholerabacillen . Munchener arztl. Intelligenzbl., 1884, p. 549. 1569. SCHOTTELIUS. Zum mikroskop. Nachweis von Cholerabacillen in Dejektio- nen. Deutsche med. Wochenschr., 1885, No. 14. 1570. KLEIN. British Med. Journ., vol. i., 1885. 1571. Lancet, London, vol. ii., 1885. Ibid., vol. i., 1885. 1572. Proc. Roy. Soc., London, vol. xxxviii., 1885. 1573. The bacteria of Asiatic cholera. London, 1889. 1574. FINKLER UNO PRIOR. Untersuchungen liber Cholera nostras. Deutsche med. Wochenschr., 1884, No. 36. 1575. Ueber die Kommabacillen. KOlnische Zeit., 1884 (November llth). 1576. • Forschung tiber Cholerabakterien. Erganzungshefte /.urn Centralbl. fiir allg. Gesundheitspflege, Bd. i., Heft 5 and 6. 1577. MILLER. Kommaformiger Bacillus aus der Mundhohle, Deutsche med. Wochenschr., 1885, No. 9. 1578. EMMERICH. Die CholerainNeapel. Deutsche med. Wochenschr., 1884, No. 50. 1579. FLUGGE. Kritik der Emmerich'schen Untersuchungen liber Cholera. Deutsche med. Wochenschr., 1885, No. 2. 1580. NICATI ET RIETSCH. Recherchcs sur le ckolera. Arch, de Physiol. norm, et pathol., 1885, p. 72. 1581. Compt. rend. Acad. des Sc., t. xcix., p. 928. 1582. Recherches sur le cholera. Paris, 1886. 1583. KLEBS. Mittheilungen zur JEtiologie der Cholera. Correspondenzblatt fiir Schweizer Aerzte, 1885. 1584. BABES. Untersuchungen liber Koch's Kommabacillus. Virchow's Archiv, Bd. xcix., 1885, p. 148. 1585. EMMERICH. Untersuchungen liber die Pilze der Cholera asiatica. Archiv fiir Hygiene, 1885, p. 291. 1586. BUCHNER UND EMMERICH. Die Cholera in Palermo. Aerztl. Intelligenz- blatt, Munchener med. Wochenschr., 1885, No. 44. 1587. DE SIMONE. Altre richerche sul colera ; epidemic di Palermo del 1885. Giorn. internaz. delle Scienze med., 1886, No 8. 1588. TIZZONI UND CATTANI. Untersuchungen liber Cholera. Centralbl. fiir die med. Wissensch., 1886, p. 769. 1589. CUNNINGHAM. Relation of cholera to schizomycetic organisms. Scientific memoirs of the medical officers of the army of India. Calcutta, 1885. 1590. LUSTIG. Bakteriologische Studien iiber Cholera. Centralbl. fiir die med. Wissensch., 1887, Nos. 16 and 17. 1591. — _ Zeitschrift fiir Hygiene, Bd. iii., 1887, p. 146. 1592. GAFFKY. Bericht liber die Thatigkeit der zur Erforsclmng der Cholera im Jahre 1883 nach Egypteu und Indien entsandten Kommission. Unter Mit- wirkung von Dr. R. Koch. Arbeiten aus dem K. Gesundheitsamte, Ber- lin, Bd. iii., 1887. 838 BIBLIOGRAPHY. 1593. FERRAN. Sur 1'action pathogene et prophylactique du bacillus-virgule Compt. rend. Acad. des Sc., t. c., p. 959. 1594. Revendication de la priorite de la decouverte des vaccins du cholera asiatique faite sous les auspices de la municipalite de Barcelone. Barcelone, 1888. 1595. VAN ERMENGEM. Die Ferran'schen Impfungen. Deutsche med. Wochen- schr., 1885, No. 29. 1596. BITTER. Ueber Fermentausscheidung von Vibrio Koch und Vibrio proteus. Inaug. Diss., Milnchen, 1886. 1597. BUJWID. Eine chemische Reaktion fur die Cholerabakterien. Zeitschrift fiir Hygiene, Bd. ii., 1887, p. 52. 1598. Centralbl. fur Bakteriol., Bd. iii., 1888, p. 169. 1599. Neue Methoden zum Diagnosticiren und Isoliren der Cholerabakte- rien. Centralblatt fur Bakteriol., Bd. iv., 1888, p. 494. 1600. WEISSER UND FRANK. Mikroskopische Untersuchungen von an Cholera asiatica verstorbenen Indiern. Zeitschr. filr Hygiene, Bd. i., 1886, p. 379. 1601. ALI-COHEN. Zur Bedeutung des sog. Cholerarothes. Fortschr. der Med., 1887, No. 17. 1602. Ibid., 1888, No. 6. 1603. ALMQUIST. Einige Bemerkungen uber die Methoden der Choleraforschung. Zeitschrift fur Hygiene, Bd. iii., 1887, p. 281. 1604. BRIEGER. Ueber die Entstehung des Cholerarothes sowie liber Ptomaine aus Gelatine. Deutsche med. Wochenschr. , 1887, No. 22. 1605. Zur Kenntniss des Stoffwechselprodukte des Cholerabacillus. Berliner klin. Wochenschr., 1887, p. 8L7. 1606. Choleraroth und Cholerablau. Berliner klin. Wochenschr., 1887, p. 500. 1607. Ueber die Cholerafarbstoffe. Virchow's Archiv, Bd. ex., 1887, p. 614. 1608. CANESTRINI E MORPURGO. Resistenza del bacillus komma in culture vecchie al calore. Atti di R. Institute di Scienze, Lettere ed Arti, t. v., 1887. 1609. STERNEEHG. The thermal death-point of pathogenic microorganisms. Am. Journ. of the Med. Sc., 1887 (July) ; also in Rep. of Com. on Disinfectants, A. P. H. A., 1887, p. 138. 1610. DUNHAM. Zur chemischen Reaktion der Cholerabakterien. Zeitschrift fiir Hygiene, Bd. ii., 1887, p. 337. 1611. HUEPPE. Ueber Fortschritte in der Kenntniss der Ursachen der Cholera asiatica. Berliner klin. Wochenschr. , 1887, Nos. 9-12. 1612. Ueber Thierversuche bei Cholera asiatica. Berliner klin. Wochenschr., 1887, No. 22. 1613. Einige Bemerkungen liber Thierversuche bei Cholera asiatica. Deutsche med. Wochenschr., 1887, No. 30. 1614. Sur la virulence des parasites du cholera. Compt. rend. Acad. des Sc., t. cviii., 1889, p. 105. 1615. Zur ^Etiologie der Cholera asiatica. Prager med. Wochenschr., 1889, No. 12. 1616. KITASATO. Die Widerstandfahigkeit der Cholerabakterien gegen dasEintrock- nen und Hitze. Zeitschrift fiir Hygiene, Bd. v., 1888, p. 134. Ibid., Bd. vi., 1889, p. 11. 1617. Ueber das Verhalten der Typhus- und Cholerabacillen zu saure- und alkalihaltigen Nahrboden. Zeitschrift fiir Hygiene, Bd. iii., 1888, p. 404. 1618. Das Verhalten der Cholerabakterien im menschlichen Koth. Ibid., Bd. v., 1888, p. 487. BIBLIOGRAPHY. 1619. KITASATO. Das Verhaltcn der Cholerabakterieu in der Milch. Ibid., Bd. v., 18S8, p. 491. 1620. Zeitschrift fiir Hygiene, Bd. vi., 1889, p. 1. 1621. BOLTON. Ueber das Verhalten verschiedener Bakterienarten im Trinkwasser. Zeitschrift fur Hygiene, Bd. i., 1886, p. 76. 1622. ROY, BROWN, AND SHERRINGTON. Preliminary report on the pathology of cholera Asiatica. Proc. Roy. Soc., London, vol. xli., p. 173. 1623. SALKOWSKI. Ueber das Choleraroth und das Zustandekommen der Cholera- reaktion. Virchow's Archiv, Bd. ex., 1887, p. 366. 1624. WEICHSELBAUM. Ueber JEtiologie der Cholera. Wien, 1887. 1625. TIZZONI UND CATTANI. Untersuchungen uber Cholera. Centralbl. fiir die med. Wissensch., 1887, No. 8 ; ibid., No. 26 ; ibid., No. 29 ; ibid., No. 33 ; ibid., Nos. 39 and 40 ; ibid., No. 51. 1626. VINCENZI. Ueber intraperitonaale Einspritzung von Koch'schen Komma- bacillen bei Meerschweinchen. Deutsche med. Wochenschr., 1887, Nos. 17 and 26. 1627. WASSILJEW. Die Desinfektion der Choleradejektionen in Hospi'iilern. Zeit- schr. fiir Hygiene, Bd. iii., 1837, p. 237. 1628. ZASLEIN. Ueber den praktischen Nutzen der Koch'schen Plattenkultureu in der Choleraepidemie des Jahres 1886 in Genua. Deutsche Med.-Zeitung, 1887, p. 389. 1629. Was wSchst aus alten Cholerakulturen ? Ibid., No. 52. 1630. Beitrag zur chemischen Reaktion des Cholerabacillus. Ibid., No. 72. 1631. Ueber die Varietaten des Koch'schen Kommabacillus. Deutsche Med.- Zeitung, 1888, Nos. 64 and 65. 1632. SIRENA E ALLESSI. Azione della creolina sulbacillo-virguladi Koch. Riforma med., 1888, Nos. 257 and 258. 1633. LOEWENTHAL. Experiences biologiques et therapeutiques sur le cholera. Compt. rend. Acad. des Sc., t. cvii.. 1888, p. 1169. 1634. Sur la virulence du bacille cholerique et 1'action que le salol exerce sur cette virulence. Compt. rend. Acad. des Sc., t. cTiii., 1889, p. 192. 1635. HESSE. Unsere Nahrungsmittel als Nahrboden fiir Typhus und Cholera. Zeitschr. fiir Hygiene, Bd. v., 1889, p. 527. 1636. BERCKHOLTZ. Untersuchungen liber den Einfluss des Eiutrockuens auf die Lebensfahigkeit der Cholerabacillen. Arbeiten aus dem K. Gesundheits- amte, Bd. v., 1888. 1637. GAMELEIA. Ueber Priiventivinipfung gegen Cholera asiatica. Compt. rend. Acad. des Sc., 1888 (Aug. 20th). 1638. Sur la vaccination cholerique. Compt. rend. Soc. de BioL, 1889, No. 38. 1639. Ueber die Resistenz der Kaninchen gegenilber den Cholerabakterien. Centralbl. fiir Bakteriol., Bd. ix., 1891, p. 807. 1640. HOVORKA UNO WINKLER. Ein neues Unterscheidungsmerknwl zwischen dem Bacillus choleras asiaticae Koch und dem von Finkler und Prior entdeckten Bacillus. Allgem. "Wiener med. Zeitung, 1889. 1641. NEUHAUSS. Ueber die Geisseln an den Bacillen der asiatischen Cholera. Cen- tralbl. fur Bakteriol., Bd. v., 1889, p. 81. 1642. PKTRI. Reduktion von Nitraten durch die Cholerabakterien. Centralbl. fiir Bakteriol., Bd. v., 1889, p. 561. 1643. Ueber die Verwerthung der rothen Salpetrigsiiure-Indolreaktion zur Erkennung der Cholerabakterien. Arbeiten aus dem K. Gesundheitsamte, Bd. vi., p. 1. 840 BIBLIOGRAPHY. 1644. PFEIFFER UND NOCHT. Ueber das Verhalten der Choleravibrionen im Tau- benkorper. Zeitschrift fur Hygiene, Bd. vii., 1889, p. 259. 1645. PPUHL. Ueber die Desinfektion von Typhus- und Choleraausleerungen mit Kalk. Zeitschrift fur Hygiene, Bd. vi., 1889, p. 97. 1646. UFFELMANN. Die Dauer der Lebensfiihigkeit von Typhus- und Cliolerabacillen in Fakalmassen. Central bl. fiir Bakteriol., Bd. v., 1889, pp. 497, 529. 1647. DOWDESWELL. Note sur les flagella du microbe du cholera. Ann. de Micro- graphic, vol. ii., 1890, No. 8. 1648. Sur quelques phases du developpement du microbe du cholera. Ibid. , No. 12. 1649. DE GIAXA. Le bacille du cholera dans le sol. Ann. de Micrographie, 1890. 1650. Sur 1'action desinfectante du blanchiment des murs au lait de chaux. Ann.de Micrographie, 1890, p. 305 1651. KARLINSKY. Zur Kenntniss der Tenacitilt der Choleravibrionen. Central bl. fur Bakteriol., Bd. viii., 1890, p. 40. 1652. SCHILLEK. Zum Verhalten der Erreger der Cholera und des Unterleibstyphus in dem Inhalt der Abtrittsgruben und Abwasser. Arbeiten aus dem K. Gesundheitsamte, Bd. vi., 1890, Heft 2. 1653. SIRENA. Sulla resistenza vitale del bacillo virgola nelle acque. Riforma med., 1890, No. 14. 1654. WINTER ET LESAGE. Contribution si 1'etude du poison cholerique. Bull. med., 1890, p. 328. 1655. BOER. Ueber die Leistungsfahigkeit mehrer chemischer Desinfektionsmittel bei einigen fur den Menschen pathogenen Bakterien. Zeitschrift filr Hygiene, Bd. ix., 1890. 1656. BRUCE. Bemerkung liber die Virulenzsteigerung des Choleravibrio. Cen- tralbl. filr Bakteriol., Bd. ix., 1891, p. 786. 1657. CUNNINGHAM. On some species of choleraic comma bacilli occurring in Cal- cutta. The scientific memoirs by the medical officers of the Army of India, partvi., Calcutta, 1891. 1658. KAUPE. Untersuchungen uber die Lebensdauer der Cliolerabacillen in mensch- lichen Koth. Zeitschrift fur Hygiene, Bd. ix., 1890. 1659. MANFREDI UND SERAFINI. Ueber das Verhalten von Milzbrand- und Clio- lerabacillen in reinen Quarz- und reinen Marmorboden. Archiv fill- Hygiene, Bd. xi., p. 1. 1660. SHAKESPEARE. Report on cholera in Europe and India. Washington, 1890. SPIRILLUM OF FINKLER AND PRIOR. 1661. PINKLER UND PRIOR. Untersuchungen tiber Cholera nostras. Deutsche med. Wochenschr., 1834, No. 36. 1662. Forschungen uber Cholerabaciilen. Ergiinzungshefte zum Central - blatt fur allgemeine Gesundheitspflege, Bd. i., 1885, Hefte 5 und 6. 1663. BUCHNER. Ueber die Koch'schen und Finkler-Prior'schen Konimabacilleu. Sitzungsber. der Gesellsch. fur Morphol. und Physiol. in Mllnchen, 1885, . P. 21- 1664. Archiv fur Hygiene, 1885, p. 361. 1665. GRUBER. Ueber die als '; Kommabacillen " bezeichneteu Vibrionen von Koch und Finkler-Prior. Wiener med. Wochenschr., 1885, p. 262. 1666. SMITH. Spirillum Finkler and Prior in hepatized lung tissue. Med. News, Philadelphia, 1887, p. 536. 1667. FRANCK. Ueber Cholera nostras. Zeitschr. fiir Hygiene, Bd. iv., 1888, p. 205. BIBLIOGRAPHY. 841 16C8. FIRTSCH. Untersuchungen ilber Variationserscheinuugen bei Vibrio Proteus. Archiv fur Hygiene, Bd. viii., 1883, p. 369. 1669. KARTULIS. Zur .Etiologie der Cholera nostras, etc. Zeitschr. ftir Hygiene, Bd. vi., 1889, p. 62. 1670. Dr MATTEL Iljmethodo Schottelius nella diagnoSi batterioscopica del colera asiatico et del colera nostras. Bull. R. Accad. med. di Roma, 1888-89, No. 1. SPIRILLUM TYROGENUM. 1671. DENEKE. Ueber eine neue, den Choleraspirillen ahnliche Spaltpilzart. Deut- sche med. Wochenschr. , 1885, No. 3. 1672. FLUGGE. Die Mikroorganismen. 2d ed., 1886, p. 386. SPIRILLUM METSCHNIKOVI. 1673. GAMELEIA. Vibrio Metschnikovi (n. sp.) et ses rapports avec le microbe du cholera asiatique. Ann. de 1'Institut Pasteur, vol. ii., 1883, p. 482. 1674. . Vibrio Metschnikovi, son mode naturelle d'infection. Ibid., p. 552. 1675. Vibrio Metschnikovi, vaccination chimique. Ibid., vol. Hi., p. 542. 1676. Exaltation de la virulence. Ibid., p. 609. 1677. Localisation intestinale. Ibid., p. 625. 1678. PFEIFFEK. Ueber dea Vibrio Metschnikoff und sein Verhaltniss zur Cholera asiatica. Zeitschr. ftir Hygiene, Bd. viii., 1889, p. 347. XVI. BACTERIA IN DISEASES NOT PROVED TO BE DUE TO SPECIFIC MICROORGANISMS. ALOPECIA. 