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ATLAS AND ESSENTIALS 
 
 OF 
 
 BACTERIOLOGY 
 
 BY 
 
 Prof. K. B. LEHMANN 
 
 CHIEF OP THE HYGIENIC INSTITUTE IN WUrZBURG 
 AND 
 
 Dr. EUDOLF NEUMANN 
 
 ASSISTANT IN THE HYGIENIC INSTITUTE IN wtJRZBURG 
 
 WITH 63 CHROMO-LITHOGRAPHIC PLATES, COMPRISING 
 558 FIGURES, AND NUMEROUS ENGRAVINGS 
 
 NEW YORK 
 WILLIAM WOOD AND COMPANY 
 
• • • • 
 
.97 
 
 LIST OF PLATES. 
 
 Plate 1.-— Micrococcus pyogenes a aureus. (Ros.) Lehm 
 and Neura. 
 (Staphylococcus pyogenes aureus. Rosenbach.) 
 Plate 2. —Micrococcus pyogenes y albus. (Ros. ) 
 
 (Staphylococcus pyogenes albus. Rosenbach.) 
 
 Micrococcus pyogenes /3 citreus. (Ros.) 
 (Stai:^ylococcus pyogenes citreus. Rosenbach. ) 
 
 Micrococcus candicans. Flilgge. 
 Plate 3. —Micrococcus agilis. Ali Cohen. 
 
 Micrococcus gonorrhoeae. Neisser, Bumm. 
 
 Streptococcus meningitidis cerebrospinalis. (Weichs. ) 
 Lehm. and Neum. 
 Plate 4. — Micrococcus roseus. (Bumm.) Lehm. and Neum. 
 Plate 5.— Streptococcus lanceolatus. Gamaleia. 
 
 (Diplococcus pneumoniae. A. Fraenkel.) 
 Plate 6. — Streptococcus pyogenes. Rosenbach. 
 Plate 7. — Micrococcus tetragenus. Koch, Gaffky. 
 Plate 8.— Micrococcus luteus. Cohn em. Lehm. and Neum. 
 
 Sarcina pulmonum. Virchow, Hauser. 
 Plate 9. — Sarcina flava. De Bary em. Lehm. and Stuben- 
 
 rath. 
 Plate 10. —Sarcina aurantiaca. Flligge. 
 Plate 11. — Sarcina cervina. Stubenrath. 
 
 Sarcina pulmonum. Virchow. 
 
 Sarcina erythromyxa. Krai. 
 
 Sarcina lutea. Fliigge. 
 
 Sarcina aurantiaca. Flugge. 
 
 Sarcina rosea. Schroeter em. Zimmermann. 
 
 Micrococcus badius. Lehm. and Neum. 
 
 Sarcina canescens. Stubenrath. 
 
 13566 
 
IV 
 
 LIST OF PLATES. 
 
 Plate 12. —Bacterium pneumonias. Friedlander. 
 Plate 13. — Bacterium acidi lactici. Hiippe. 
 
 (Lactic acid bacillus. ) 
 Plate 14.— Bacterium coli commune. Escherich. 
 Plate 15. — Bacterium coli commune. Escherich. 
 Plate 16.— Bacterium typhi. Eberth, Gaffky. 
 
 (Typhoid bacillus.) 
 Plate 17. — Bacterium typhi. Eberth, Gaffky. 
 Plate 18. — Bacterium septicsemise hsemorrhagicae. Hiippe. 
 
 (Chicken cholera, rabbit septicaemia, etc. ) 
 Plate 19. — Bacterium mallei. Loffler. 
 
 (Glanders bacillus.) 
 Plate 20. — Corynebacterium diphtherise. (Loffler.) Lehm. 
 and Neum. 
 (Diphtheria bacillus.) 
 Plate 21. — Bacterium latericium. Adametz. 
 
 Bacterium hsemorrhagicum. (Kolb.) Lehm. and Neum. 
 (Morbus Werlhofii. ) 
 Plate 22. Bacterium putidum. (Flugge.) Lehm. and 
 
 Neum. 
 Plate 23.— Bacterium syncyaneum. (Ehrenb.) Lehm. and 
 Neum. 
 (Bacillus cyanogenes Flugge. Blue milk. ) 
 Plate 24. — Bacterium syncyaneum. (Ehrenb.) Lehm. and 
 Neum. 
 (Bacillus cyanogenes Flugge. Blue milk.) 
 Plate 25. —Bacterium prodigiosum. (Ehrenb.) Lehm. and 
 
 Neum. 
 Plate 26. —Bacterium kiliense. (Breunig and Fischer.) 
 Lehm. and Neum. 
 (Kiel water bacillus. ) 
 Plate 27. — Bacterium janthinum. Zopf. 
 
 Plate 28. — Bacterium fluorescens. (Flugge.) Lehm. and 
 Neum. 
 (Bacillus fluorescens liquefacieus Flugge, ) 
 Plate 29. — Bacterium pyocyaneum. (Flilgge. ) Lehm. and 
 Neum. 
 (Green pus.) 
 Plate 30. — Bacterium Zopfii. Kurth. 
 
LIST OF PLATES. V 
 
 Plate 31. — Bacterium Zopfii. Kurth. 
 