1679. ROBINSON. Pathologic und Therapie der Alopecia areata. Monatsh. fur prakt. Dermatol., 1888, Nos. 9-16. 1680. KASAULI. Zur Lehre von der Alopecia areata. Wratschr., 1888, Nos. 39-40. 1631. VAILLARD UND VINCENT. Sur une pseudo-pelade de nature microbienne. Ann. de 1'Institut Pasteur, 1890, p. 446. 1682. VON SEHLEN. Zur Frage nach den Ursachen der Alopecia areata. Tagebl. der 62. Vers. Deutscher Naturf . und Aerzte, Heidelberg, 1890, p. 584. BERI-BERI. 1682. DE LACERDA. O microbio do Beriberi. Rio de Janeiro, 1887, pp. 200, 5 plates 1681. PEKELHARING. De Beri-Beri in Atjeh. Weekblad v. h. Ned. Tijdschr. v. Geneesk., 1887, No. 25. 1685. Ueber Beri-Beri vom Standpunkte der ^Etiologie und Therapie beur- theilt. X. Internal. Med. Congress. Centralbl. filrBakteriol., Bd. ix., 1891, p. 580. 1686. PEKELHARING UND WINKLER. Mitth. ilber die Beri-Beri. Deutsche med. Wochenschr., 1887, p. 845. 1687. Recherches sur la nature et la cause du beri-beri et sur les moyens de le combattre. Faites par ordre du gouvernment ueerlandais. Utrecht, 1888. 1688. ALi-ConEN. Het outstaan van varieteiten bij bacterifin inzonderheit bij den Beri-Beri-Mikrokokken. Overgedrukt nit het Nederlandisch Tijdschrift voor Geneeskunde, 1888. 842 BIBLIOGRAPHY. 1689. EYKMANX. Verslag over de onderzoekingen verricht in het Laboratorium voor Pathologische Anatomic en Bacteriologie te Weltevreden, gedurende het jaar 1888. BRONCHITIS. 1690. LUMNITZER. Centralbl. fiir Bakteriol., Bd iii., 1888, p. 621. 1691. PICCHINI. Rivista clinica, 1889, p. 121. CARCINOMA. 1692. BALLANCE AND SHATTUCK. Report on cultivation experiments with malig- nant new growths. Brit. Med. Journ., 1887, p. 929. 1693. FREIRE. Deutsche med. Wochenschr., 1888, p. 14. 1694. RAPPIN. Recherches sur 1'etiologie des tumeurs malignes. Nantes, 1887. 1695. SCHILL. Ueber den regelmiissigen Bef und von Doppelpunktstabschen in car- cinomatosen und sarcomatosen Geweben. Deutsclie med. Wochenschr., 1887, p. 1034. 1696. SCHEURLEN. Die ^Etiologie des Carcinoms. Deutsche med. Wochenschr., 1887, p. 1033. 1697. Zur Carcinomfrage. Deutsche med. Wochenschr., 1888, No. 30. 1698. BAUMGARTEN. Ueber Scheurlen's Carcinombacillus. Centralbl. filr Bak- teriol., Bd. iii., 1888 1699. VAN ERMENGEM. Etiologie du cancer. Bull, de la Soc. beige de Microscopic, 1888 (March 31st). 1700. LAMPIASI-RUBINO. Sulla natura parasitaria del tumori cancerosi. La Riforma medica, 1888. 1701. MAKARA. Untersuchungen ilber die yEtiologie des Carcinoms. Deutsche med. Wochenschr., 1888, No. 31. 1702. NEPVEU. Contribution a 1'etude des baeteries dans les tumeurs. Gazette hebdom. de Med. et de Chir., 1888, No. 18. 1703. PFEIFFER. Der Scheurlen'sche Krebsbacillus ein Saprophyt. Deutsche med. Wochenschr., 1888, No. 11. 1704. ROSENTHAL. Untersuchungen iiber das Vorkommen von Mikroorganismen in Geschwiilsten, namentlich Carcinomen, mit besonderer Berucksichtigkeit des Scheurlen'schen Carcinombacillus. Zeitschrif t f iir Hygiene, Bd. v., 1888, p. 161. 1705. SENGER. Studien zur ^Etiologie des Carcinoms. Berliner klin. Wochenschr. , 1888, No. 10. 1706. THOMA. Ueber eigenartige parasitare Organismen in den Epithelzellen der Carcinome. Fortschr. der Med., 1889, p. 413. 1707. SJOBRING. Ein parasitarer protozoaartiger Organismus in Carcinomen. Fort- schr. der Med., 1890, No. 14. CHANCROID. 1708. BENDER. Das Ulcus molle. Centralbl. fur Bakteriol., Bd. iii., 1888, pp. 10, 53, 81. 1709. DE LUCA. II micrococco dell' ulcera molle. Mouatshef te f tir prakt. Dermatol. , 18S6, p. 430. 1710. DUCREY. Experimentelle Untersuchungeu ilber den Ansteckungsstoff des weichen Schankers und ilber die Bubonen. Monatshef te fur prakt. Derma- tol. Bd. ix., No. 9. BIBLIOGRAPHY. 843 CHOLERA INFANTUM. 1711. PANUM. Das putride Gift, die Bakterien, die putride Itifektion oder Intoxica- tion und Septicamie. Arch, fur path. Anat. und Phys., Bd. ix. , 1874. 1712. BOOKER. A study of some of the bacteria found in the dejecta of infants af- flicted with summer diarrhea. Trans. Ninth Internat. Med. Congress, vol. iii., 1887. 1713. - — A study of some of the bacteria found in the faeces of infants affected with summer diarrho3a. Trans. Am. Pediatric Soc., vol. i., 1889, p. 198. 1714. JEFFRIES. A contribution to the study of the summer diarrhoeas of infancy. Trans. Am. _ Pediatric Soc., vol. i., 1889, p. 249. 1715. BAGINSKY. Ueber Gahrungsvorgange im Kindlichen Darmkaual, etc. Deut- sche med. Wochenschr. , 1888, Nos. 20 and 21. 1716. Ueber Cholera infantum. Archiv fiir Kinderheilkunde, Bd. xii., Hefte 1 imd 2. 1717. Ueber Cholera infantum. Berliner klin. Wochenschr., 1889, Nos. 46, 47, and 49. 1718. ESCIIERICII. Die Gahrungsvorgange im kindlichen Darmkanal. Deutsche med. Wochenschr., 1888, No. 24. 1719. TOMKINS. Bacteriological researches in connection with summer diarrhoea. Brit. Med. Journ., 1888, p. 417. 1720. VAUGHAN. Experimental studies on some points connected with the causation and treatment of the summer diarrhoeas of infancy. Medical News, Phila., 1888 (June 9th). CHOLERA NO8TRAS. FlNKLER AND PllIOR. Op. cit. (No. 1661) FRANCK. Op. cit. (No. 1667). KARTULIS. Op. cit. (No. 1669). CONJUNCTIVITIS. 1721. SATTLER. Untersuchungen liber das Trachom. Bericht tiber die Ophthalmo- logencongress zu Heidelberg, 1882. 1722. Ueber die Naturder Jequirity- Ophthalmic. Zehender's klin. Monatsbl., 1883 (June). 1723. SATTLER ET DE WECKER. L'ophthalmie jequiritique. Paris, 1883. 1724. DE WECKER. Die jequirity'sche Ophthalmic. Klin. Monatsbl. f ilr Augen- - heilkunde, Jahrg. 20 und 21. 1725. CORNIL ET BERLIOZ. Sur I'empoisonnement par le jequirity. Compt. rend. Acad. des Sc., 1883. 1726. VENNEMANN ET BRUYLANTS. Le jequirity et son principe pathogene. Brus- sels, 1884. 1727. GIFFORD. Beitrag zur Lehre von der sympathischen Ophthalmic. Archiv fiir Augenheilkunde, Knapp und Schweigger, Bd. xvii., 1886, p. 14. 1728. Ueber das Vorkommen von Mikroorgauismcn bei Conjunctivitis ecze- matosa und anderen Zustanden der Bindehaut und Cornea. Ibid., Bd. xvi., 1866, p. 197. 1729. KNAPP. Versuche ilber die Einwirkung von Bakterien auf Augenopera- tionswunden. ArchfV fiir Augenheilkunde, Bd. xvi., 1886, p. 167. 844 BIBLIOGRAPHY. 1730. FRANKE. Ueber den Xerosebacillus und seine atiologische Bedeutung. Tagebl. die 59. Versamml. Deutscher Naturf. und Aerzte zu Berlin, 1886, p. 223. 1731. FRANKEL, EUG., UND FKANKE. Ueber den Xerosebacillus und seine atiolo- gische Bedeutung. Archiv filr Augenheilkunde, Bd. xvii., 1887, p. 176. 1732. KAKTULIS. Zur yEtiologie der agyptischen katarrhalischen Conjunctivitis. Centralbl. fur Bakteriol., Bd. i., 1887, p. 289. 1733. WEEKS. Xerosis conjunctivse bei Sauglingen und bei Kindern. Archiv fiir Augenheilkunde, Bd. xvii., 1887, p. 193. 1734. Der Bacillus des aku ten Bindehautkatarrhs. Ibid., p. 318. 1735. MONTI. Richerche batteriologiche sulla xerosi congiuntivale e sulla panoftal- mite. Archivio per le Scienze mediche, Bd. xi., 1887, No. 4. 1736. ANDREWS. Contagious conjunctivitis ; its causes, prevention, and treatment. Trans. New York Academy of Med., 1886, p. 317. 1737. GOLDSCHMIDT. Zur ^Etiologie des Trachotns. Centralbl. fur klin. Med., 1887, No. 18. 1738. KUCHARSKY. Bakteriologisches liber Trachom. Centralbl. fur prakt. Augen- heilk., 1887, p. 225. 1739. SCIILAFKE. Der Trachomakokkus. Centralbl. fiir Bakteriol. , Bd. ii., 1887, p. 45. 1740. ERNST. Ueber den Bacillus xerosis und seine Sporenbildung. Zeitschr. fiir Hygiene, Bd. iv., 1888, p. 25. 1741. SCHREIKER. Ueber die Bedeutung der sogenannten Xerosebacillen. Fortschr. der Med., 1888, p. 650. 1742. SCHMIDT. Ueber die Mikroorganismen beim Trachom, etc. Inaug. Diss., Petersburg, 1887. 1743. Beobachtungen tlber Culturen und Impfungen von Trachom-Mikro- organismus. Russkaja Medizina. Abstract in Jahresbericht der Ophthal- mologie, 1887, p. 200. 1744. WITTRAM. Bakteriologische Beitrilge zur ^-Etiologie des Trachoms. Inaug. Diss., Dorpat, 1889. 1745. SHOIJGOLOWICZ. Zur Frage von dem Mikroorganismus des Trachoms. St. Petersburger med. Wochenschr., 1890, Nos. 28 and 30. 1746. NOISZEWSKI. Der Mikroorganismus des Trachoms. Gazeta lekarska, 1890, No. 50 (Polish). CORYZA. 1747. KLEBS. Allgm. Path. Jena, 1886, p. 326. 1748. HAJEK. Die Bakterien bei der akuten und chronischen Coryza, -sowie bei der Ozsena, und deren Beziehung zu den genannten Krankheiten. Berliner klin. Wochenschr., 1888, No. 33. 1749. PASQUALE. Ulterior! richerche sugli streptococchi delle mucose a contribuo dell' eziologia della corizza. Giornale internaz. delle Scienze med., 1890. 1750. SCHROTTER UND WiNKLER. Beitrag zur Pathologie der Coryza. Wien, 1890. 1751. PAULSEN. Mikroorganismen in der gesunden Nasenhohle und beim akuten Schnupfen. Centralbl. fur Bakteriol., Bd. viii., 1890, p. 344. CYSTITIS. 1752. SCHOTTELIUS UND RsiNHOLD. Ueber Bakteriurie. Centralbl. fiir klin. Med., 1886, p. 635. BIBLIOGRAPHY. 845 1753. MICOLI. Osservazioni cliniche e batteriologiche iatorno ad alcuni casi di cistite e di catarro vesicale. Giornale la Rivista clinica, 1886 (Novern 1754. RovaiNG. Om Blarebetandelsernes Atiologi, Pathogenes og Behandling Kjobenhavn, 1889, 280 pp. Abstract iii Baumgarten's Jahresbericht, Bd v., 1889, p. 356. 1755. KROGIUS. Sur un bacille pathogene (Urobacillus liquefaciens septicus) trouve dans les urines pathologiques. La Semaine med., 1890, No. 81. 1756. GUYON. Berliner klin. Wochenschr., 1888, No. 32. 1757. SCHNITZLER. Zur ^Etiologie der akuten Cystitis. Centralbl. filr Bakteriol., Bd. viii.,1890,p. 789. 1758. LUNDSTROM. Die Zersetzung von Harnstoff durch Mikroben und deren Be- ziehungen zur Cystitis. Abstract in Centralbl. fur Bakteriol., Bd. ix., 1891, p. 672. 1759. BUMM Zur ^Etiologie der puerperalen Cystitis. Verhandlung der Deutschen Gesellschaft filr Gynakologie, 1886, p. 162. CEREBRO-SPINAL, MENINGITIS. 1760. BANTI. Meningite cerebrale. Esame batterioscopico. La Sperimentale, 1886. 1761. Pneumococco o diplococco capsulato ? Sperimentale, 1889 (Feb ). 1762. Fol E BORDONI-UFFREDUZZI. Sulla meningite cerebrospinale epidemica. Giornale della R. Accad. di Medicina, 1886, Nos. 3 and 4. 1763. Ueber Bakterienbef unde bei Meningitis cerebrospinalis und die Bezie- hung derselben zur Pneumonic. Deutsche med. Wochenschr., 1886, p. 249. 1764. Weitere Mittheilungen iiber den sog. " Meningokokkus." Ibid., p. 568. 1765. Sull' eziologia della meningite cerebro-spinale epidemica. Arch, per le Sci. mediche, vol. xi., 1887, No. 19. 1766. Ueber die JStiologie der Meningitis cerebrospinalis epidemica. Zeit- schrift fur Hygiene, Bd. iv., 1888, p. 67. 1767. GOLDSCHMIDT. Ein Beitrag zur JStiologie der Meningitis cerebrospinalis. Centralbl. fur Bakteriol., Bd. ii., 1887, No. 22. 1768. NEUMANN UND SCHAFFER. 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Zur Kenntniss der schweren Erytheme und der multiplen Hautgan- gran. Fortschr. der Med., 1888, No. 7. GRANULOSA FONGOIDES. 1810. AUSPITZ. Ein fall von Granulosa f ungoides. Vierteljahresschr. fur Dermat. und Syph., 1885, p. 123. 1811. RINDFLEISCH. Mykosis f ungoides. Deutsche med. Wochenschr., 1885, p. 233. HYDROPHOBIA. 1812. PASTEUR. Nouvelle communication sur la rage. Ann de Med. veterin., 1884. 1813. Lettre sur la rage. Ann. de 1'Institut Pasteur, vol. i., 1887, p. 1. 1814. Lettre & M. Duclaux. Ann. de 1'Institut Pasteur, vol. ii., 1888, p. 117. 1815. 8ur la methode de prophylaxie de la rage apres morsure. Compt. rend. Acad. des Sc., t. cviii., 1889, p. 1228. 1816. PASTEUR, CHAMBERLAND, Roux ET THUILLIER. Nouveaux faits pour servir a la connaissance de la rage. Compt. rend. Acad. des Sc., t. xcv., 1882, No. 24. 1817. PASTEUR, CHAMBERLAND ET Roux. Methode pour prevenir la rage apres morsure. Compt. rend. Acad. des Sc., 1885 (October 26th). 1818. GIBIER. These de doctorat, July 26th, 1884. 1819. BERT. Contribution a 1'etude de la rage. Compt. rend. Acad. des Sc., 1882. 1820. FOL. Sur un microbe, dout la presence parait liee & la virulencera bique. Compt. rend. Acad. des Sc., 1885, No. 24. 1821. BABES. Untersuchungen iiber die Hundswuth. Centralbl. filr die med. Wissensch., 1887, No. 37. 1822. Studien ttber die Wuthkrankheit. Virchow's Archiv, Bd. ex., 18S7. 848 BIBLIOGRAPHY. 1823. BARDACH. Sur la vaccination intensive des chiens inocules de la rage par trepanation. Ann. de 1'Institut Pasteur, vol. i., 1887, p. 84. 1824. Nouvelles recherches sur la rage. Ibid., vol. ii., 1888, p. 9. 1825. STERNBERG. Experiments on the temperature destructive of the virus of hydrophobia. In Rep. of the Com. on Disinfectants of the A. P. H. A. (Concord, 1888), p. 147. 1826. ERNST. An experimental research upon rabies. Am. Journ. of the Med. Sc. 1887, p. 321. 1827. VON FRISCH. 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Pasteur's system, containing a trans- lation of all his communications on the subject, the technique of his method, and the latest statistical results. London, 1887, 231 pp. 1836. ULL.MANN. Ein Beitrag zur Frage ilber den Werth der Pasteur'schen Schutz. impfungen am Menschen. Wiener med. Blatter, 1887, p. 1260. 1837. VULPIAN. Nouvelle statistique des personnes qui ont ete traitees a 1'Institut Pasteur, etc. Compt. rend. Acad. des Sc., 1887 (January 28th). 1838. GALTIER. Nouvelles experiences sur 1'inoculation antirabique en vue de pre. server les animaux mordus par les chiens enrages. Compt. rend. Acad. des Sc., t. cvi., 1888, p. 1189. 1839. Persistance de la virulence rabique dans les cadavres enfouis. Ibid., cvii., p. 364. 1840. Nouvelles experiences tendant a demontrer 1'efflcacite des injections intraveineuses de virus rabique, etc. Ibid., cvii., 1888, p. 798. 1841. HELMAN. Etudes sur les formes furieuses et paralytiques de la rage chez les lapins. 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Versuch einer quantitativen Bestimmung der Mikroorganis- men im Boden von Kirchhofen. Inaug. Diss. St Petersburg 1887. 2144. SMOLENSKI. Bakteriologische Untersuchungen des Bodens im Lager der Avantgarde bei Krasnoje Selo. Wratsch., 1887, No. 10. 2145. ADAMETZ. Ueber die niederen Pilze der Ackerkrume. Inaug. Diss., Leipzig, 1886. 2146. KRAMER. Die Bakteriologie in ihren Beziehungen zur Landwirthschaft. Wien, 1890. 2147. FRANKLAND, G. C. UND P. F. Ueber einige typische Mikroorganismen im Wasser und Boden. Zeitschrift filr Hygiene, Bd. vi., 1889, p. 373. 2148. The nitrifying process and its special ferment. Pro*;. R. Soc., London, vol. xlvii.,1890*, p. 296. 2149. GRANCIIER UND RICHARD. Ueber den Einfmss des Bodens auf die Krank- heitserreger. Centralbl. filr Bakteriol., Bd. vii., 1889, p. 578. 2150. REIMERS. Ueber den Gehalt des Bodens an Bakterien. Zeitschrift filr Hy- giene, Bd. vii., 1889, p. 307. 2151. SACHSSE. Die Mikroorganismen des Bodens. Chemisches Centralbl., Bd. ii., 1889, Hefte 4 und 5. 2152. DE GIAXA. Le bacille du cholera dans le sol. Ann. de Micrographie, 1890. 71 863 BIBLIOGRAPHY. 2153. WINOGRADSKY. Recherches sur les organismes de la nitrification. Ann. de 1'Institut Pasteur, 1890, p. 213; ibid., p. 257; ibid., p 760. Ibid., 1891, p. 92. 2154. DOWD. A study of the hygienic condition of our streets. Medical Record, New York, 1890, p 700. 2155. MANFREDI UNO SERAFINI. Ueber das Verhalten von Milzbrand- und Cholera- bacillen in reinera Quartz- und reinem Marmorboden. Archiv fur Hygiene, Bd. xi., p. 1. 2156. JORDAN AND RICHARDS, ELLEN H. Investigations upon nitrification and the nitrifying organism. State Board of Health of Mass. : Rep. on Purification of Sewage and Water, vol. ii., 1890, p. 864. 2157. PROSKAUER. Untersuchung des Bodens des alten Charite-Kirchhofes. Zeit- schr fur Hygiene, Bd. xi., 1891, p. 3. IV. BACTERIA OF THE SURFACE OF THE BODY AND OF EXPOSED MUCOUS MEMBRANES. 2158. BORDONI-UFPREDUZZI. Ueber die biologischen Eigenschaften der normalen Hautmikrophyten. Fortschr. der Med., 1886, No. 5. 2159. KUMMELL, FORSTER, FtJRBRiNGER. Disinfection of the hands. See Nos. 508, 530, 536. 2160. MAGGIORA. Contribute allo studio dei microfiti della pelle umana normale e specialraente del piede. Giorn. della R. Societa: d'Igiene, 1889. 2161. UNNA UND SEHLEN. Flora dermatologica. Monatsh. fiir prakt. Dermat., x., 1890. p 485 Ibid, xi 1890, p. 471. Ibid., xii., 1891, p. 249. 2162. FICK. Ueber Mikroorganismen im Conjunktivalsack. Wiesbaden, 1887. 2163. G A YET. Asepsie methodique. LaSemaine med., 1887, p. 199. 2164. FELSER. Die Mikroorganismen des Conjunktivalsackes und die Antisepsis derselben. Abstract in Centralbl. filr Bakteriol., Bd. v., 1889, p. 321. 2165. GOMBERT. Recherches experimentales sur les microbes des conjonctives, Paris, 1889. 2166. HAJEK. Ueber Ozsena. Munchener med. Wochenschr., 1887, No. 47. 2167. REIMANN. Ueber Mikroorganismen im Nasensecret bei Ozsena. Inaug. Diss., Wilrzburg, 1887. 2168. VON BESSER. Ueber die Bakterien der normalen Luftwege. Ziegler's Bei- trage, Bd. iv 1889, p. 331. 2169 WEIBEL Untersuchungen tiber Vibrionen. Centralbl. fiir Bakteriol., Bd. ii.,1887, p. 465. 2170. - — Ibid Bd. iv , 1888, p. 225. 2171. ROHRER Bakteriologische Beobachtungen bei Affektionen des Ohres und des Nasen-Rachenraumes. Centralbl. fur Bakteriol., Bd. iii., 1888, p. 644. 2172. THOST. Pneumoniekokken in der Nase. Deutsche med. Wochenschr., 1886, p. 161. 2173. WRIGHT. Nasal bacteria in health. New York Med. Journ., 1889 (July 27th). 2174. PAULSEN. Mikroorganismen in der gesunden Nasenhohle und beim akuten Schnupfen. Centralbl. fur Bakteriol., Bd. viii., 1890, p. 344. 2175. STERNBERG. A fatal form of septicaemia in the rabbit produced by the sub- cutaneous injection of human saliva. Johns Hopkins Univ. Stud. Biol. Lab., Baltimore, vol. ii., 1882, p. 183. 2176. A contribution to the study of the bacterial organisms commonly found upon exposed mucous membranes, etc. Proc. Am. Assn. Adv. Sc., vol. xxx., 1881, p. 83. BIBLIOGRAPHY. 863 2177. VIGNAL. Recherches sur les microorganismes de la bouche. Arch, de Physiol. norm, et path., 1886, No. 8. 2178. MILLER. Zur Kenntniss der Bakterien der Mundhohle. Deutsche med. Wochenschr., 1884, No. 47. 2179. The microorganisms of the human mouth. Philadelphia, 1890, 364 pp. 2180. BIONDI. Die pathogenen Mikrcorganismen des Speichels. Zeitschrift fur Hygiene, Bd. ii , 1887, p. 194. 2181. KREIBOHM. Ueber das Vorkommen pathogener Mikroorganismen im Mund- secrete. Inaug. Diss., Gottingen, 1889. 2182. NETTER. Microbes pathog&nes contenus dans la bouche de sujets sains, etc. Revue d'Hygiene, 1889, No. 6. 2183. PODBIELSKY. Untersuchungen der Mikroben der Mundhohle von Erwach- senen und Kindern im gesunden Zustand. 124 pp. 8vo, Katzan, 1890. Russian. Abstract in Centralbl. far Bakteriol., Bd. ix., 1891, p. 617. 2184. SANARELLI. Der menschliche Speichel und die pathogenen Mikroorganismen der Mundh5hle. Centralbl. fur Bakteriol., Bd. x., 1891, p. 817. 2185. DODERLEIN. Ueber das Vorkommen von Spaltpilzen in der Lochien des Uterus und der Vagina gesunder und kranker Wochnerinnen. Archiv fur Gynakol., Bd. xxi., 1887, p. 412. 2186. WINTER. Die Mikroorganismen im Genitalkanal der gesunden Frau. Zeit- schrift fur Geburtshulfe und Gynakol., Bd. xiv., 1888, Heft 2. 2187. VON OTT. Zur Bakteriologie der Lochien. Archiv fur Gynakol., Bd. xxxi., 1888, p. 436. 2188. CZERNIENSKY. Zur Frage der Puerperalerkrankungen. Archiv filr Gynakol., Bd. xxxiii., 1888, p. 73. 2189. STEFFECK. Bakteriologische Begrilndung der Selbstinfektion. Zeitschr. fur Geburtshulfe und Gynakologie, Bd. xx., p. 339. V. BACTERIA OF THE STOMACH AND INTESTINE. 2190. FALKENHEIM. Ueber Sarcine. Archiv fur experim. Pathologic und Pharma- kologie Bd. xix., 1885, p. 1. 2191. DE BARY. Beitrag zur Kenntniss der niederen Mikroorganismen im Magen- inhalt. Archiv filr experim. Pathol. und Pharmakol., Bd. xx., 1886, p. 243. 2192. MILLER. Ueber einige gasbildende Spaltpilze des Verdauungstraktus, ihr Schicksal im Magen und ihre Reaktion auf verschiedene Speisen. Deutsche med. Wochenschr., 1886, No. 8. 2193. VAN PUTEREN. Ueber die Mikroorganismen im Magen von SSuglingen. Wratch., 1888, No. 22. Abstract in Zeitschr. filr Mikroskopie, Bd. v., 1888, p 539. 2194. STRAUS ET WURTZ. De 1'action du sue gastrique sur quelques microbes pathogenes. Arch, de Med. exper. et d'Anat. pathol., 1889, No. 3. 2195. KURLOW UND WAGNER. Ueber die Wirkung des menschlichen Magensaftes auf pathogenen Mikroorganismen. Wratsch., 1689, p. 926. 2196. CAPITAN ET MORAN. Recherches sur les microorganismes de 1'estomac Compt. rend. Soc. de Biol., 1889, p. 25. 2197. ABELOUS. Recherches sur les microbes de I'estomac a 1'etat normal, et leur action sur les substances alimentaires. Compt. rend. Soc. de Biol., 1889, p 86. 864 BIBLIOGRAPHY. 2198. RACZTNSKI. Zur Frage liber die Mikroorganismen des Verdauungskanals. Inaug. Diss., St. Petersburg, 1888. Abstract in Centralbl. fiir Bakteriol., Bd. vi., 1889, p. 112. 2199. HAMBURGER. Ueber die Wirkung des Magensaftes auf pathogene Bakterien. Centralbl. filr klin. Med., 1890, No. 24. 2200. KIANOWSKI. Zur Frage ilber die antibakteriellen Eigenschaften des Magen- saftes. Wratsch., 1890, Nos. 38-41. Abstract in Centralbl. filr Bakteriol., Bd. ix., 1891, p. 420. 2201. NOTHNAGEL. Niedere Organismen in den menschlichen Darmentleerungen, Zeitschr. fur klin. Med., Bd. iii., 1884. 2202. BRIEGER. Ueber Spaltungsprodukte der Bakterien. Zeitschr. ftlr physiolog, Chemie, Bd. viii. and ix. 2203. STAHL. Mikroorganismen in den Darmentleerungen. Verhandlungen des 3 Congr. f Sir innere Med., 1884. 2204. BIENSTOCK. Ueber die Bakterien der Faces. Zeitschr. filr klin. Med., 1884, Bd. viii. 2205. KUISL. Beitrage zur Kenntniss der Bakterien im normalen Darmtraktus. Inaug. Diss., Munchen, 1885. 2206. ESCHERICH. Die Darmbakterien des Sauglings. Stuttgart, 1886, 177 pages. Also, Fortschr. der Med., 1885, Nos. 16 and 17. 2207. BeitrSge zur Kenntniss der Darmbakterien. Milnchenermed. Wochen- schr., 1886, Nos. 1, 43, and 46. 2208. Ueber Darmbakterien im Allgemeinen und diejenigen der Sauglinge im besonderen, etc. Centralbl. fiir Bakteriol., Bd. ii., 1887, Nos. 24 and 25. 2209. SUCKSDORP. Das quantitative Vorkommen von Spaltpilzen im menschlichen Darmkanale. Archiv fiir Hygiene, Bd. iv., 1886. 2210. BAGINSKY. Zur Biologic der normalen Milchkothbakterien. Zeitschr. far physiol. Chemie, Bd. xii., p. 434; Bd. xiii., p. 352. 2211. BOOKER. A study of some of the bacteria found in the dejecta of infants af- flicted with summer diarrhoea. Trans. Ninth Internat. Med. Cong., vol. iii. 2212. Second communication. Trans, of the Am. Pediatric Soc., vol. i., 1889, p. 198. 2213. JEFFRIES. The bacteria of the alimentary canal, especially in the diarrhoea of infancy. Boston Med. and Surg. Journ., 1888, Sept. 6th. 2214. — — — • A contribution to the study of the summer diarrhoeas of infancy. Trans. Am. Pediatric Soc., vol. i., 1889, p. 249. 2215. STERNBERG. Report on the etiology and prevention of yellow fever. Wash- ington 1891, p. 115. 2216. DE GIAXA. Del quantitative di batteri nel contenuto del tubo gastrico-enterico di alcuni animali. Giorn. intern, delle Sci. med., 1888. VI. BACTERIA OF CADAVERS AND OF PUTREFYING MATERIAL FROM VARIOUS SOURCES. 2217. COHN. Untersuchungen iiber Bakterien. Beitr. zur Biol. der Pflanz., Bd. i., 1872, p. 202. 2218. FLUGGE. Fermente und Mikroparasiten. Handb. der Hygiene von Petten- kofer und von Ziemssen, 1883, p 112. 2219. BRIEGER. Ueber giftige Produkte der Faulnissbakterien. Berliner klin. Wochenschr., 1884, No. 14. 2220. HAUSER. Ueber Faulnissbakterien. Leipzig, 1885. BIBLIOGRAPHY. 805 2221. HACSER. Ueber das Vorkommen von Mikroorganismen im lebenden Gewebe gesunder Thiere. Archiv fur exper. Pathol. und Pharmakol., Bd. xx., 1885, p. 162. 2222. ZWEIFEL. Gibt es im gesuaden lebenden Organismus Faulnisskeime ? Tagebl. der 58. Versamml. Deutscher Naturforscher und Aerzte in Strass- burg, 1885, p. 303. 2223. HUEPPE. Ueber die Beziehungen der Faulniss zu den Infektionskrankheiten. Berliner klin. Wochenschr., 1887, p. 721. 2224. SCHRANK. Untersuchungen ilber den im Hilhnerei die stinkende Faulniss hervorrufenden Bacillus. Wiener med. Jahrb. , 18S8, p. 303. 2225. STRASSMANN CJND STRECKER. Bakterien bei der Leichenfaulniss. Zeitschr. fUr Medicinalbeamte, 1888, No. 3. 2226. FACKE. Ueber die Entwicklung von Stickstoff bei Faulniss. Abstract in Centralbl. fur Bakteriol., Bd. iii., 1888, p. 588. 2227. STERNBERG. Report on etiology and prevention of yellow fever. Washing- ton, 1891, p. 121. VII. BACTERIA IN ARTICLES OF FOOD. MILK 2228. LISTER. The cause of putrefaction and lactic fermentation. The Pharm. Journ., 1887. 2229. HUEPPE. Untersuchungen tlber die Zersetzungen der Milch durch Mikro- organismen. Mitth. aus dem K. G-esundheitsamte, Bd. ii., 1884. 2230. - Deutsche med. Wochenschr., 1884, No. 48. 2231. DUCLAUX. Memoire sur le lait. Ann. de 1'Institut agronomique, 1882. 2232. Chimie Biologique, chapitre lix. (Lait, crgme, et beurre). Paris, 1883. 2233. Le lait. Etudes chimiques et microbiologiques. Paris, 18S7, 336 pp. 2234. SCHMIDT-MUHLHEIM. Untersuchungen ilber fadenziehenden Milch. Archiv f lir die ges. Physiol., Bd. xxvii., pp. 490-510. 2235. KERN. Ueber ein neues Milchferment aus dem Kaukasus. Bull, de la Soc. Imp. des Naturalists de Moskau, 1881, No. 3. 2236. Dispora caucasica, eine neue Bakterienform. Biolog. Centralbl., Bd. ii., p. 137. 2237. HEYDUCK. Ueber Milchsauregahrung. Wochenschr. filr Brauerei, 1887, No. 17. 