 Plate 32. —Bacterium vulgare ^ mirabilis. (Hauser. ) Lehm. 
 and Neum. 
 (Proteus mirabilis Hauser.) 
 Plate 33. — Bacterium vulgare. (Hauser.) Lehm. and 
 Neum. 
 (Proteus vulgaris Hauser. ) 
 Plate 34. — Bacterium erysipelatus suum. (Loffler.) Mi- 
 gula. 
 (Hog erysipelas. ) 
 
 Bacterium murisepticum. (Flligge.) Migula, 
 
 (Mouse septicaemia. ) 
 Plate 35. — Bacillus megatherium. De Bary. 
 Plate 36.— Bacillus subtilis. F. Cohn. 
 
 (Hay bacillus, ) 
 Plate 37.— Bacillus subtilis. F. Cohn. 
 
 (Hay bacillus.) 
 Plate 38. — Bacillus anthracis. F. Cohn and R. Koch, 
 
 (Anthrax bacillus. ) 
 Plate 39. — Bacillus anthracis. F. Cohn and R. Koch. 
 
 (Anthrax bacillus. ) 
 Plate 40. — Bacillus anthracis. F. Cohn and R. Koch. 
 
 (Anthrax bacillus. ) 
 Plate 41. — Bacillus mycoides. Fliigge. 
 
 (Root bacillus.) 
 Plate 42. Bacillus mycoides. Flugge. 
 
 (Root bacillus.) 
 
 Bacillus butyricus. Hlippe. 
 
 (Butyric acid bacillus.) 
 Plate 43.— Bacillus vulgatus. (Flugge.) Migula. 
 
 (B. mesentericus vulgatus Flugge. Potato bacillus. ) 
 Plate 44.— Bacillus mesentericus. (Flugge.) Lehm. and 
 Neum. 
 
 (B. mesentericus fuscus Fliigge.) 
 Plate 45.— Bacillus tetani. Nicolaier. 
 
 (Tetanus bacillus.) 
 Plate 46.— Bacillus Chauvoei of French writers. 
 
 (Rauschbrand.) 
 Plate 47.— Bacillus oedematis maligni. Koch. 
 
VI 
 
 LIST OF PLATES. 
 
 iberculosi 
 
 is. (Koch.) 
 
 Lehm, and 
 
 (Koch. ) 
 
 Buchner, 
 
 
 (Koch. ) 
 
 Buchner. 
 
 
 (Koch.) 
 
 Buchner. 
 
 
 (Koch.) 
 
 Buchner. 
 
 
 (Koch.) 
 
 Buchner. 
 
 . 
 
 Plate 48. — Mycobacterium 
 Neum. 
 (Tubercle bacillus. ) 
 Plate 49. — Vibrio cholera). 
 
 (Comma bacillus. ) 
 Plate 50. — Vibrio cholera3. 
 
 (Comma bacillus. ) 
 Plate 51. — Vibrio choleras. 
 
 (Comma bacillus.) 
 Plate 52. — Vibrio cholerse. 
 
 (Comma bacillus.) 
 Plate 53. — Vibrio choleraB. 
 
 (Comma bacillus.) 
 
 Vibrio Metschnikovii. Gamaleia. 
 
 Vibrio proteus. Buchner. 
 
 (Vibrio Finkler. Author. ) 
 Plate 54. — Vibrio albensis. Lehm. and Neum. 
 
 (Fluorescent Elbe vibrio. ) 
 Plate 55. — Vibrio danubicus Heider. 
 
 Vibrio berolinensis Rubner. 
 
 Vibrio aquatilis Gunther. 
 Plate 56. — Vibrio proteus. Buchner. 
 
 (Vibrio Finkler. Author.) 
 Plate 57. — Spirillum rubrum. v. Esmarch. 
 
 Spirillum concentricum. Kitasato. 
 Plate 58. — Spirillum serpens. (E. O. Mijller. ) 
 Neum. 
 
 Spirilla from nasal mucus. 
 
 Spirillum undula. Ehrenberg. 
 
 Vibrio spermatozoides. Loffler. 
 
 Spirochsetes of the mucus from the gums. 
 
 Spirillum Obermeieri Virchow. 
 
 (Recurrens spirilla. ) 
 Plate 59.— Leptothrix epidermidis. Biz. 
 Plate 60.— Oospora farcinica. Sauv. and Rad. 
 
 (Farcin de boeuf . ) 
 Plate 61. — Oospora chromogenes. (Gasparini.) 
 Neum. 
 
 (Cladothrix dichotoma Autorum non Cohn. ) 
 
 Lehm. and 
 
 Lehm. and 
 
LIST OF PLATES. 711 
 
 Plate 62.— Oospora bovis. (Harz. ) Sauv. and Rad. 
 
 (Actinomyces. ) 
 Plate 63. — Mycobacterium leprae. (Arm. Hansen.) Lehm. 
 and Neum. 
 
 (Leprosy bacillus.) 
 
 Bacterium influenzae. R. Pfeiffer. 
 
 (Influenza bacillus. ) 
 
 Bacterium pestis (Kitasato, Yersin) . Lehm. and Neum. 
 
 (Plague bacillus. ) 
 
 Bacteria in soft chancre. 
 
LIST OF ABBEEVIATIONS. 
 
 A. H. = Archiv flir Hygiene, Munich. Oldenbourg since 1883. 
 
 A. G. A. = Arbeiten aus dem kaiserlichen Gesundheitsamt, 
 Berlin, Springer, since 1885. 
 
 A. K. = Arbeiten aus dem bakteriologischen Institut der tech- 
 nishen Ilochschule zu Karlsruhe. Edited by Klein and 
 Migula, since 1894. 
 
 A. P. — Annalcs de 1' Institut Pasteur, Paris, Masson, since 1887. 
 
 C. B. = CentralblattfiirBakteriologieuudParasitenkunde, Jena, 
 Fischer. Since 1894 this publication has been divided into 
 two parts : 
 
 C. B., Part I., devoted to medico-hygienic questions. 
 
 C. B., Part II., devoted to zymotechnical, agricultural, and 
 phytopathological studies. 
 
 Z. H. = Zeitschrift fiir Hygiene, Leipsic, Veit, since 1886. 
 
 Fltigge = Fliigge : Die Mikroorganismen, second edition, Leip- 
 sic, 1886. 
 
 Kitt, B, K. = Kitt : Bakterienkunde fiir Tieraerzte, second edi- 
 tion, Vienna, 1893. 
 
 Zimmermann 1 and 2 = 0. E. R. Zimmermann : Die Bakterien 
 unserer Trink- und Nutzwasser, Chemnitz, Part I., 1890; 
 Part II.. 1894. 
 
Tab. 1, 
 
Explanation of Plate 1. 
 
 Micrococcus pyogenes a aureus. Eosenbach, Leh- 
 
 mann and Neumann. 
 
 (Staphylococcus aureus Eos.) 
 
 I. Gelatin stick culture, six days at 22°. 
 II. Agar streak culture, five days at 22°. 
 
 III. Agar stick culture, five days at 22°. Stick canal. 
 
 IV. Agar stick culture, five days at 22°. Surface. 
 V. Agar plate culture (natural size), six days at 22°. 
 
 Superficial and deep colonies. 
 
 VI. Agar plate, six days at 22"". x60. Superficial 
 small colony. 
 
 VII. Gelatin plate (natural size), four days at 22°. 
 Superficial and deep colonies. 
 VIII. Gelatin plate, four days at 22"". x 60. Superficial 
 and deep colonies. 
 
 IX. Potato culture, six days at 22°. 
 X. Microscopical preparation ( X 1, 000) of agar cul- 
 ture, two days at 22°. 
 
 XI. Microscopical preparation; individual cocci, be- 
 fore and after division. xlj500. 
 
 XX. 
 
Explanation of Plate 2. 
 
 Micrococcus pyogenes y albus. Eosenbach. 
 
 (Staphylococcus albus.) 
 
 I. Agar streak culture, four days at 22°. 
 II. Gelatin stick culture, fi-ve days at 22°. 
 
 Micrococcus pyogenes /5 citreus. Eosenbach. 
 (Staphylococcus citreus.) 
 
 III. Agar streak culture, six days at 22°. 
 
 Micrococcus candicans. Flligge. 
 
 IV. Gelatin stick culture, six days at 22°. 
 V. Gelatin plate, eight days at 22°. 
 
 VI. Gelatin plate, six days at 22°. Left side, super- 
 ficial colony ; right side, deep colony, x 50. 
 VII. Potato culture, ten days at 22°. 
 VIII. Microscopical preparation of agar culture (xTOO), 
 two days. 
 
Tab. 2. 
 
 \^ 
 
 LithAlSt V y RpirMiold Vliiiubcii 
 
Tab. 3. 
 
 LiihAnsr.v. K Reirhhold . Miinchm 
 
Explanation of Plate 3. 
 
 Micrococcus agilis. Ali-Cohen. 
 
 I. Gelatin stick culture, six days at 22°. 
 II. Gelatin plate, seven days at 22°. x 50. On right 
 side, superficial colony; on left side, deep- 
 seated colony. 
 
 III. Agar plate, seven days at 22°. Natural size. 
 
 IV. Microscopical preparation (x600) from an agar 
 
 culture two days old. The individual cocci 
 vary greatly in size, and are more irregular than 
 appears in the plate. 
 V. Potato culture, ten days at 22°. 
 
 Micrococcus gonorrhce^. Neisser, Bumm. 
 
 VI. Smear preparation from gonorrhoeal pus. x 1, 000. 
 The large blue cells are pus cells. 
 
 VI. a. Smear preparation from gonorrhoeal pus. x 
 1,200. Semi-schematic. 
 
 VI. h. Diplococcus gonorrhoeae much enlarged. Sche- 
 matic. 
 
 Streptococcus meningitidis cerebrospinalis. 
 (Weichselbaum) Lehmann and Neumann. 
 
 VII. Smear preparation from meningeal exudation ; pus 
 cells with transversely divided diplococci. (Cop- 
 ied from Jaeger: Zschr. f. Hyg., Vol. XIX., 
 PI. VI., Pig. 3.) About X 1,200. 
 
 VIII. Microscopical preparation; pure culture, forma- 
 tion of tetrads. Aboutx 1,200. (Copied from 
 Jaeger: Zschr. f. Hyg., Vol. XIX., PI. VII., 
 Fig. 6.) 
 
 VI. a Yl. b 
 
Explanation of Plate 4. 
 