2238. LINDNER. Ueber ein neues, in Malzmaischen vorkommendes Milchsaure bil- dendes Ferment. Wochenschr. filr Brauerei, 1887, No. 23. 2239. LOFFLER. Ueber Bakterien in der Milch. Berliner klin. Wochenschr., 1887, Nos. 33 and 34. 2240. ADAMETZ. Ueber einen Erreger der schleimigen Milch, Bacillus lactis vis- cosus. Milchzeitung, 1889, p. 941. 2241. Die Bakterien normaler und abnormaler Milch. Oesterr. Monatsschrift filr Thierheilk. und Thierzucht, Bd. xv., 1890, No. 2. 2242. Untersuchungen ilber Bacillus lactis viscosus, eine weitverbreiteten milchwirthschaftlichen Schadling. Berliner landwirthschaftliche Jahr- bucher, 1891. 2243. BAGINSKY. Rothe Milch. Deutsche Medicinalztg., 1889, No. 9. 2244. CNOPF. Spaltpilzuntersuchungen in der Kuhmilch. Tagebl. der 62. Ver- samtnlung Deutscher Naturf. und Aerzte in Heidelberg, 1889, p. 493. 2245. FOKKER. Ueber das Milchsaureferment. Fortschr. der Med., 1890, p. 401. 866 BIBLIOGRAPHY. 2246. GROTENFELT. Studien liber Zersetzungen der Milch. Fortschr. der Med., 1889, p. 41. Ibid., p. 121. 2247. KABHHEL. Ueber das Ferment der Milchsauregahrung in der Milch. Allgem. Wiener med. Zeitung, 1889, Nos. 52 and 53. 2248. MENGE. Ueber rothe Milch. Centralbl. filr Bakteriol., Bd. vi., 1889, p. 593, 2249. SCROLL. Beitrage zur Kenntniss der Milchzersetzung durch Mikroorganis- men. I. Ueber blaue Milch. Fortschr. der Med., 1889. 2250. II. Ueber Milchsauregahrung. Ibid., 1890, p. 41. 2251. KREUGER. Beitrage zum Vorkommen pyogener Kokken in Milch. Centralbl. filr Bakteriol., Bd. vii., 1890, pp. 425, 464, 493. 2252. ERNST. How far may a cow be tuberculous before her milk becomes danger- ous as an article of food ? Am. Journ. of the Med. Sci., 18S9 (Nov.). 2253. HIRSCIIBERGER. Experimentelle Beitrage zur Infektiositat der Milch tuber- kulOser Kline. Archiv filr klin. Med., Bd. xliv., 18S9, p. 500. 2254. SEDGWICK AND BATCHELDER. A bacteriological examination of the Boston. milk supply. Boston Med. and Surg. Journ., 1892, p. 25. 2255. HEIM. Versuche liber blaue Milch. Arbeiten aus dem K. Gesundheitsamte, Bd. v., p. 518. 2256. CONN. Ueber einen bittere Milch erzeugenden Mikrokokkus Centralbl. fiir Bakteriol., Bd. ix., 1891, p. 653. BREAD, BUTTER, CHEESE, MEATS, BEER, ETC. 2257. JOHNE. Ein mikroskopisch-bakteriologischer Beitrag zur Frage der Fleisch- vergiftungen. Ber. liber das Veterinarwesen im Konigr. Sachsen, 1886, p. 40. 2258. EHRENBERG. Ueber einige in einem Falle von sog. " Wurstvergiftung " aus dem schadlichen Materiale Faulnissblasen, sowie liber einige, durch die Thatigkeit eines besonderen, im gleichen Materiale auf gefundenen Bacillus gebildete Zersetzungsprodukte. Zeitschrift filr physiol. Chemie, Bd. xi., 1887. 2259. NAUWERCK. "Wurstvergiftung. Med. Correspondenzbl. des wilrttemberg. arztl. Landesvereins, 1886, No. 20. 2260. BENECKE. Ueber die Ursachen der Veriinderung, welche sich wiihrend des • Reifungsprocesses im Emmenthaler Kase vollziehen. Centralbl. fiir Bakte- riol., Bd. ii., 1887, No. 18. 2261. PEUCH. Note sur la contagion de la tuberculose par le lait non bouilli et la viande crue. Revue veterin., 1888, p. 649. 2262. GARTNER. Ueber die Fleischvergif tung in Frankenhausen am Kyff hauser und den Erreger derselben. Correspondenzbl. des allg. arztl. Vereins von Thii- ringen, 1888, No. 9. £263. DE VRIES. Ueber blauen Kase. Petersen's Milchzeitung, Bel. xvii., 1888, Nos. 44 and 45. 2264. ADAMETZ. Bakteriologische Untersuchungen liber den Reifuugsprocess des- Kase. Landwirthsch. Jahrbiicher, 1889, p. 227. 2265. JSRGENSEN. Die Mikroorganismen der Gahrungsindustrie, 2d ed. , 1889. 2266. KRATSCHMER UND NIEMILOWICZ. Ueber eine eigeiithiimliche Brotkrankheit. Wiener klin. Wochenschr., 1889, No. 30. 2267. VAN LAER. Note sur les fermentations visqueuses. Abstract in Centralbl. fiir Bakteriol., Bd. vii., 1890, p. 308. 2268. PETERS. Die Organismen des Sauerteigs und ihre Bedeutung fiir die Brot- gahrung. Botan. Zeitung, Jahrg. xlvii., 1889, Nos. 25-27. BIBLIOGRAPHY. 867 2269. BERNHEIM. Die parasitiiren Bakterieu der Cerealieu. Milncheuer med. Wo- chenschr., 1888, p. 748. 2270. BUCHNER. Notiz betreffend die Frage des Vorkommens von Bakterien im normalen Pflanzeagewebe. Mlinchener med. Wochenschr., 1888, No. 52. 2271. FERNBACH. De 1'absence des microbes dans les tissus vegetaux. Ann. de 1'Institut Pasteur, 1888, p. 567. 2272. HILTNER. Die Bakterien der Futtermittel und Saamen. Laudwirthsch. Ver- suchsstationen, Bd. xxxiv., 1887, p. 391. 2273. Di VESTEA. De 1'absence des microbes dans les tissus vegetaux. Ann. de Tlnstitut Pasteur, 1888, p. 670. 2274. FAZIO. I microorganismi nei vegetati usati f reschi nell' alimentazione. Rivista internazionale d'Igiene, 1890, Nos. 1-3. 2275. KREUGER. Bakteriologisch chemische Untersuchung kasiger Butter. Cen- tralbl. fur Bakteriol., Bd. vii., 1890, pp. 425, 464, 493. 2276. BEU. Ueber den Einfluss des Raucherns auf die Filulnisserreger bei der Kon- servirung von Fleischwaaren. Centralbl. filr Bakteriol., Bd. viii., 1890, pp. 513, 545. 2277. FORSTER. Ueber den Einfluss des Raucherns auf die Inf ektiositat des Flei- sches perlsiichtiger Rinder. Mlinchener med. Wochenschr., 1890, No. 16. 2278. FREUDENREICH. Sur quelques bacteries produisant le boursouflement des fromages. Ann. de Micrographie, t. ii., 1890, No. 8. 2279. GAFFKY UND PAAK. Ein Beitrag zur Frage der sogenannten Wurst- und Fleischvergiftung. Arbeiten aus dem K. Gesundsheitsamte, Bd. vi., 1890, Heft 2. 2280. UFFELMANN. Verdorbenes Brot. Centralbl. fur Bacteriol., Bd. viii., 1890, p. 481. 2281. POPOFP. Sur un bacille anaerobic de la fermentation pannaire. Ann. de 1'Institut Pasteur, 1890, p. 674. 2282. ZIEDLER. Beitrilge zur Kenntniss einiger in Wiirze und Bier vorkommenden Bakterien. Wochenschr. fur Brauerei, 1890, Nos. 47, 48. 2283. KASTNER. Experimentelle Beitrage zur Infektiositat des Fleisches tubercu- loser Rinder. Milnchener med. Wochenschr., 18?9, Nos. 84, 35. 2284. KRATSCHMER UND NIEMILOWICZ. Ueber eine eigenthiimliche Brotkrankheit. Wiener klin. Wochenschr., 1889, No. 30. 2285. STEINHEIL. Ueber die Infektiositat des Fleisches bei Tuberculose. Munch- ener med. Wochenschr., 1889, Nos. 40, 41. VIII. NON-PATHOGENIC MICROCOCCI. 2286. Micrococcus flavus liquefaciens. FLUGGE, Die Mikroorganismen, 2d ed., p. 174. 2287. Micrococcus flavus desidens. FLUGGE, ibid., p. 177. 2288. Micrococcus agilis. ALI-COHEN, Centralbl. fur Bakteriol., Bd. vi., p. 33. 2289. Micrococcus fuscus. ADAMETZ, Die Bakterien der Nutz und Trinkwasser, Vienna, 1888. 2290. Diplococcus citreus conglomeratus. BUMM, Der Mikroorganismus der Gon. Schleimhauterkrankungen. Wiesbaden, 1885, p. 17. 2291. Diplococcus citreus liquefaciens. TOMMASOLT, Monatshefte f ilr prakt. Derma- tol., Bd. ix., p. 56. 2292. Diplococcus flavus liquefaciens tardus. TOMMASOLI, op. cit. (No. 2291). ?3293. Diplococcus fluorescens fujtidus. KLAMANN, Allgemeine medizin. Central- zeitung, 1887, p. 1347. 868 BIBLIOGRAPHY. 2294. Diplococcus luteus. ADAMETZ, Die Bakterien der Nutz und Trinkwasser, Vienna, 1888. 2295. Diplococcus roseus. BUMM, Der Mikroorganismus der Gou. Schleimhaut- erkrankungen, 1885, p. 25. 2296. Micrococcus cremoides. ZIMMERMANN, Die Bakterien unserer Nutz- und Trinkwasser. Chemnitz, 1890. 2297. Micrococcus roseus. EISENBERG, Bakteriologische Diagnostik, 3ded., p. 408. 2298. Micrococcus aurantiacus. ADAMETZ, op. cit. (No. 2289). TILS, Zeitschr. fur Hygiene, Bd. ix., p. 301. 2299. Micrococcus cerasinus siccus. ADAMETZ, op. cit. (No. 2289). 2300. Micrococcus versicolor. FLUGGE, Die Mikroorganismen, 2d ed., 1886. TILS, Zeitschr. fur Hygiene, Bd. ix., p. 299. 2301. Micrococcus of Dantec. DANTEC, Etude de la. morue rouge, Ann. de 1'In- stitut Pasteur, t. v., 1891, p. 659. 2302. Micrococcus carneus. ZIMMERMANN, op. cit. (No. 2296). 2303. Micrococcus cinnabareus. FLUGGE, op. cit. (No. 2300), p. 174. 2304. Micrococcus cereus albus. PASSET, Fortschr. der_Medicin, Bd. iii., 1885. TILS, Zeitschr. fur Hygiene, Bd. ix., p. 300. 2305. Micrococcus cereus flavus. PASSET, op. cit. (No. 2304). 2306. Micrococcus citreus. ADAMETZ, op. cit. (No. 2294). TILS, op. cit. (No. 2304). 2307. Micrococcus fervidosus. ADAMETZ, op. cit. (No. 2294). TILS, op. cit. (No. 2304). 2308. Micrococcus flavus tardigratus. FLUGGE, op. cit. (No. 2300), p. 175. 2309. Micrococcus luteus. ADAMETZ, op. cit. (No. 2294). TILS, op. cit. (No. 2304). 2310. Micrococcus violaceus. ADAMETZ, op. cit. (No. 2294). 2311. Staphylococcus viridis flavescens. GUTTMANN, Virchow's Archiv fiir path. Anat., Bd. cvii., p. 261. 2312. Micrococcus ochroleucus. PROVE, Beitrage xur Biologic der Pflanzen, Bd. iii., p. 409. 2313. Micrococcus acidi lactici liquefaciens. KRUGER, Centralbl. fiir Bakteriol., Bd. vii., p. 464. 2314. Micrococcus aGrogenes. MILLER, Deutsche med. Wochenschr., 1886, No. 8. 2315. Micrococcus albus liquefaciens. VON BESSER, Beitrage zur path. Anat., etc., Bd. vi., p. 346. 2316. Micrococcus foetidus. KLAMANN, Allg. med. Centralzeitung, 1887, p. 1344. 2317. Micrococcus radiatus. FLUGGE, op. cit. ^No. 2300), p. 176. 2318. Diplococcus albicans amplus. BUMM, op. cit. (No. °295). 2319 Micrococcus candicans. FLUGGE, op. cit. (No. 2300). TILS, op. cit. (No. 2298), p. 299. 2320. Micrococcus candidus. TILS, op. cit. (No. 2298), p. 299. 2321. Micrococcus acidi lactici. MARPMANN, Ergilnzungshefte des Centralbl. fiir allg. Gesundheitspflege, Bd. ii., Heft 2, p. 22. 2322. Micrococcus lactis viscosus. CONN, Centralbl. fiir Bakteriol., Bd. ix., p. 653. 2323 Streptococcus acidi lactici. MARPMANX, op. cit. (No. 2321), p. 121. 2324. Micrococcus aquatilis. BOLTON, Zeitschrift fiir Hygiene, Bd. i., p. 94. 2325. Micrococcus concentricus. ZIMMERMANN, op. cit. (No. 2296). 2326. Micrococcus cumulatus tenuis. VON BESSER, op. cit. (No. 2315), p. 347. 2327. Micrococcus plumosus. ADAMETZ, op. cit. (No. 2294). 2328. Micrococcus rosettaceus. ZIMMERMANN, op. cit. (No. 2296). 2329. Micrococcus urece. PASTEUR, Compt. rend. Acad. des Sc., t. Ixxxiii., 1876. VON JACKSCII, Zeitschrift fiir physiol. Chemie, Bd. v., 1881. LEPINE ET Roux, Compt. rend. Acad. des. Sc., t. ci., 1835. LEUBE UND GRASSER, Virchow's Archiv, Bd. c., p. 556. BIBLIOGRAPHY. 869 2330. Micrococcus ureae liquefaciens. FLUGGE, op. cit. (No. 2300), p. 170. 2331. Micrococcus viticulosus. FLUGGE, op. cit. (No. 2300), p. 178. 2332. Diplococcus albicans tardissimus. BOMM, op. cit. (No. 2295). TOMMASOLI, Monatsheft filr prakt. Dermatol., Bd. ix., p. 54. 2333. Diplococcus albicans tardus. TOMMASOLI, op. cit. (No. 2332), p. 49. 2334. Staphylococcus albus liquefaciens. ESCHERICH, Die Darmbakterien des Sau- glings. Stuttgart, 18S6, p. 88. 2335. Micrococcus ovalis. ESCHERICH, op. cit. (No. 2334), p. 90. 2336. Diplococcus coryzse. HAJEK, Berliner klin. Wochenschr., 1888, No. 83. 2337. Micrococcus Finlayensis. STERNBERG, Report on etiology and prevention of yellow fever, Washington, 1891, p. 219. 2338. Micrococcus of Freire. STERNBERG, op. cit. (No. 2337), p. 163. 2339. Streptococcus coli gracilis. ESCHERICH, op. cit. (No. 2334). 2340. Streptococcus acidi lactici. GROTENFELT, Fortschr. der Med., Bd. vii., p. 124. 2341. Streptococcus giganteus urethrae. LUSTGARTEN UND MANNEBERG, Viertel- jahresbericht fur Dermatol. und Syph., 1887, p. 918. 2342. Streptococcus albus. TILS, op. cit. (No. 2298), p. 302. 2343. Streptococcus vermiformis. TILS, op. cit. (No. 2298), p. 302. 2344. Streptococcus brevis. VON LINGELSHEIM, Zeitschr. filr Hygiene, Bd. x., p. 331. 2345. Streptococcus cadaveris. STERNBERG, op. cit. (No. 2837), p. 218. 2346. Streptococcus Havaniensis. STERNBERG, op. cit. (No. 2337), p. 219. 2347. Streptococcus liquefaciens. STERNBERG, op. cit. (No 2337), p. 219. 2348. Micrococcus tetragenus versatilis. STERNBERG, op. cit. (No. 2337), p. 164. 2349. Pediococcus albus. LINDNER, Die Sarcineorganismen der Gahrungsgewebe, Berlin, 1888. 2350. Pediococcus acidi lactici. LINDNER, op. cit. (No. 2349). 2351. Pediococcus cerevisise. LINDNER, op. cit. (No. 2349). 2352. Micrococcus tetragenus mobilis ventriculi. MENDOZA, Centralbl. fur Bakte- riol.,Bd. vi., p. 506. 2353. Micrococcus tetragenus subflavus. VON BESSER, op. cit. (No. 2315), p. 347. 2354. Sarcina aurantiaca. Mitth. aus dem K. Gesundheitsamte, Bd. ii. 2355. Sarcina lutea. EISENBERG, op. cit. (No. 2297), p. 15. 2356. Sarcina flava. LINDNER, op. cit. (No. 2349). 2357. Sarcina rosea. LINDNER, op. cit (No. 2349). 2358. Sarcina alba. EISENBERG, op. cit. (No. 2297), p. 26. 2359. Sarcina Candida. LINDNER, op. cit. (No. 2349). 2360. Sarcina pulmonum. HAUSER, Deutsches Archiv fiir klin. Medizin, Bd. xlii., p. 131. 2361. Sarcina ventriculi. FALKENHAIM, Archiv fur exper. Pathol. und Pharmakol. , Bd. xix., p. 339. 