 Micrococcus roseus. (Bumm) Lehmann and Neumann. 
 
 I. Gelatin stick culture, twenty days at temperature 
 of room. 
 II. Agar streak culture, thirty days at temperature of 
 room. The white reflex on the right side is 
 not always so pronounced. 
 
 III. Agar stick culture, ten days 22°. Puncture canal. 
 
 IV. Agar stick culture, ten days 22°. Surface. 
 
 V. Agar plate, twelve days at 22°. x50. Above, a 
 
 superficial, below, a deep-seated colony. 
 
 VI. Agar plate. More delicate structure. Fourteen 
 
 days at 22°. x 50. Above, a superficial colony, 
 
 below, deep-seated colonies. 
 
 VII. Gelatin plate, eight days at 22°. x 50. Superficial 
 
 and deep colonies. 
 VIII. Microscopical preparation from agar culture (x 
 1,000), three days. The cocci are dividing. 
 IX. Potato culture of diplococcus roseus placed on an 
 anthrax culture, ten days at temperature of 
 room. 
 X. Potato culture, twenty days at temperature of 
 room. 
 
Tab. 4. 
 
Tab. 5. 
 
Explanation of Plate 5. 
 
 Streptococcus lanceolatus. Gamaleia. 
 
 (Diplococcus pneumoniae A. Fraenkel.) 
 
 (Pneumococcus.) 
 
 I. Gelatin stick culture, ten days at 22°. 
 II. Agar streak culture, four days at 37°. 
 
 III. Agar stick culture, four days at 37°. Puncture 
 
 canal. 
 
 IV. Agar stick culture, four days at 37°. Surface. 
 V. Agar plate, three days at 37°. Natural size. 
 
 VI. Agar plate, three days at 37°. x50. Superfi- 
 cial colony. The dark colony is situated near 
 the surface. 
 VII. Agar plate, three days at 37°. x50. Deep-seated 
 colonies. 
 VIII. Gelatin plate, eight days at 22°. The upper col- 
 ony superficial, the two lower ones deep seated. 
 IX. Smear preparation from pneumonia sputum. 
 
 X 1,000. 
 X. Pure culture from agar plate three days old. x 
 1,000. 
 XI. Microscopical preparation. 
 
 (a) Diplococci, single and arranged in chains. 
 High magnifying power. 
 
 (b) Diplococci surrounded with gelatinous cap- 
 sule. 
 
 ; . ^ ! 
 
 I • 
 
 XI. 
 
Explanation of Plate 6. 
 
 Streptococcus pyogenes. Eosenbach. 
 
 I. Agar streak culture, ten days at 37°. 
 II. Gelatin stick culture, six days at 22°. The col- 
 ony is not often found in such a vigorous state. 
 
 III. Agar stick culture, six days at 37°. Puncture 
 
 canal. 
 
 IV. Agar stick culture, six days at 37°. Surface. 
 V. Gelatin plate, six days at 22°. 
 
 VI. Gelatin plate, six days at 22°. x70. Somewhat 
 abnormal shape with ragged edges. The larger 
 colonies superficial, the smaller ones deep. 
 VII. Gelatin plate, six days at 22°. x 70. More fre- 
 quent form. Upper one superficial, lower one 
 deep. 
 VIII. Agar plate, eight days at 37°. x50. Larger colony 
 superficial, smaller colonies deep. 
 
 IX. Microscopical preparation from a bouillon cul- 
 ture, two days at 37°. X 700. The individual 
 cocci are usually more regularly rounded. 
 X. Microscopical preparation from an agar culture, 
 two days. Shorter chains. xljOOO. 
 
 XI. Microscopical preparation. Called streptococcus 
 conglomeratus. Smear preparation from the 
 blood of the spleen from a case of scarlatina. 
 Copied from Kurth (Kaiserl. Gesundheits- 
 amt, Vol. VII.). 
 XII. Streptococci chains, before and during division. 
 High magnifying power. 
 
 V 
 
 V 
 
 XII. 
 
Tab. 6. 
 
 
 
 ^^^^^^_ 
 
 
 
 .,.: 
 
 
 
 # 
 
 
 e 
 
 
 
 e 
 
 i 
 vn. 
 
 
 •* 
 
 1 
 
 VI. 
 
 LiiiuuiiL.v. r itcicimuKi, Muartiea 
 
Tab. 7, 
 
 LiilijViist V Y RcirhhnUl.Miifirheti 
 
Explanation of Plate 7. 
 
 Micrococcus tetra genus. Koch, Gaffky. 
 
 I. Agar streak culture, five days at 37°. 
 II. Gelatin stick culture, ten days at 22°. Puncture 
 canal. The " nail-head" shape is characteristic. 
 
 III. Gelatin stick culture, ten days at 22°. Surface. 
 
 The color is too brown in the plate ; should have 
 been white. 
 
 IV. Agar stick culture, six days at 37°. The puncture 
 
 does not always turn out so vigorous. 
 V. Agar stick culture, six days at 37°. Surface. 
 VI. Agar plate, five days at 37°. Natural size. 
 VII. Gelatin plate, eight days at 22°. In nature the 
 
 colonies are pure white. Natural size. 
 VIII. Gelatin plate, eight days at 22°. x60. The larger 
 colony is superficial, the smaller ones are deep. 
 IX. Microscopical preparation from an agar culture 
 (x800) two days old. We do not always find 
 tetrads alone. There are numerous individual 
 cocci. 
 X. Potato culture, seven days at 37°. 
 XI. Microscopical appearances. Tetrads before, dur- 
 ing, and after division highly magnified. 
 
 XI. 
 
Explanation of Plate 8. 
 
 Micrococcus LUTEus. Cohen with Lehm. andNeum. 
 I. Gelatin stick culture, six days Sit 22°. 
 II. Gelatin plate, three days at 22°. x 50. On right 
 side superficial, on left side deep-seated colony. 
 
 III. Microscopical preparation (x 1,000) from an agar 
 
 plate two days old. The micrococci are often 
 aggregated into tetrads. 
 
 IV. Agar plate (natural size), five days at 22°. 
 
 The colonies are sometimes more yellow. 
 V. Potato culture, six days at 22°. Sometimes, has a 
 dull lustre. 
 
 Sarcina pulmonum. Virchow, Hauser. 
 
 VI. Gelatin stick culture, twenty days at 22°. In 
 reality the puncture is grayer in color. 
 VII. Agar streak, twenty days at 22°. 
 VIII. Gelatin plate, twenty days at 22°. On the right, 
 superficial colony ; on the left, deep-seated one. 
 IX. Potato culture, twenty days at 22°. 
 
Tab. 8. 
 
Tab. 9. 
 
Explanation of Plate 9. 
 
 Sakcina flava. De Bary with Lehm. and Stubenrath. 
 
 I. Gelatin stick culture, ten days at 22°. 
 II. Agar streak culture, six days at 22°. 
 
 III. Agar stick culture, six days at 22°. Puncture 
 
 canal. 
 
 IV. Agar stick culture, six days at 22°. Surface. 
 V. Gelatin plate, five days at 22°. Natural size. 
 
 VI. Gelatin plate, five days at 22°. x60. Superficial 
 colony. 
 VII. Agar plate, six days at 22°. Natural size. 
 VIII. Agar plate, six days at 22°. x60. Upper colony 
 superficial, lower colony deep seated. 
 IX. Potato culture, ten days at 22°. 
 X; Microscopical preparation. Pure culture from an 
 agar plate, x 1? 000. Stained with f uchsin and 
 decolorized with acetic acid. 
 XI. Microscopical preparation. Pure culture from 
 bouillon. Unstained. xljOOO. 
 XII. Sarcina in the shape of bales (regular combination 
 
 of individual packages). 
 XIII. Sarcina in heaps of packages (irregular mass of 
 single regular or irregular packages). 
 
 xn. XIII. 
 
Explanation of Plate 10. 
 
 Sarcina aurantiaca. riiigge. 
 
 I. Gelatin stick culture, six days at 22°. 
 II. Agar streak culture, five days at 22°. The color 
 is not so red in all cases, usually it is a bright 
 orange. Likewise in the agar stick and potato 
 cultures. 
 
 III. Agar stick culture, six days at 22°. Puncture 
 
 canal. 
 
 IV. Agar stick culture, six days at 22°. Surface. 
 
 V. Gelatin plate, five days at 22°. Natural size. 
 The gray rim around the colony indicates the 
 depression. 
 VI. Gelatin plate, five days at 22°. x60. A young 
 colony. The gray ring indicates the zone of 
 depression. 
 VII. Agar plate, five days at 22°. Natural size. 
 VIII. Agar plate, five days at 22°. x 60. Upper colony 
 superficial, lower colonies deep seated. The 
 superficial colonies are usually opaque toward 
 the middle. 
 IX. Potato culture eight days old. 
 X. Microscopical preparation. Pure culture of agar. 
 X 1,000. Colored with fuchsin and decolor- 
 ized with acetic acid. 
 XI. Microscopical preparation. Pure culture from 
 bouillon. xl?O0O. Unstained. Semi-sche- 
 matic. 
 
 10 
 
Tab. 10. 
 
 i. — - 
 
 V 
 
 1 
 
 
 tf 
 
 Lz 
 
 ' vu- 
 
 
 IX 
 
Tab. 11 
 
Explanation of Plate 11. 
 
 Sarcin^ Diverse. 
 
 I. Sarcina cervina Stubenrath. Agar streak culture, 
 fifteen days at 22'', isolated from gastric con- 
 tents. 
 II. Sarcina pulmonum Virchow. Agar streak cul- 
 ture, fifteen days at 37°. 
 
 III. Sarcina erythromyxa Krai. Agar streak culture, 
 
 thirty days at 22°, isolated from beer. 
 
 IV. Sarcina lutea Fliigge. Agar streak culture, ten 
 
 days at 22°, isolated from stomach. 
 V. Sarcina aurantiaca Fliigge. Agar streak culture, 
 
 ten days at 22°, isolated from dough. 
 VI. Sarcina rosea Schroeter and Zimmermann. Agar 
 streak culture, twenty-five days at 22°, isolated 
 from " weissbeer. " 
 VII. Micrococcus badius Lehman and Neumann. Agar 
 streak culture, fifteen days at 22°, isolated from 
 the atmosphere. 
 VIII. Sarcina canescens Stubenrath. Agar streak cul- 
 ture, twenty days at 22""^ isolated from stomach. 
 