2362. Micrococcus amylovorus. BURRILL, Proc. Am. Assoc. Adv. Sc., vol. xxix., 1880, p. 583 ; Am. Naturalist, vol. xv., 1881, p. 527. ARTHUR, Report New York Agric. Exper. Station, 1884, p. 357 ; Proc. Am. Assn. Adv. Sc., vol. xxxiv. , 1885, p. 295 ; History and biology of pear blight, Proc. Acad. Nat. Sc., Philadelphia, 1886. 2363. Ascococcus Billrothii. COHN, Beitrage zur Biologic der Pflanzen. FL GGE, op. cit. (No. 2300), p. 184. 2364. Leuconostoc mesenteroides. CIENKOWSKI, Die Gallertbildungen des Zucker- riibensaftes, Charkow, 1878. ZOPF, Die Spaltpilze, 2ded., p. 45. 870 BIBLIOGRAPHY. IX. NON-PATHOGENIC BACILLI. 2365. Bacterium luteum. ADAMETZ, op. cit. (No. 2294). 2366. Bacillus aurantiacus. FKANKLAND, Zeitschr. fur Hygiene, Bd. vi., p. 390. 2367. Bacillus brunneus. ADAMETZ, op. cit. (No. 2294). 2368. Bacillus aureus. ADAMETZ, op. cit. (No. 2294). TOMMASOLI, Monatsheft fur prakt. Dermatol., Bd. ix., p. 57. 2369. Bacillus flavocoriaceus. ADAMETZ, op. cit. (No. 2294). 2370. Bacillus berolinensis Indicus. CLASSEN, Centralbl. fiir Bakteriol., Bd. vii.,. p. 13. 2371 . Bacillus constrictus. ZIMMERMANN, op. cit. (No. 2296). 2372. Bacillus fluorescens aureus. ZIMMERMANN, op. cit. (No. 2296). 2373. Bacillus fluorescens longus. ZIMMERMANN, op. cit. (No. 2296). 2374. Bacillus fluorescens teuuis. ZIMMERMANN, op. cit. (No. 2296). 2375. Bacillus fluorescens non-liquefaciens. EiSENBEiia, op. cit. (No. 2297), p. 145. 2376. Bacillus fluorescens putidus. FLUGGE, op. cit. (No. 2300), p. 288. 2377. Bacillus erythrosporus. FLUGGE, op. cit. (No. 2300), p. 288. 2378. Bacillus viridis pallescens. FRICK, Virchow's Archiv fiir path. Anat., Bd, cxvi., p. 292. 2379. Bacillus virescens. FRICK, op. cit. (No. 2378), p. 292. 2380. Bacillus iris. FRICK, op. cit. (No. 2378), p. 292. 2381. Bacillus fuscus. ZIMMERMANN, op. cit. (No. 2296). 2382. Bacillus rubefaciens. ZIMMERMANN, op. cit. (No. 2296). 2383. Bacillus striatus flavus. VON BESSER, op. cit. (No. 2315), p. 249. 2384. Bacillus subflavus. ZIMMERMANN, op. cit. (No. 2296). 2385. Bacillus cyanogenus. FLUGGE, op. cit. (No. 2300), p. 291. HUEPPE, Mitth. aus dem K. Gesundheitsamte, Bd. ii., p. 335. NEELSEN, Beitrage zur Biol. derPflanzen, Bd. iii., Heft 2. HEIM, Arbeitenaus dem K. Gesundheitsamte, Bd. v., p. 518. JORDAN, Rep. Mass. State Board of Health, 1890, Purifica- tion of Water and Sewage, vol. ii., p. 883. 2386. Bacillus fuscus limbatus. SCBTEIBENZUBER, Allgemeine Wiener med. Zeitung,r 1889, p. 171. 2387. Bacillus latericeus. ADAMETZ, op. cit. (No. 2294). 2388. Bacillus spiniferus. TOMMASOLI, op. cit. (No. 2368), p. 58. 2389. Bacillus rubescens. JORDAN, op. cit. (No. 2335), p. 835. 2390. Bacillus allii. GRIFFITHS, Proc. Roy. Soc., Edin., vol. xv., p. 40. 2391. Bacillus fulvus. ZIMMERMANN, op. cit. (No. 2296). 2392. Bacillus helvolus. ZIMMERMANN, op. cit. (No. 2296). 2393. Bacillus ochraceus. ZIMMERMANN, op. cit. (No. 2296). 2394. Bacillus plicatilis. ZIMMERMANN, op. cit. (No. 2296). 2395. Bacillus janthinus. ZOPF, Die Spaltpilze, Breslau, 1884, p. 62. PLAGGE TJND PROSKAUER, Zeitschrif t f iir Hygiene, Bd. ii., p. 463. JORDAN, op. cit. (No. 2385), p. 840. 2396. Bacillus violaceus Laurentius. JORDAN, op. cit. (No. 2385), p. 838. 2397. Bacillus tremelloides. TILS, Zeitschrift fiir Hygiene, Bd. ix., p. 292. 2398. Bacillus cuticularis. TILS, op. cit. (No. 2397), p. 293. 2399. Flesh-colored bacillus. TILS, op. cit. (No. 2397), p. 294. 2400. Bacillus arborescens. FRANKLAND, op. cit. (No. 2366), p. 379. 2401. Bacillus citreus cadaveris. STRASSMAN UND STRECKER, Zeitschrift fiir Medi- cinalbeamte, 1888, No. 3. 2402. Bacillus membranaceus amethyst inus. EISENBERG, op. cit. (No. 2297), p. 421. 2403. Ascobacillus citreus. TOMMASOLI, op. cit. (No. 2332), p. 60. BIBLIOGRAPHY. 871 2404. Bacillus cceruleus. SMITH, The Medical News, 1887, rol. ii., p. 758. 2405. Bacillus fluorescens liquefaciens. FLUGGE, op. cit. (No, 2300), p. 239. STERN- BEBG, op. cit. (No. 2337), p. 208. 2406. Bacillus fluorescens liquefaciens minutissimus. TOMM ASOLI, op. cit. (No. 2332),. p. 57. 2407. Bacillus fluorescens nivalis. SCHMOLCK, CentralbL fur Bakteriol., Bd. iv.,. p. 545. 2403. Bacillus lactis erythrogenes. GROTENFELT, Fortschr. der Med., 1889, No. 2, p. 41. 2409. Bacillus glaucus. ADAMETZ, op. cit. (No. 2294). 2410. Bacillus lividus. PLAGGE UND PROSKAUER, Zeitschrift fur Hygiene, Bd. ii., p. 463. EISENBERG, op. cit. (No. 2297), p. 81. 2411. Bacillus Indicus. EISENBERG, op. cit. (No. 2297), p. 79. 2412. Bacillus prodigiosus. EHRENBERG, Verhandl. der Berliner Acad., 1839. SCHOTTELIUS, Biologische Untersuchungen ilber den Micrococcus prodir- giosus, 185 pp., Leipzig, 1887. 2413. Bacillus mesentericus ruber GLOBIG, Zeitschrift filr Hygiene, Bd. iii., p. 3221. 2414. Bacillu^ pyocyanus ft. ERNST, Zeitschrift fur Hygiene, Bd. ii., 365. 2415. Bacilluo mycoides roseus. SCROLL, Fortschr. der Med., Bd. vii., p. 46. 2416. Bacillus rosaceum metalloides. DOWDESWELL, Ann. de Micrographie, vol. ii.r p. 310. 2417. Bacillus viscosus. FRANKLAND, op. cit. (No. 2366), p. 391. 2418. Bacillus violaceus. FRANKLAND, op. cit. (No. 2366), p. 394. 2419. Bacillus sulfureum. HOLSCIIEWNIKOFF, Fortschr. der Med., Bd. vii., p. 202. 2420. Bacillus rubidus. EISENBERG, op. cit. (No. 2297), p 88. TILS, op. cit. (No^. 2397), p. 307. 2421 Bacterium termoof Vignal. VIGNAL, Archiv de Physiol., t. viii., 1886, p. 842.. 2422. Bacillus buccalis minutus. VIGNAL, op. cit. (No. 2422), p 365.. 2423. Bacillus of Canestrini. CANESTRINI, Atti Soc. Ven, -Trent. Sci. Nat., xii., p.. 134. 2424. Bacillus ubiquitus. JORDAN, op. cit. (No. 2385), p. 830. 2425. Bacillus candicans. FRANKLAND, op. cit. (No. 2366), p. 398. 2426. Bacillus albus. EISENBERG, op. cit. (No. 2497), p. 171. 2427. Bacillus acidi lactici (Hueppe). HUEPPE, Mittheilungen aus dem K. Gesund- heitsarate, Bd. ii., p. 337. GROTENFELT, Fortschr. der Medicin, Bd. viii,, p. 121. 2428. Bacillus limbatum acidi lactici. MARPMANN, op. cit. (No. 2321), p. 122. 2429. Bacillus lactis pituitosi. LOFFLER, Berliner klin. Wochenschr., 1887, p. 631.. 2430. Bacillus aerogenes. MILLER, Deutsche med. Wochenschr., 1886, No. 8. 2431. Bacterium aerogenes. MILLER, op. cit. (No. 2430). 2432. Heliobacterium a<5rogeues. MILLER, op. cit. (No. 2430). 2433 Bacillus aquatilis sulcatus, Nos. 1, 2, 3, 4, and 5. WEICHSELBAUM, Das oster- reichische Sanitatswesen, 1839, Nos. 11-23. 2434. Bacillus multipediculus. FLUGGE, op. cit. (No. 2300), p. 323. 2435. Bacillus cystiformis. CLADO, Bull, de la Soc. d'Anat. de Paris, 1887, p. 339.. 2436. Bacillus hepaticus fortuitus. STEUNBERG, op. cit. (No. 2337), p. 205. 2437. Bacillus intestinus motilis. STERNBERG, op. cit. (No. 2337), p. 205. 2438. Bacillus caviae fortuitus. STERNBERG, op. cit. (No. 2337), p. 206. 2439. Bacillus coli similis. STERNBERG, op. cit. (No. 2337), p. 218. 2440. Bacillus flliformis Havaniensis. STERNBERG, op. cit, (No. 2337), p. 21L 2441. Bacillus Martinez. STERNBERG, op. cit. (No. 2337), p. 214. 872 BIBLIOGRAPHY. 2442. Bacillus epidermidis. BIZZOZKRO, Virchow's Archiv, Bd. xcviii., p. 455. BOR- DONI-UFFREDUZI, Fortschritt der Med., 1886, p. 156. 2443. Bacillus nodosus parvus. LUSTGARTKN, Vierteljahresschr. filr Derm. und Syph. 1887, p. 914. 2444. Bacillus hyacinthi septicus. HEINZ, Centralbl. filr Bakteriol., Bd. v., p. 535. 2445. Bacterium gliscrogenum. MALERBA, Giornale intern delle Sci. med., Naples, 1888, fasc. li. 2446. Bacillus ovatus minutissimus. TOMMASOLI, op. cit. (No. 2332), p. 59. 2147. Capsule bacilli of Smith SMITH, Centralbl. fiir Bakteriol., Bd. x., p. 181. 2448. Bacillus putrificus coli. Bacillus subtilis simulans Nos. 1 and 2. BIENSTOCK, Zeitschr. f ur klin. Med., Bd. viii. 2449. Bacillus striatus albus. VON BESSER, op. cit. (No. 2315), p. 349. 2450 Bacillus stolonatus. ADAMETZ, op. cit. (No. 2294). 2451. Bacillus ventriculi. RACZYNSSKY, Diss. der militarisch-medicinischen Akad., St. Petersburg, 1888. 2452. Bacillus Zopfli. KUUTH, Bot. Zeitung, 1883. CROOKSHANK, Manual of bac- teriology, 3d ed., p. 344. 2453. Bacterium Ziirnianum. ADAMETZ, op. cit. (No. 2294). 2454. Bacillus of Colomiatti. KUSCHBERT UND NEISSER, Breslauer arztliche Zeit- schrift, 1883, No. 4. FRANKEL UND FRANKE, Knapp-Schweiger's Archiv, Bd. xvii.,p 176. 2455. Bacillus scissus. FRANKLAND, op. cit. (No. 2366), p. 398. 2456. Bacilli of Fulles. FULLES, Zeitschrift fur Hygiene, Bd. x., p. 250. 2457. Bacillus phosphorescens gelidus. FORSTER, Centralbl. filr Bakteriol., Bd. ii., p. 337. 2458. Phosphorescent bacilli of Katz. KATZ, Centralbl. filr Bakteriol., Bd. ix. 2459. Bacillus phosphorescens Indicus. FISCHER, Zeitschrift fur Hygiene, Bd. ii., p. 54 ; Centralbl. fur Bakteriol., Bd. iv., p. 89. 2460. Bacillus phosphorescens indigenus. FISCHER, Centralbl. filr Bakteriol., Bd. iii., p. 105. 2461. Bacillus circulans. JORDAN, op. cit. (No. 2385), p. 831. 12462. Bacillus superficialis JORDAN, op. cit. (No. 2385), p. 833. 2463. Bacillus reticularis JORDAN, op. cit. (No. 2385), p 834. 2464. Bacillus hyalinus. JORDAN, op. cit. (No. 2385), p 835. 2465. Bacillus cloacae. JORDAN, op. cit. (No. 2385), p. 836. 2466. Bacillus delicatulus. JORDAN, op. cit. (No. 2385), p. 837. 2467. Bacillus aquatilis. FRANKLAND, op. cit. (No 2366), p. 382. 2468. Bacillus diffusus. FRANKLAND, op. cit. (No. 2366), p. 396. 2469. Bacillus liquidus. FRANKLAND, op. cit. (No. 2366), p. 383. 2470. Bacillus vermicularis. FRANKLAND, op. cit. (No. 2366), p. 384. 2471. Bacillus nubilus. FRANKLAND, op. cit. (No. 2366), p. 387. 2472. Bacillus pestifer. FRANKLAND, Phil. Trans , vol. clxxviii., p. 27 Jc 2473. Bacillus filiformis. TILS, op. cit. (No. 2397), p. 294. 2474. Bacillus devorans. ZIMMERMANN, op. cit. (No. 2296). 2475. Bacillus gracilis. ZIMMEIIMANN, op. cit. (No. 2296). 2476. Bacillus guttatus. ZIMMERMANN, op. cit. (No. 2296). 2477. Bacillus implexus. ZIMMERMANN, op. cit. (No. 2296). 2478. Bacillus punctatus. ZIMMERMANN, op. cit. (No. 2296). 2479. Bacillus radiatus. ZIMMERMANN, op. cit. (No. 2296). 2480. Bacillus radiatus aquatilis. ZIMMERMANN, op. cit. (No. 2296). :2481. Bacillus vermiculosus. ZIMMERMANN, op. cit. (No. 2296). 2482. Bacillus aerophilus. FLUGGE, op. cit. (No. 2300), p. 321. BIBLIOGRAPHY. 873 2483. Bacillus my coides. FLUGGE, op. cit. (No. 2300), p. 334. ZIMMERMANN, op. cit. (No. 2296). 2484. Bacillus mesentericus vulgatus. VIGNAL, Le bacille mesentericus vulgatus,. Paris, 1889— an extended monograph. FLUGGE, op. cit. (No. 2300), p. 322. 2485. Bacillus mesentericus fuscus FLUGGE, op. cit. (No. 2300), p. 321. TILS, op. cit. (No. 2397), p. 310. 2486. Bacillus megatherium. DE BARY, Vergl. Morphologic und Biol. der Pilze, Leipzig, 1884. TILS, op. cit. (No. 2397), p. 312. 2487. Bacillus albus putidus. ADAMETZ, op. cit. (No. 2294). 2488. Bacillus brassicae. POMMER, Mitth. des botanischen Inst. zu Gratz, Bd. i., p 95. 2489. Bacillus butyricus of Hueppe. HUEPPE, Mitth. aus dem K. Geshundheits- amte, Bd. ii., 1884. 2490. Bacillus gasoformans. EISENBERG, op. cit. (No. 2297), p. 107. 2491. Bacillus carabiformis. KACZYNSKY, Diss. der militar-med. Akad. in St. Peters- burg, 1888. 2492. Bacillus graveolens. BORDONI-UFFREDUZZI, Fortschr. der Med., 1886, p. 157. 2493. Bacillus carotarum. A. KOCH, Habilitationsschrift, Gottingen, 1888. 2494. Bacillus inflatus. A. KOCH, op. cit. (No. 2493). 2495. Bacillus ramosus. EISENBERG, op. cit. (No. 2297), p. 126. 2496. Bacillus subtilis. PRAZMOWSKI, Untersuchungen fiber die Entwickluiigsge- schichte und Fermentwirkung einiger Bakterien, Leipzig 1880. FLUGGE, op cit. (No. 2300), p. 315. VIGNAL, op. cit. (No. 2421), p. 344. BABES, Journ. d'Anat., 1884, p. 41. 2497. Bacillus subtilis similis. STERNBERG, op. cit. (No. 2337), p. 210. 2498. Bacillus leptosporus. L. KLEIN, Centralbl. f ur Bakteriol. , Bd. vi., p 345. 2499. Bacillus sessilis. L. KLEIN, op. cit. (No. 2498), p. 378. 2500. Bacillus allantoides. L. KLEIN, op. cit. (No. 2498), p. 383. 2501. Bacillus of Scheiirlen. SCHECRLEN, Deutsche med. Wochenschr. , 1888, p. 1033. SENGER, Berl. klin. Wochenschr., 1888, p. 186. VAN ERMENGEM, Bull. Soc. beige de Microscopic, seances de janv. 28 et du mars 31, 1888. PFEIFFER, Deutsche med. Wochenschr., 1888, No. 11. ROSENTHAL, Zeit- schr fur Hygiene, Bd. v., p. 161. 2502. Bacillus lactis albus. LOFFLER, Berliner klin. Wochenschr., 1887, p. 630 2503. Bacillus liodermos. LOFFLER, op. cit. (No. 2502), p. 630. 2504. Bacillus ulna. FLUGGE, op. cit. (No. 2300), p 329. 2505. Bacillus ulna of Vignal. VIGNAL, op. cit. (No. 2421), p. 360. 2506 Bacillus liquefaciens. EISENBERG, op. cit. (No. 2297), p. 112. 