 11 
 
Explanation of Plate 12. 
 
 Bacterium pneumonia. Friedlander. 
 
 (Friedlander's pneumonia bacillus.) 
 
 I. Agar streak culture, four days at 22°. 
 II. Gelatin stick culture, ten days at 22°. 
 
 III. Agar stick culture, four days at 22°. Puncture 
 
 canal. 
 
 IV. Agar stick culture, four days at 22°. Surface. 
 V. Gelatin plate, three days at 22°. Natural size. 
 
 VI. Agar plate, two days at 22°. x60. The brown, 
 
 whetstone-shaped colony is deep seated. 
 VII. Gelatin plate, three days at 22°. x 50. Above, 
 
 superficial colony ; below, deep-seated one. 
 VIII. Agar plate, four days at 22°. Natural size. The 
 
 delicate gray colonies and the smallest ones are 
 
 deep seated. One colony has been colored too 
 
 yellow. 
 IX. Microscopical preparation. Pure culture ( x 800) 
 
 from an agar plate. Stained with fuchsin. 
 X. Microscopical preparation. Smear preparation 
 
 from sputum. X 800. Fuchsin stain. 
 XI. Potato culture, six days. 
 
 13 
 
Tab. 12. 
 
Tab. 13. 
 
Explanation of Plate 13. 
 
 Bacterium acidi lactici. Fltigge. 
 
 (Lactic-acid bacillus. ) 
 
 I. Gelatin stick culture, five days at 22°. In nature 
 the puncture canal is a little whiter. 
 II. Agar streak culture, five days at 22°. 
 
 III. Agar stick culture, three days at 22°. Puncture 
 
 canal. 
 
 IV. Agar stick culture, three days at 22°. Surface. 
 V. Agar plate, three days at 22°. Natural size. 
 
 VI. Agar plate, three days at 22°. x 50. Upper col- 
 ony superficial, lower ones deep seated. Vide 
 PI. 14, VII. 
 VII. Gelatin plate, two days at 22°. 
 VIII. Gelatin plate, two days at 22°. x50. Upper col- 
 ony superficial, lower colonies deep seated. 
 The superficial colony may vary extremely in 
 its growth. Vide PL 15, IV., VII. ; PI. 16, 
 IX., VIII.; PI. 17, I., II. 
 IX. Microscopical preparation. Pure culture from an 
 
 agar colony, x 800. 
 X. Potato culture, six days at 22°. The air bubbles 
 on the surface often cover it completely. 
 
 13 
 
Explanation of Plate 14. 
 
 Bacterium coli commune. Escherich. 
 
 I. Gelatin stick culture, ten days at 22°. 
 II. Gelatin streak culture, four days at 22°. In na- 
 ture, transparent and iridescent like mother-of- 
 pearl. VideTl. 16, VI. 
 
 III. Agar streak culture, four days at 22°. Vide PI. 
 
 16, V. 
 
 IV. Agar stick culture, two days at 22°. Puncture 
 
 canal. 
 V. Agar stick culture, two days at 22°. Surface. 
 
 VI. Agar plate, four days at 22°. x60. Deep-seated 
 colonies. VideVL 13, VI. 
 
 VII. Agar plate, four days at 22°. x60. A part of a 
 superficial colony. During growth occasionally 
 exhibits forms like bacillus acidi lactici. Vide 
 PI. 13, VI. ; PI. 17, v., VI. ; PI. 18, IV. ; PI. 
 12, VIII. 
 VIII. Agar plate, three days at 22°. Natural size. 
 
 IX. Potato culture, five days at 22°. May also ap- 
 pear of a lighter or darker color. 
 X. Bacteria with long flagella from bacterium bras- 
 sicse acidse. xljOOO. Stained according to 
 Loffler's method. 
 
 XI. Bacteria with flagella, from the bacterium of pig- 
 eon diphtheria. xljOOO. Stained by Loffler's 
 method. 
 XII. Bacteria with one flagellum, rarely with two fla- 
 gella, from bacterium of the deer plague. 
 X 1,000. Stained by Loffler's method. 
 
Tab. 14. 
 
 viu. 
 
Tab. 15. 
 
 VTl 
 
 VTII 
 
Explanation of Plate 15. 
 
 Bacterium coli commune. Escherich. 
 
 I. Gelatin plate, eight days at 22°. x60. Coli culti- 
 vated from pus. Deep-seated colonies. Ab- 
 normal shapes. 
 II. Gelatin j)late, four days at 22°. Natural size. 
 
 III. Gelatin plate, one day at 22°. x 90. Superficial 
 
 colony. Vide PL 13, VIII. ; PL 16, VIII. 
 
 IV. Gelatin plate, four days at 22°. x 60. Superficial 
 
 colony. Vide PL 16, IX. ; PL 17, I., II. 
 V. Gelatin plate, four days at 22°. x60. Deep- 
 seated colony. 
 VI. Gelatin plate, ten days at 22°. x90. Superficial 
 
 colony. 
 VII. Gelatin plate, ten days at 22°. x90. Superficial 
 colony. 
 VIII. Microscopical preparation. Pure culture from an 
 agar plate, x 500. 
 IX. Bacteria of various kinds of coli. x 1,000. Great 
 differences in size. 
 
 • /• V 
 
 IX. 
 
 16 
 
Explanation of Plate 16. 
 
 Bacterium typhi. Eberth, Gaffky. 
 
 (Typhoid bacillus.) 
 
 I. Agar stick culture, three days at 22°. Puncture 
 
 canal. 
 II. Agar stick culture, three days at 22°. Surface. 
 
 III. Gelatin stick culture, eight days at 22°. Punc- 
 
 ture canal. 
 
 IV. Gelatin stick culture, eight days at 22°. Surface. 
 y. Agar streak culture, four days at 22°. Vide PI. 
 
 14, III. 
 VI. Gelatin streak culture, three days at 22°. Vide 
 
 PI. 14, II. 
 VII. Gelatin plate, one and a half days at 22°. Deep- 
 seated colony. Vide PI. 15, V. ; PI. 13, VIII. 
 VIII. Gelatin plate, one and a half days at 22'^'. Super- 
 ficial colony. Vide PI. 15, III. ; PI. 13, VIII. 
 IX. Gelatin plate, four days at 22°. Superficial col- 
 ony. Vide PI. 15, IV., VII. 
 
 16 
 
Tab. 16. 
 
 M 
 
 va 
 
 □ □ 
 
 L\. 
 
 VTU 
 
Tab. 17. 
 
Explanation of Plate 17. 
 
 Bacterium typhi. Eberth, Gaffky. 
 
 (Typhoid bacillus.) 
 
 I. Gelatin plate, eight days at 22°. x90. Superfi- 
 cial colony. Vide PI. 15, VII., VI. 
 II. Gelatin plate, eight days at 22°. xl50. Su- 
 perficial colony. 
 
 III. Gelatin plate, four days at 22°. Natural size. 
 
 IV. Agar plate, four days at 22°. Natural size. 
 
 V. Agar plate, four days at 22°. x60. Deep-seated 
 
 colonies. 
 VI. Agar plate, four days at 22°. x60. Superficial 
 colonies. 
 VII. Potato culture, five days at 22°. 
 VIII. Microscopical preparation. Pure culture from 
 agar plate, x 1,000. 
 IX. Microscopical preparation. Bacilli with flagella. 
 Copied from Fraenkel and Pf eiffer : " Atlas d. 
 Bakterienkunde," Plate 54, Fig. 111. 
 X. Microscopical preparation. Long thread, thickly 
 studded with flagella. x 1, 500. Loffier' s stain. 
 XI. Microscopical preparation of bacterium typhi 
 murium Loftier, with flagella and capsule. 
 X 1,500. Stained by Loffler's method. 
 
 17 
 
Explanation of Plate 18. 
 
 Bacterium sEPTic^MiiE hemorrhagica. Htippe. 
 
 (Fowl cholera, rabbit septicaemia, etc.) 
 
 I. Gelatin stick culture, seven days at 22°. 
 
 II. Agar streak culture, seven days at 22°. 
 
 III. Agar plate, five days at 22°. Natural size. 
 
 IV. Agar plate, five days at 22°. x60. Superficial 
 
 colony. Vide PI. 17, YI. ; 14, YII. ; 13, VI. 
 V. Agar plate, five days at 22°. x60. Deep-seated 
 colonies. 
 VI. Gelatin plate, five days at 22°. Natural size. 
 VII. Gelatin plate, five days at 22°. x90. Deep-seated 
 colonies. 
 VIII. Gelatin plate, five days at 22°. x 90. Superficial 
 colony. Vide PL 17, I.; PL 16, IV., VIII.; 
 PL 15, IV., III., VII. ; PL 13, VIII. 
 IX. Microscopical preparation, x 1,000. Pure culture 
 from an agar plate. 
 X. Individual bacteria. Highly magnified. Sche- 
 matic. 
 
 
 18 
 
Tab. 18. 
 
Tab. 19. 
 
Explanation of Plate 19. 
 
 Bacterium mallei. Loffler. 
 
 (Glanders.) 
 
 I. Gelatin stick culture, six days at 22°. 
 II. Agar streak culture, six days at 37°. The mid- 
 dle white line is not always so pronounced. 
 
 III. Agar stick culture, three days at 37°. Puncture 
 
 canal. 
 
 IV. Agar stick culture, three days at 37°. Surface. 
 V. Gelatin plate, five days at 22°. Natural size. 
 
 VI. Gelatin plate, four days at 22°. x60. Upper 
 
 colony superficial, lower colonies deep seated. 
 VII. Agar plate, two days at 22°. x60. Upper col- 
 ony superficial, lower colonies deep seated. 
 VIII. Microscopical preparation. Pure culture. X 800. 
 Puchsin stain. 
 IX. Potato culture, two days at 37°. 
 X. Potato culture, twenty days at 37°. 
 XI. Individual bacteria. Highly magnified. In some 
 places the staining fluid is absorbed poorly or 
 not at all. 
 
 XI. 
 
 X» 
 
Explanation of Plate 20. 
 
 CoRYNEBACTERiuM DIPHTHERIA. (Lo£9.er) Lehmann 
 and Neumann. 
 
 (Diphtheria bacillus.) 
 
 I. Glycerin-agar stick culture, twenty days at 22°. 
 
 Puncture canal. 
 II. Glycerin-agar streak culture, eight days at 22°. 
 III. Glycerin-agar stick culture, twenty days at 22°. 
 