2507 Bacillus mai'dis. PALTATJF UND HEIDER, Medicinische Jahrbilcher, 1889, No. 8. EISENBERG, op. cit. (No. 2297), p. 119. 2508 Proteus sulfureus LINDENBORN UND HOLSCHEWNIKOFF, Fortschr. der Med., Bd. vii., p. 201. 2509. Bacillus thermophilus. MIQUEL, Ann de Micrographie, 1888, p. 4. 2510. Bacillus tumescens. A. KOCH, op. cit. (No 2493). ZOPF, Die Spaltpilze, Breslau, 1884, p. 61. 2511. Bacillus buccalis maximus. MILLER The microorganisms of the human mouth, Phila., 1890, p. 73 (English translation from Ger. ed.). 2512. Leptothrix buccalis of Vignal. VIGNAL, op. cit. (No 2421), p. 337. 2513 Bacillus b, Bacillus/, and Bacillus^' of Vignal. VIGNAL., op. cit. (No. 2421). 2514. Bacillus Havaniensis liquefaciens. STERNBERG, op. cit. (No. 2337). p. 215. 2515 Bacillus liquefaciens communis. STERNBERG, op. cit. (2337), p. 209. 2516 Bacillus muscoides. LIBORIUS, Zeitschr. filr Hygiene, Bd. i., p. 163. •'874 BIBLIOGRAPHY. •2517. Bacillus polypiformis. LIBORIUS, op. cit. (No. 2516), p. 162. 2518. Bacillus solidus. LUDERITZ, Zeitschr. fur Hygiene, Bd. v., p. 152. .2519. Bacillus butyricus. PASTEUR, Compt. rend. Acad. des Sc., lii., 1861. TRECUL, Compt. rend. Acad. desSc., Ixi., 1865; ibid., Ixv., 1867. VAN TIEGHEM, Compt. rend. Acad. des Sc., Ixxxviii., 1879; ibid., Ixxxix., 1879; ibid., 1884 (No. 6). TAPPEINER, Fortschr. der Med., Bd. i., p. 151 ; Bd. ii., pp. 377 and 416. PRAZMOWSKI, op. cit. (No. 2496). '2520. Clostridium fcetidum. LIBORIUS, op. cit. (No. 2516), p. 160. 2521. Bacillus liquefaciens magnus. LUDERITZ, op. cit. (No. 2518), p. 146. 2532. Bacillus liquefaciens parvus. LUDERITZ, op. cit. (No. 2518), p. 148. 2523. Bacillus radiatus. . LUDERITZ, op. cit. (No. 2518), p 149. 2524. Bacillus spinosus. LUDERITZ, op. cit. (No. 2518), p. 153. 2525. Bacillus anaerobicus liquefaciens. STERNBERG, op. cit. (No. 2337), p. 214. X NON-PATHOGENIC SPIRILLA 2526. Spirillum sputigenum. MILLER, Deutsche med. Wochenschr., 1884, No. 47. "2527. Spirillum dentium. MILLER, op. cit. (No. 2511). 2528. Spirillum plicatile. KOCH, Beitrag zur Biol, der Pflanzen, Bd. ii., p. 420. :2529. Spirillum rugula. PRAZMOWSKI, op. cit. (No. 2496). VIGNAL, op. cit. (No. 2421) p. 347. '2530. Spirillum sanguineum. COHN, Beitrag zur Biol. der Pflanzen, Bd. i., p. 169. 2531. Spirilla of Weibel. WEIBEL, Centralbl. fur Bakteriol., Bd. iv. '2532. Spirillum concentricum. KITASATO, Centralbl. fiir Bakteriol., Bd. iii., p. 73. :2533. Spirillum rubrum. VON ESMARCH, Centralbl fllr Bakteriol., Bd. i., p. 225. 2534. Spirillum of Smith. SMITH, Centralbl filr Bakteriol., Bd. x., p. 178. "2535. Spirillum of Miller. MILLER, Deutsche med. Wochenschr., 1884, Nos. 34 and 48. XL LEPTOTRICHE^E AND CLADROTRICH^E. '3536. Crenothrix Kilhniana. COHN, Max Schultz's Archiv, Bd. iii. ZOPP, Die Spaltpilze, 2d ed. , p. 67. 2537. Beggiatoa alba. ZOPF, op. cit. (No. 2536), p. 71. 2538. Beggiatoa roseo persiciua. ZOPF, op. cit. (No. 2536), p. 73. LANKESTER, Quar Journ. Mic. Sci., vol. xiii., 1873 ; ibid., vol. xvi., 1876. 2539. Beggiatoa mirabilis. ZOPF, op. cit. (No. 2536), p. 74. 2540. Phragmidiothrix multiseptata. ENGLER, Bericht der Kommission zur Erfor- schung deutscher Meere, 1881. 2541. Cladothrix dichotoma. COHN, Beitrag zur Biol. der Pflanzen, Bd. i., Heft 3. CIENKOWSKI, zur Morph. der Bakterien, St. Petersburg, 1876. ZOPF, op. cit. (No. 2536), p. 77. 5542. Cladothrix Foersteri. COHN, op. cit. (No. 2541), Bd. i., Heft 3. XII. ADDITIONAL SPECIES OF BACTERIA, NOT CLASSIFIED. .2543 WINOGRADSKY. Recherchcs sur les organismes de la nitrification, Ann. de 1'Institut Pasteur, t. iv., 1890, p. 213; 2e Memoire, ibid., p 257; 3e Me- moire, ibid., p. 760 ; 4e Memoire, ibid., t. v., 1891, p. 93 ; 5e Memoire, ibid., t. v., p. 575. :2544. FRANKLAND, PERCY F. AND GRACE C. The nitrifying process and its spe- cific ferment. Proc. of the Royal Soc. of London, vol. xlvii., 1890, p. 296. :2545 JORDAN, E. O., AND RICHARDS, ELLEN H. Investigations upon nitrification and the nitrifying organism. Rep. Mass. State Board of Health, Purifi- cation of Sewage and Water, vol. ii., 1890, p. 865. BIBLIOGRAPHY. 875 •2546. MUNTZ. De la formation des nitrates dans la terre. Compt. rend. Acad. des Sc , t. cxii., No. 20. 2547. WARINGTON. On nitrification. Journ. of the Chem. Soc., Lond., 1890, p. 485. 2548. Streptococcus conglomeratus. KURTH, Trans. Ninth Interuat. Med. Cong., Berlin, 1891, p. 335. '2549. RUSSELL. Untersuchungen liber im Golf von Neapel lebende Bakterien. Zeifschr. fur Hygiene, Bd. xi., 1891, p. 190. 2550. Bacillus capsulatus mucosus. FASCHING, S. B. K. Akad. Wiss., Wiener Cen- tralbl., Ib91, p. 295. '2551. Bacillus of potato rot. KRAMER, Bakteriologische Untersuchungen uber die Nassfaille der Kartoffelknollen. Oesterreichisches landwirthschaft. Cen- tralbl., 1891, p. 11. •2552. Bacillus vacuolosis. STERNBERG, op. cit. (No. 2337), p. 208. 2553. Bacillus of Dantec. DANTEC, Etude de la morue rouge, Ann. de 1'Institut Pasteur, t. v., 1891, p. 659. 2554. Bacillus Havaniensis. STERNBERG, op. cit. (No. 2337), p. 207. 2555. Bacillus amylozyma. PERDRIX, Sur les fermentations produites par un mi- crobe, anaerobic de 1'eau. Ann. de 1'Institut Pasteur, t. v., 1891, p. 287. 2556. Bacillus rubellus. OKADA, Centralbl. filr Bakteriol., Bd. xi., 1892, p. 1. 2557. Bacterium ureoe. JAKSCH, Zeitschr. fur phys. Chem., Bd. v., p. 395. LEUBE UND GRASSER, Virchow's Archiv, Bd. c., p. 556. 2158. Sarcina mobilis. MAUREA, Centralbl. fur Bakteriol., Bd. xi., 1892, p. 228. •2159. POHL. Centralbl. fur Bakteriol., Bd. xi., 1892, p. 142. 2160. Bacillus butyricus of Botkin. BOTKIN, Zeitschr. fur Hygiene, Bd. xi., 1892, p. 421. 2561. MIQCEL. Etude sur la fermentation ammoniacale et sur les ferments de 1'uree. Ann. de Micrographie, vols. ii., iii., iv., and v. (1889-1892). 2562. BOVET. Contribution a 1'etude des microbes de 1'intestin gre"le. Ann. de Mi- crographie, vol. iii., 1891, p. 353. •2563. FREUDENREICH. Sur un nouveau bacille trouve dans les fromages boursoufles. Ann. de Micrographie, vol. iii., 1891, p. 161. 2564. Sur quelques bacteries produisent le boursouflement des frommages. Ann. de Micrographie, vol. ii., 1890, p. 353. 3565. GUILLEBEAU. Description de deux nouveaux microbes du lait filant. Ann. de Micrographie, vol. iv., 1892, p. 225. '2566. Bacillus denitrificans. GILTAY ET ABERSON, Arch. Neerlandaises Sci. exact. et nat., xxv., 1891, p. 341. :2567. Bacillus cyano-fuscus. BEYERINCK, Bot. Ztg., 1891, vol. xlix., Nos. 43-47. 2568. Description of a pus-producing bacillus obtained from earth. BOLTON, Am. Journ. Med. Sc., June, 1892. :2569. VAUGHAN. A bacteriological study of drinking water. Am. Journ. Med. Sc., 1892, civ., p. 107. 2570. WELCH AND NTTTTALL. A gas-producing bacillus (Bacillus afirogenes capsu- latus, nov. spec.) capable of rapid development in the blood vessels after death. Bull, of the Johns Hopkins Hospital, July, 1892. •2571. CANON UND PrELiCKE. Berliner klin. Wochenschr., No. 16, 1892. 2572. BRANNAN AND CHEESMAN. A study of typhus fever, clinical, pathological, and bacteriological. Medical Record, New York, June 25th, 1892. •2573. MENGE. Uebereinen Mikrokokkus mit Eigenbewegung. Centralbl. fur Bak- teriol., Bd. xii., p. 49(1892). 2574. STERNBERG. Op, cit. (No 2337), p. 216. 876 BIBLIOGRAPHY. SUPPLEMENT TO BIBLIOGRAPHY. 2575. PFUHL. Beitrag zur .iEtiologie der Influenza. Centralbl. fiir Bakteriol., Bd. xi., 1892, p. 398. 2576. FIOCCA. Ueber einen im Speichel einiger Hausthiere gefundenen, dem In- fluenzabacillus ahnlichen Mikroorganismus. Centralbl. filr Bakteriol., Bd. xi., 1892, p. 406. 2577. STERN. Ueber Desinfektion des Darmkanales. Zeitschrift fiir Hygiene, Bd. xii., 1892, p. 88. 2578. ROBB AND GHRISKY. The bacteria in wounds and skin stitches. Johns Hopkins Hosp. Bull., vol. iii., 1892, p. 37. 2579. BRIEGER, KITASATO TTND WASSERMANN. Ueber Immunitat und Giftfesti- gung. Zeitschrift fur Hygiene, Bd. xii., 1892, p. 138. 2580. FREUDENREICH. De 1'antagonism des bacteries. Ann. de Micrographie, vol. ii., 1889, p. 1. 2581. PARK. Diphtheria and allied pseudo-membranous inflammations. Medical Record, New York, July 30th, 1892. 2582. STERNBERG. Practical results of bacteriological researches. Am. Journ. Med. Sc., vol. civ., No. 1 (July, 1892). INDEX. Abrin, pathogenic action of, and immu- nity from, 259 Abscesses, formation of, 217, 264 Acetone, germicidal value of, 189 Acetic acid, production of, 130 germicidal value of, 174 Acids, production of, 129 germicidal action of, 172 Acne contagiosa of horses, bacillus of, 478 Atsroscope of Maddox, 543 ; of Hesse, 545 Agar-agar jelly, 43 methods of filtering, 44 Agar-gelatin, 43 Agua coco as a culture medium, 39 Air, bacteria in, 541 Alcohol, germicidal value of, 189 Alkalies, germicidal value of, 175 Alopecia, bacteria in, 514 Alum, antiseptic value of, 180 Aluminum acetate, antiseptic value of, 180 Ammonia, germicidal value of, 176 Ammonium carbonate, germicidal value of, 180 Anaerobic bacilli, pathogenic, 482 non- pathogenic, 687 cultivation of, 78 Aniline oil, antiseptic value of, 190 dyes, germicidal value of, 189 Anthrax, 327 bacillus, chemical products of, 333 bacillus, toxalbumin of, 332 bacillus, formation of spores, 331 Antiseptics, action of, 156 comparative value of, 178 Antiseptic value, method of determin- ing, 156, 157 Antitoxine of pneumonia, 257, 308 of tetanus, 256 Antitoxines, 256 Arnold's steam sterilizer, 54 Arsenious acid, germicidal value of, 175 Arthrospores, 116 Ascobacillus citreus, 634 Ascococcus, generic characters, 17 Billrothii, 618 Johnei, 315 72 Aseptol, germicidal value of, 190 Atmospheric bacteria, 541 methods of collecting, 541-560 general results of researches made, 550 list of species found, 552 Miquel's method of collecting, 546 ; Petri's method, 548 soluble filter for collection of, 549 Attenuation of virulence, 123, 233 by antiseptic agents, 124 by cultivation in blood of immune animal, 125 by heat, 124 Bacilli, general characters of, 18 morphology of, 23 Bacillus, generic characters, 18 acidiformans, 449 acidi lactici, 645 of acne contagiosa of horses, 478 aBrogenes capsulatus, 731 aerophilus, 672 of Afanassiew, 469 agilis oitreus, 733 albus, 645 albus anafirobiescens, 729 albus cadaveris, 473 albus putidus, 675 allantoides, 680 allii, 629 alvei, 477 amylozyma, 718 anae"robicus liquefaciens, 693 anthracis, 328 aquatilis, 666 aquatilis sulcatus No. I., 646 aquatilis sulcatus No. II., 647 aquatilis sulcatus No. III., 647 aquatilis sulcatus No. IV., 648 aquatilis sulcatus No. V. , 648 arborescens, 633 argenteo-phosphorescens No. I., 659 argenteo-phosphorescens No. II., 659 argenteo-phosphorescens No. III., 660 argen teo- pi i osphorescens 1 iq uef a- cieiis, 661 aurantiacus, 620 878 INDEX. Bacillus aureus, 621 of Babes and Oprescu, 435 of Belfauti and Pascarola, 417 beriolinensis Indicus. 621 of Booker, 443-446, 463 of Bovet, 724 brassicae, 675 brunneus, 620 buccalis fortuitus, 685 buccalis maximus, 683 buccalis minutus, 643 butjricus, 688 butyricus of Botkin, 721 butyricus of Hueppe, 675 cadaveris, 492 canalis capsulatus, 476 canalis parvus, 476 candicans. 644 of Canestrini . 643 of Canon and Pielicke, 731 capsulatus, 431 capsulatus mucosus, 716 carabiformis, 676 carotarum, 676 caviae fortuitus, 650 cavicida, 425 cavicida Havaniensis, 425 of Cazal and Vaillard, 435 cholera? gallinarum, 408 chromo-aromaticus, 475 circulans, 663 citreus cadaveris, 633 cloaca? , 065 coeruleus, 635 coli communis, 439 coli similis, 650 of Colpmiatti, 656 constrictus, 622 coprogenes parvus, 424 coprogenes fretidus, 468 crassus sputigenus, 426 cuniculicida, 408 cuniculicida Havaniensis, 450 cuticularis, 632 cyanogenus, 626 cyaneo phosphorescens, 661 cyano fuscus, 727 cystiformis, 649 of Dantec, 717 delicatulus, 666 of Demme, 465 denitrificans, 727 dentalis viridans, 471 devorans, 669 diffusus, 667 diphtheria?, 360 diphtheria? columbrarum, 367 diphtheria? vitulorum, 368 endocarditidis capsulatus, 464 endocarditidis griseus, 463 enteritidis, 429 epidermidis, 651 erysipelatos suis, 420 erythrosporus, 624 figurans, 729 Bacillus filiformis, 669 filiformis Havaniensis, 650 of Fiocca, 456 flavocoriaceus, 621 flavescens, 721 fluorescens aureus, 622 fluorescens liquefaciens, 635 fluorescens liquefaciens minutissi- mus, 636 fluorescens longus, 622 fluorescens nivalis, 636 fluorescens noii-liquefaciens, 623 fluorescens putidus, 624 fluorescens tenuis, 623 foetidus oza?na?, 466 No. I. of Fulles, 657 No. II. of Fulles, 657 fulvus, 629 f uscus, 625 fuscus limbatus, 628 gallinarum. 430 gasoformans, 676 of Gessner, 475 gingiva? pyogenes, 471 glaucus, 637 gracilis, 670 gracilis anaerobiescens, 728 gracilis cadaveris, 733 granulosus, 711 graveolens, 676 of grouse disease, 429 of Guillebeau, a, b, c, 725 guttatus, 670 halophilus, 716 Havaniensis, 718 Havaniensis liquefaciens, 686 helvplus, 630 heminecrobiophilus, 481 hepaticus fortuitus, 649 Hessii, 727 of hog cholera, 413 hyacinthi septicus, 651 hyalinus, 665 hydrophilus fuscus, 432 implexus, 671 incanus, 720 Indicus, 637 indigogenus, 476 inflatus, 677 of influenza, 371 intestinus motilis, 649 of intestinal diphtheria in rabbits, 368 invisibilis, 729 iuunctus, 721 iris, 625 janthinus, 631 of Jeffries, 448 of Kartulis, 477 of Koubasoff , 405 lactis albus, 680 lactis erythrogenes, 636 lactis pituitosi, 645 latericeus, 628 of Laser, 434 I*DEX. 879 Bacillus leporis lethalis, 458 leprse, 394 leptosporus, 679 of Lesage, 464 of Let ze rich, 466 limosus, 713 limbatus acidi lactici, 645 liodermos, 680 liquefaciens, 682 liquefaciens comraunis, 686 liquefaciens magnus, 690 liquefaciens parvus, 690 liquidus, 667 litoralis, 714 lividus. 637 of Loeb, 437 of Lucet, 436 of Lumnitzer, 467 maidis. 682 malar iae of Klebs and Tommasi-Cru- deli, 523 mallei, 396 marinus, 714 Martinez. 651 of measles, 731 megatherium, 674 membranaceus amethystinus, 634 meningitidis purulent*, 474 mesentericus fuscus, 674 mesentericus ruber, 639 mesentericus vulgatus, 673 multipediculus, 648 murisepticus 420 murisepticus pleomorphus, 460 muscoides, 687 mycoides, 673 mycoides roseus, 640 Neapolitanus, 439 necrophorus, 468 of Nocard, 406 nodosus parvus, 651 nubilus, 668 ochraceus, 630 oedematis afirobicus, 465 cedematis maligni, 488 of Okada, 479 ovatus miimtissimus, 652 oxytocus perniciosus, 469 pestifer, 669 phosphorescens gelidus, 658 phosphorescens Indicus, 662 phosphorescens iudigenus, 663 plicatus. 630 pneumouiae, 296 pneumosepticus, 431 polypiformis, 687 prodigiosus, 638 pseudotuberculosis, 470 pulpae pyogenes, 471 punctatus, 671 of purpura haemorrhagica, 480, 48L putrificus coli, 654 pyocyanus, 454 pyocyanus fi, 639 pyogenes fcetidus, 427 Bacillus pyogenes soli, 728 radiatus, 691 radiatus aquatilis, 671 ramosus, 677 reticularis, 664 of rhinoscleroma, 404 rosaceum metalloides, 640 of Roth, 479 rubefaciens, 625 rubellus, 719 rubescens, 629 rubidus, 642 salivarius septicus, 298 sauguinis typhi, 732 saprogenes II., 469 Schafferi, 725 of Scheurlen, 680 of Schimmelbusch, 466 of Schou. 468 scissus, 656 septicaemias hasmorrhagicae, 408 septicus acuurinatus, 472 septicus agrigenus, 419 septicus keratomalaciae, 472 septicus sputigenus, 298 septicus ulceris gangraenosi, 472 septicus vesicae, 475 sessilis, 679 smaragdinus foetidus, 430 smaragdino- phosphorescens, 658 solidus, 688 spiniferus, 628 spinosus, 693 stolonatus, 654 stoloniferus, 720 striatus albus, 654 striatus flavus, 626 subflavus. 626 subtilis, 677 subtilis simulans No. I., 654 subtilis simulans No. II., 654 sulfureum, 641 superficialis. 664 of swine plague, 417 of symptomatic anthrax, 493 tenuis sputigenus, 433 tetani, 482 thalassophilus, 711 thermophilus, 682 of Tommasoli, 467 tremelloides, 632 of Tricomi, 473 tuberculosis, 375 tuberculosis gallinarum, 392 tumescens, 683 typhi abdominalis, 346 typhi murium, 434 typhosus, 346 ubiquitus, 644 ulna, 681 ulna of Vignal, 681 of Utpadel, 477 vacuolosis, 717 varicosus conjunctiva, 474 venenosus, 729 880 INDEX. Bacillus venenosus breyis, 730 venenosus invisibilis, 730 venenosus liquefaciens, 730 ventriculi, 655 vermicularis, 668 vermiculosus, 673 b of Vignal, 684 /of Vignal, 685 j of Vignal, 685 violaceus, 641 violaceus Laurentius, 631 virescens, 624 viridis pallescens, 624 viscosus, 640 Bacillus coli communis in peritonitis, 447 tuberculosis, spore formation (?), 379; thermal death point, 380 tuberculosis ; staining methods, 377 typhi abdominalis, flagella of, 347 typhi abdominalis, in spleen, 341 ; in faeces, 341 ; in water, 350 typhi abdominalis; chemical products of, 344 Bacteria in the air, 541 Bacterial cells, chemical composition of, 117 Bacteriological diagnosis, 735 Bacterium afirogenes, 646 coli commune, 439 gliscrogenum, 652 lactis aerogenes, 447 luteum, 620 termo of Vignal, 642 tholoideum, 475 ureae. 720 Zopfii, 656 Zuruianum, 656 Baumgarten, classification of, 12 Beggiatoa, sulphur grains in, 24 alba, 705 mirabilis, 706 roseo persicina, 705 Benzene, germicidal value of, 190 Benzoic acid, germicidal value of, 175 Beri-beri, bacteria in, 514 Bibliography, 735 to 876 Biological characters, modifications of, 122 Biskra button, micrococcus of, 318 Blood serum, collection of, 37 cultures upon, 75 germicidal value of, 198, 229 sterilization of, 5~> Booker's bacilli, 443-446 Boracic acid, germicidal value of, 174 Bouillon, 41 Bread paste, 49 Brieger's bacillus, 425 Bright's disease, micrococci in, 319 Bromine, germicidal value of, 170 Bronchitis, bacteria in, 467, 515 Buchner's method of cultivating anae- robic bacteria, 83 Bilffelseuche, bacillus of, 408 Butter, bacteria in, 590 Butyric acid, production of, 130 germicidal value of. 175 Cadaverin, 139 Cadavers, bacteria in, 585 Calcium hydroxide, germicidal value of, 176 chloride, antiseptic value of, 180 hypochlorite, germicidal value of , 180 Camphor, germicidal value of, 190 Capsule bacilli of Smith, 652 Carbolic acid, germicidal value of, 191 Carbon dioxide, action of, 16G Carbonic oxide, action of, 166 Carcinoma, bacteria in, 515 Catarrhal inflammations, 220 Caucasian milk ferment, 132 Cerebro-spinal meningitis, bacteria in, 518 Chancroid, bacteria in, 515 Charbon, 327 symptomatique, bacillus of, 493 Cheese, bacteria in, 590 Chemiotaxis, 235 Chloral hydrate, antiseptic value of, 181 Chlorine, antiseptic and germicidal value of, 169 Chloroform, antiseptic value of, 169 Cholera in ducks, bacillus of, 413 infantum, bacteria 4n, 515 infantum, Proteus vulgaris in, 4."»9 nostras, bacteria in, 515 ptomaines, 142 Cholin, 140 Chromic acid, germicidal value of, 173 Chronic infectious diseases, bacilli in, 374 Citric acid, germicidal value of, 174 Cladothrix, morphology of, 24 intricata, 708 dichotoma, 706 Foersteri, 707 Cladotricheae, 703 general characters of, 19 Classification. 10 biological, 14-16 morphological, 13 of pathogenic bacteria, 533 of Baumgarten, 12 of Cohn, 11 of Davaine, 10 of Dujardin, 10 of Ehrenberg, 10 of Hoffmann, 10 of Hueppe, 16 of Niljreli, 11 of Robin, 3 of Sachs, 11 of Zopf, 12 Clathrocystis roseo- persicina, 705 Clostridium. morphology of, 23 foetidum, 689 Coal-tar products, germicidal value of, 139 Coffee infusion, germicidal value of, 193 Cohn, classification of, 1 1 Colon bacillus, 439 INDEX. 881 Colonies of bacteria, general characters of, 71 Comma bacillus of Koch, 500 Conjunctivitis, bacteria in, 517, 574 bacilli in. 474. 477 pus cocci in, 282 Contact preparations. 27 Corrosive sublimate, germicidal value of, 182 Coryza, bacteria in, 517 Cotton air filter, 4 Crenothrix Kilhuiana, 703 Creolin, germicidal value of, 192 Cresol, germicidal value of, 193 Croupous pneumonia, bacteria in, 288 etiology of, 288-296 Culture media. 37 Cultures in liquid media, 60 in solid media, 67 Cupric sulphate, germicidal value of, 181 Cystitis, bacilli in, 475, 517 Darmbacillus of Sehottelius, 468 Davaine, classification of , 10 Davaine's septicaemia, bacillus of, 408 Uecolorization, 28 Demme, bacillus of, 465 Deneke, spirillum of, 511 Dengue, bacteria iu, 519 Defensive proteids, 260 Desiccation, effect of, 151 Diagnosis, bacteriological, 735 Dimensions of bacteria, 20 Diplococcus albicans amplus, 603 coryzae, 608 citreus liquefaciens, 595 citreus conglomerate, 594 fluorescens foatidus, 596 flavus liquefaciens tardus, 595 intercellularis meningitidis, 310 luteus, 596 pneumonia. 298 of pneumonia in horses, 322 roseus, 596 Disinfectants, general account of the ac- tion of. 156 Disinfection, practical directions for, 201 by steam, 203 of clothing, 202 of ships, 203 of the sick-room, 202 in diphtheria, 210 of excreta, 206 Disiufektol, germicidal value of, 194 Diphtheria, bacteria in, 356 experiments on animals, 359, 362 bacillus, toxalbumin of, 364 bacillus, immunity from, 364 Diphtheritic inflammations, 219 Double staining, 28 Drop cultures, 62 Dujardin, classification of, 10 Eczema epizp5tica, bacteria in, 519 Eggs, bacteria in, 591 Ehrenberg, classification of, 10 Ehrlich's solution, 29 Ehrlich-Weigert solution, 29 Electricity, action of, upon bacteria, 154 Emmerich's bacillus. 439 Empyema, bacteria in, 520 Endocarditis, ulcerative, 271 bacteria in, 520 micrococci in, 320 ^^^ streptococcus pyogenes in, 2<4\278 Enzymes, tryptic, 129 Erysipelas, etiology of, 274, 277 Erythema, bacteria in, 521 nodosum, 465 Von Esmarch's roll tubes, 74 Esmarch's method of cultivating anafro- bic bacteria, 82 Essential oils, germicidal value of, 194 Ether, germicidal value of, 194 Eucalyptol, germicidal value of, 195 Excreta, disinfection of , 201, 206 Experiments upon animals, 94 Faeces, bacteria in, 583, 584 Farcy in cattle, 406 Fermentation, putrefactive, 134 of urea, 132 viscous, 133 Ferments, soluble, 136 Ferric chloride, germicidal value of, 182 Ferrous sulphate, germicidal value of, 181 Finkler and Prior, spirillum of, 509 Fiocca, bacillus of, 456 Fixing, upon cover glass, 26 Flagella, methods of staining, 32 of bacilli, 24 of bacteria, 112 Flesh-colored bacillus, 632 Flesh peptone gelatin, 41 solution, 41 Foot and mouth disease, bacteria in, 519 Formic acid, germicidal value of, 175 Foul brood, bacillus of, 477 Fowl cholera, bacillus of, 408 Frankel's method of cultivating anaero- bic bacteria, 80 Freezing, action of, upon bacteria, 145 Friedlander's bacillus, 296 method of staining tubercle bacilli, 30. Gabbett's method of staining tubercle ba- cilli, 30 Gallic acid, germicidal value of, 175 Gases, action of, upon bacteria, 161 Germicides, action of, 156 Germicidal value, methods of determin- ing, 158 Gibier, bacillus of, 453 Glanders, bacillus of, 396 bacillus, staining of, 397 diagnosis of, 401 Glycerin, germicidal value of, 195 -agar, 43 882 INDEX. Gold chloride, germicidal value of, 182 Gonococcus, 283 Gram's method, 29 Granuloma fungoides, bacteria in, 521 Green pus, bacillus of, 454 Grouse disease, bacillus of, 429 Growth, conditions of, 118 Hsematococcus bovis, 322 Hail, bacteria in. 559 . Hands, disinfection of, 205 Heat, action of, upon bacteria, 145 moist, action of, 146 dry, action of, 146 Helicobacterium aerogenes, 646 Heterogenesis, 5 Historical, 3 Hoffmann, classification of, 10 Hog cholera, bacillus of, 4i3 erysipelas, bacillus of, 420 Hot-air ovens, 52 Hydrant water, bacteria in, 561 Hydrochloric acid, germicidal value of, 173 Hydrofluoric acid, germicidal value of, 171 Hydrogen peroxide, antiseptic and ger- micidal value of, 165 Hydrophobia, bacteria in, 521 Hydrosulphuric acid, action of, 167 formation of, 134 Hydroxylamin, germicidal value of, 195 Ice, bacteria in, 560 Icterus, bacteria in, 523 . Immunity, 226 acquired, 232 from injection of filtered cultures, 234 theories of, 237 Impftetanus bacillus, 417 Incubating ovens, 86 oven of D'Arsonval, 82 oven of Roux, 93 Infection, channels of, 223 mixed. 222 secondary, 221 Inflammations of mucous membranes, pus cocci in. 281 Influenza, bacteria in, 370 Infusoria, 3 Injections into the eye, 97 into the circulation. 96 into peritoneal cavity, 96 into the intestine, 97 Intestine, bacteria in, 580, 582 Involution forms, 23 Iodine, germicidal value of, 169 trichloride, germicidal value of, 170 lodoform, germicidal value of, 170 Iron, sulphate of, antiseptic value, 182 Jeffries' bacilli, 448 Jequirity solution, as culture medium, 47 Karliusky's method of filtering agar, 45 Kartulis, bacillus of, 477 Koch's plate method, 72 method of staining flagella, 32 syringe, 95 Kiihne's method of staining bacteria in tissues, 34 Lactic acid, germicidal value of, 174 fermentation, 588 Lake water, bacteria in, 560 Lanolin, germicidal value of, 196 Lead chloride, germicidal value of. 182 nitrate, germicidal value of, 182 Leprosy, bacillus of, 394, 523 Leptothrix buccalis of Vignal, 684 Leptotricheae, 703 general characters of, 19 Lesage, bacillus of, 464 Leuconostoc, generic characters, 17 mesenteroides, 619 Liborius' met hod of cultivating anaerobic bacteria, 83 Light, action of, upon bacteria, 151 Liquid media, cultures in, 60 Liquefaction of gelatin, 70, 128 List of bacteria described, 737 Lochial discharge, bacteria in, 578 Loffler's solution, 29 method of staining flagella, 32 Lustgarten, bacillus of, 402 Malachite green, germicidal value of, 190 Malaria, 523 Malic acid, germicidal value of, 175 Malignant pustule, 347 Malignant oedema, bacillus of, 488 Mallein, 144 Marsh gas, formation of, 134 Mastitis, bovine, micrococcus of, 317 Mastitis in sheep, micrococci in. 320 in cows, streptococcus of, 321 Measles, bacteria in, 524 Meats, bacteria in, 590 Meatus-urinarius, bacteria of, 578 Meningitis, bacteria in, 474, 524 Mercuric chloride, germicidal value of, 182 cyanide, germicidal value of, 184 iodide, antiseptic value of, 184 Merismopedia, generic characters, 17 Methane, action of, 167 Methyl violet, germicidal value of, 189 Methylamine. 140 Methyl guanidin, 141 Metritis, puerperal, etiology of, 274 Micrococci, general characters of, 17 Micrococcus acidi lactici, 603 acidi lactici liquefaciens, C01 aerogenes, 602 agilis, 594 albicans tardus. 607 albicans tardissimus. 607 albus liquefaciens, 602 of Almquist, 325 INDEX. Micrococcus amylovorus, 618 aquatilis, 604 aquatilis invisibilis, 728 ascoforrnans, 315 aurantiacus, 597 botryogenus 3 1 5 of bovine mastitis, 317 of bovine pneumonia, 317 candicans, 603 candidus, 603 carneus, 598 cerasinus siccus, 599 cereus albus, 599 cereus flavus, 599 cinnabareus, 599 citreus, 599 concentricus, 604 cremoides, 597 cumulatus tenuis, 605 of Dantec, 598 of Demme. 319 endocarditidis rugatus, 320 fervidosus, 600 Finlayensis, 608 No. II of Fischel, 324 flavus desidens, 594 flavus liquefaciens, 593 flavus tardigradus, 600 foetidus, 604 of Forbes, 326 of Freire, 608 Freudenreichi, 726 fuscus, 594 of gangrenous mastitis in sheep, 320 gingivae pyogenes, 323 gonorrho?ae, 283 of Heydeureich, 318 of Kirchner, 324 lactis riscosus, 604 luteus, 600 of Manfredi, 316 of Manneberg, 319 ocbroleucus, 601 ovalis, 608 ovatus, 318 Pasteuri, 298 plumosus. 605 pneumonia? crouposae, 298 ; in saliva of healthy persons, 298 ; in pneu- monic sputum, 299; in meningitis, 300 in otitis media, 301 ; in ul- cerative endocarditis, 301 ; in acute abscesses. 301 of pyaemia in rabbits, 312 pyogenes tenuis, 274 radiatus. 602 rosettaceus, 605 roseus, 597 salivarius pyogenes, 311 salivarius septicus, 312 of septicaemia in rabbits, 312 subflavus, 312 tetragenus, 314 tetragenus mobilis ventriculi, 615 Micrococcus tetrageuus subflavus, 615 tetragenus versatilis, 613 of trachoma (?), 313 ureae, 606 ureae liquefaciens, 606 versicolor, 598 violaceus, 601 viticulosus, 606 Microzyma bombycis, 318 Milk, bacteria in, 588, 590 as a culture medium, 39 germicidal value of, 200 fermentation of, 589 Mil /brand, 327 Miquel's method of collecting atmo- spheric bacteria, 546 Mixed infection, 222 Modes of action of pathogenic bacteria, 215 Modification of biological characters. 123 Morphology of bacteria, 20 Motions of bacteria, 113 Mouse septicaemia, bacillus of, 420 Mouth, bacteria of, 575 list of bacteria found in, 579 Mucous membranes, bacteria of, 573 Miiller's method of staining spores, 32 Muscarin, 140 Mustard, oil of, antiseptic value, 196 Mykoprotein, 117 Nageli, classification of, 11 Naphthol, germicidal value of, 196 Nares, bacteria of, 575 Nasal catarrh, pus cocci in, 282 Neisser's method of staining sporse, 31 Nephritis, bacteria in, 466, 524 Neuridin. 139 Neurin, 140 Nitrates, reduction of, 136 Nitric acid, germicidal value of, 173 Nitrification, 13ij Nitrifying bacillus of Winogradsky, 710 Nitrifying bacilli. 709-711 Nitromonas of Winogradsky, 709 Nitrous oxide, action of, 167 Nitrous acid, germicidal value of, 173 Noma, bacilli in, 466 Nose, list of bacteria found in, 579 Nosema bombycis, 318 Nutrient gelatin, preparation of, 42 Okada, bacillus of, 479 Ophidomonas sanguinea, 705 Otitis media, bacteria in 525 Micrococcus pneumonias crouposae in, 301 pus cocci in, 281 Osmic acid, germicidal value of, 173 Osteomyelitis, 270 bacteria in, 525 Oxalic acid, germicidal value of, 174 Oxygen, action of, upon bacteria, 164 Ozaena, bacteria in, 466, 526 Ozone, action of, upon bacteria, 164 INDEX. Panhistophyton ovatum, 318 Papin's digester, 54 Parasites, facultative, 120 Parasitic bacteria, 215 Parasitism, 120 Parotitis, bacteria in, 526 Pasteur-Chamberlain filter, 57 Pasteur's solution, 46 method of inoculating rabbits for rabies, 97 Pathogenic bacteria, classification of, 533 in water, 563, 565 Pediococcus albus, 614 cerevisiae. 615 Pemphigus, bacteria in, 526 acutus, micrococcus of, 319 Penicillum glaucum, 542 Peppermint, oil of, antiseptic value, 196 Peptotoxin, 141 Peritonitis, bacteria in, 526 Bacillus coli communis in, 447 Perlsucht, 375 Petri's dishes, 73 method of collecting atmospheric bacteri», 548 Phagocytosis, 245 Phosphorescence, 137 Phosphoric acid, germicidal value of, 173 Photographing bacteria, 101 by sunlight, 105 ; by gaslight, 107; by electric light, 105 ; by calcium light, 105 Phragmidiothrix multiseptata, 706 Phylaxins, 260 Physical agents, influence of, 145 Pigment, production of , 126 Plate method of Koch, 72 Pleuritis, bacteria in, 527 Plcuro- pneumonia of cattle, bacteria in, 527 Pneumobacillus liquefaciens bovis, 470 Pneumonia, bovine, micrococcus of, 317 bovine, bacilli in, 470 in horses, diplococcus of, 322 Pneumotoxin, 257, 308 Post-mortem examination of animals, 99 Potassium arsenite, germicidal value of, 185 bichromate, germicidal value of, 185 bromide, antiseptic value of, 185 chlorate, germicidal value of, 185 cyanide, antiseptic value of, 185 hydroxide, germicidal value of, 175 iodide, germicidal value of, 185 permanganate, germicidal value of 186 Potato, preparation of, 47 paste, 49 bacillus, 673 cultures upon, 76 rot, bacillus of, 716 Pregl's method of staining bacteria in tissues, 35 Pressure regulator of Moitessier, 88 Products of vital activity, 126 Proteus capsulatus septicus, 429 hominis capsulatus, 427 of Karlinsky, 460 lethalis, 462 mirabilis, 460 septicus, 462 sulfureus, 682 Zenkeri, 462 vulgaris, 457 Pseudodiplococcus pneumonia;, 323 Pseudo-diphtheritic bacillus, 365 Ptomaines, 139 Purpura haamorrhagica, bacilli in, 480. 481. 527 Pus, formation of, 218, 263 Putrefaction, 134 Putrefying material, bacteria in, 585 Putrescin, 140 Pyocyanin, 127 Pyogenic bacteria, 263 Pyoktanin, germicidal value of, 189 Quinine sulphate, germicidal value of, 186 Rabbit septicaemia, bacillus of, 408 Rain water, bacteria in, 559 Ranvier's moist chamber, 63 Rauschbrandbacillus 493 Relapsing fever, spirillum of, 497 Reproduction by binary division, 113 by spores, 114 rapidity of, 114 Rhinoscleroma, bacteria in. 527 bacillus of, 404 Ricin, pathogenic action of, and immunity from 259 Rinderseuche, bacillus of. 408 River water, bacteria in, 559 Robin, classification of , 3 Roll tubes of Von Esmarch, 74 Roth, bacilli of, 479 Rotzbacillus. 396 Rouget, bacillus of, 420 Roux's method of cultivating anaerobic bacteria, 79 Sachs, classification of, 11 Salicylic acid, germicidal value of, 174 Saliva, bacteria of, 575 Saprin, 140 Saprophytes, definition of, 215 Sarcina aurantiaca, 615 alba. 617 Candida, 617 flava, 616 lutea. 616 mobilis, 720 pulmonum, 617 rosea. 616 ventriculi, 617 Sarcinse, morphology of, 22 generic characters, 17 Scarlet fever, bacteria in, 527 Scheurlen's bacillus, 680 Schweinerothlauf , bacillus of, 420 INDEX. 885 Sea water, bacteria iu, 564 Secondary infections, 221 Senile gangrene, bacilli in, 478 Septicaemia, bacilli of, 407 Sewers, bacteria in, 561 Silicate jelly, 46 Silver chloride, germicidal value of , 186 nitrate, germicidal value of, 186 Smear preparations, 26 Smith, capsule bacilli of, 652 Smoke, antiseptic value of, 196 Snow, bacteria in, 559 Sodium borate, germicidal value of, 187 carbonate, germicidal value of, 187 chloride, antiseptic value of, 187 hydroxide, germicidal value of, 176 hyposulphite, antiseptic value of, 187 sulphite, antiseptic value of, 187 Soil, bacteria in, 567, 568, 571, 572 pathogenic, bacteria in, 570 Solid culture media, 41 characters of growth in, 68 Sozins, 260 Sphaerococcus acidi lactici, 604 Spirilla, general characters of, 18 morphology of, 24 non-pathogenic, 694 Spirillum aureum, 700 cholerse Asiatic*, 500 concentricum, 701 denlium, 694 of Finkler and Prior, 509 flavum, 700 rlavescens, 700 lingua, 687 Metschnikovi, 511 of Miller, 702 nasale, 897 Obermeieri, 497 plicatile, 695 rubrum, 701 sanguineum, 696 serpens, 696 of Smith, 701 sputigenum, 694 , tenue, 697 tyrogenum, 511 voluntans, 696 a of Weibel, 698 r of Weibel, 699 Spirochaete Obermeieri, 497 Spirulina, generic characters, 18 Spontaneous generation, 4 Spores, 5 methods of staining, 31 formation of, 114 germination of, 115 resistance of, to heat, 116 thermal death-point of, 149 Staining methods, 25 upon cover glass, 25 bacteria in tissues, 33 sections of gelatin stick cultures, 36 solutions, 29 Staphylococci, characters of, 21 Staphylococcus albus liquef aciens, 607 epidermidis albus, 272 pyogeues albus, 272 pyogenes aureus, 265 ; in osteomye- litis, 270 ; in ulcerative endocardi- tis, 271 pyogenes citreus, 273 viridis flavescens, 601 Steam sterilizers, 53 disinfection by, 203 Sterilization of culture media, 50 by discontinuous heating, 51 by dry heat, 52 by filtration, 56 Sternberg's method of cultivating bac- teria in liquid media, 63 method of cultivating anaerobic bac- teria, 81 Stomach, bacteria in, 580 Streak cultures, 75 Streptococci, classification of, 275 Streptococcus albus, 610 acidi lactici, 610 articulorum, 278 bombycis, 318 of Bpnome, 325 brevis, 611 cadaveris, 611 coli gracilis, 609 conglomerate, 711 coryzje contagiosa? equoruin, 322 erysipelatos, 274 generic characters, 17 giganteus urethras, 610 Havaniensis, 612 lanceolatus Pasteuri, 298 liquefaciens, 613 longus, 274 of mastitis in cows, 321 perniciosus psittacorum, 326 pyogenes, 274 pyogenes in diphtheritic inflamma- tions, 278 pyosepticus, 325 septicus, 318 septicus liquefaciens, 323 vermiformis, 611 Structure of bacteria, 111 Sulphur grains, in genus Beggiatoa, 24 dioxide, antiseptic and germicidal value of, 168 Sulphuric acid, germicidal value of. 172 Sulphurous acid, germicidal value of, 172 Surface of body, bacteria of, 573 Swine pest, bacillus of, 413 plague, bacillus of, 417 Sycosis, bacilli in, 467 Symptomatic anthrax, bacillus of, 493 Syphilis, bacteria in, 528 bacillus of Lustgarten, 402 bacillus of Eve and Lingard, 403 Syringes for injecting bacteria into ani- mals, 95 Taunic acid, germicidal value of, 175 88(3 INDEX. Tartaric acid, germicidal value of, 175 Temperature favorable for growth, 119 Teianin, 142, 484 Tetanotoxin, 485 Tetanus antitoxin, 488 bacillus, 482 toxalbumin, 143 Tetrads, definition of, 22 Texas fever of cattle, bacteria in, 528 Thermal death-point of bacteria, 147 ; of spores, 149 Thermo- regulator of Bohr, 88 electro-magnetic, 90 of Milncke, 90 of Reichert, 88 of Rohrbeck, 86 Thermo-regulators, 86 Thymic acid, germicidal value of, 175 Thymol, antiseptic value of, 196 Thymus bouillon, 230 Tin chloride, germicidal value of, 187 Tobacco smoke, antiseptic value of, 197 Toxalbumins, 142 Trachoma, pus cocci in, 282 Trimethylamine, 140 Tubercle bacillus, cultivation of, 381 chemical products of, 3S6 method of obtaining pure culture of, 383 methods of staining, 30 thermal death-point of, 380 Tuberculin, 144, 3»7 Tuberculosis, bacillus of, 375 diagnosis of, in cows, 389 of fowls, 3^3 transmission of, 391 Turpentine, oil of, antiseptic value, 196 Typhoid bacillus, detection of, in water, 353 fever, bacillus of, 337 fever, bacillus of, in spleen of man, 340 fever, experiments on animals, 341 Typhotoxin, 141, 351 Typhus fever, bacteria iu, 528 Underclothing, bacteria of, 574 Unna's method of filtering agar, 45 Urea, fermentation of, 132 Urine as a culture medium, 39 germicidal action of, 200 Urobacilli of Miquel. 722-724 Urobacillus Duclauxi, 723 Freudenreichi, 723 Maddoxi, 724 Pasteuri 722 Schiitzenbergi, 724 Utpadel, bacillus of, 477 Vaccinia, bacteria in, 528 Vagina, bacteria of, 578 Valerianic acid, germicidal value of, 175 Varicella, bacteria in, 528 Variola, bacteria in, 528 Vibrio Metschnikovi, 511 proteus, 509 rugula, 695 Vibrion septique, 488 Virulence, recovery of, 125 loss of, 124 Viscous fermentation, 133 Vital activity, products of, 126 Water, bacteria in, 553 bacteria, methods of collection, 554, 555 bacteria, counting colonies of, 557 list of bacteria found in, 565, 566 Weigert's method of staining bacteria in tissues, 34 Well water, bacteria in, 561 Whooping cough, bacilli in, 469 Wildseuche, bacillus of, 408 Winogradsky, nitrifying bacillus of, 710 Wurmkrankheit, 406 Yellow fever, bacteria in, 529-531 Ziehl's solution, 29 Ziehl Neelsen method of staining tubercle bacilli, 30 Zinc chloride, germicidal value of, 187 sulphate, germicidal value of, 188 Zooglcya, definition of, 21 Zopf, classification of, 12 8 '""tlK *L12lS"'TJI Wow- « icn renewed. f/$o immediate re — — -^-Y^ a!96S ' .General Library University of Californi Berkeley LD 21-40m-5,'65 (F4308slO)476 YC 884 1 8 472. 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