 Surface. 
 lY. Glycerin-agar plate, eight days at 22°. x60. 
 
 Deep and superficial colonies. 
 V. Glycerin-agar plate, forty days at 22°. x 60. On 
 the left side deep-seated colonies ; on the right 
 side deep and superficial colonies. 
 VI. Glycerin-gelatin plate, twenty days at 22°. 
 
 Natural size. Superficial and deep colonies. 
 VII. Glycerin-gelatin plate, twenty days at 22°. x60. 
 On the left side deep-seated colonies; on the 
 right side superficial ones. 
 VIII. Potato culture, fourteen days at 22°. 
 IX. Microscopical preparation. Pure culture from 
 
 bouillon two days old. x TOO. 
 X. Microscopical preparation. Pure culture from 
 bouillon. Involution forms. About x 1,200. 
 XI. Individual bacteria. Highly magnified. Sche- 
 matic. 
 
 20 
 
Tab. 20. 
 
 ua^ 
 
 vm 
 
 a 
 
Tab. 21 
 
 YE. 
 
 Vffl. 
 
 LitliJ\nxt.y. F. ReichlioUi , Miiiichf 
 
Explanation of Plate 21. 
 
 Bacterium latericium. Adametz. 
 
 I. Agar streak culture, seven days at 22°. 
 II. Gelatin stick culture, fourteen days at 22°. 
 
 III. Gelatin plate, seven days at ^l"" . x60. Deep- 
 
 seated colonies on the right, superficial on the 
 left. 
 
 IV. Potato culture, thirty days at 22°. Natural size. 
 V. Agar plate, seven days at 22°. Superficial colony 
 
 on the right, deep one on the left. 
 VI. Microscopical preparation. Pure culture from 
 agar twenty-four hours. About x 800. 
 
 Bacterium h^morrhagicum. (Kolb) Lehm. and 
 Neum. (Morbus Werlhofii.) 
 
 VII. Microscopical preparation. Pure culture from 
 bouillon three days old. (Copied from Kolb : A. 
 G., Vol. VII., PI. II., Figs, 1 and 2). 
 VIII. Smear preparation from the liver of a dog. (Copied 
 from Kolb: Lo., Vol. VIL, PL in., Fig. 8.) 
 
 21 
 
Explanation of Plate 22. 
 
 Bacterium putidum (Fliigge) Lehm. and Neum. 
 
 (Bacterium fluorescens non-liquef aciens Autor. ) 
 
 I. Gelatin stick culture, three days at 22°. 
 II. Gelatin plate, twenty -four hours at 22"". x90. 
 Deep-seated colony. 
 
 III. Gelatin plate, twenty -four hours at 22°. x90. 
 
 Superficial colony. Vide PI. 13, VIII. ; PL 15, 
 III. 
 
 IV. Gelatin plate, four days at 22°. Natural size. 
 
 Appearance of colonies upon a dark background. 
 V. Potato culture, four days at 22"". Natural size. 
 
 Vide PI. 14, IX. 
 VI. Microscopical preparation. Pure culture from 
 gelatin plate, x 800. Ordinarily threads are 
 formed on agar. 
 VII. Agar plate, eight days at 22°. Natural size. 
 Appearance of the colony on a white back- 
 ground. 
 VIII. Agar plate, three days at 22°. x60. 
 IX. Bacteria with one flagellum, more rarely two fla- 
 gella. X 1,000. Stained according to Loffler's 
 method. 
 
 J^ 
 
 EL 
 
 23 
 
Tab. 22. 
 
 Lithj\iist,v. ¥ Reiohhold . Miinrhwi 
 
Tab. 23. 
 
 p 
 
 w 
 
 ''«s '^' 
 
 [ 
 
 
 vn. 
 
 ItthJtaslv. F Reichhold . Miinohen 
 
Explanation of Plate 23. 
 
 Bacterium syncyaneum. (Ehrenb.) Lehm. and Neum. 
 
 (Bacillus cyanogenes Fliigge ; blue milk. ) 
 
 I. -III. Gelatin stick cultures, six to ten days at 22°. 
 Other shades of color are also observed. 
 IV. Agar stick culture, ten days at 37°. 
 V. Bouillon culture, four days at 37°. 
 VI. Milk culture, three days at 37°. upon non-steril- 
 ized milk. 
 VII. Microscopical preparation. Pure culture from 
 
 agar plate, x 800. 
 VIII. Microscopical preparation. Pure culture. Fla- 
 gella stained with Loffler^s mordant. 
 IX. Bacteria with flagella ; one or more at a pole, x 
 1,000. Stained by Loffler's method. 
 
 H 
 
 IX. 
 
 23 
 
Explanation of Plate 24. 
 
 Bacterium syncyaneum. (Ehrenb.) Lelun. and Neum. 
 
 (Bacillus cyanogenes Fliigge ; blue milk. ) 
 
 I.-III. Potato cultures, three to ten days at 22°. 
 
 Potatoes of different kinds inoculated with the 
 
 same culture. The differences in color may be 
 
 still more manifold. 
 
 IV. Agar plate, three days at 22°. Natural size. 
 
 V. Agar plate, three days at 22° . x 60. On the right 
 
 deep-seated, on the left superficial colonies. 
 VI. Gelatin plate, three days at 22°. Natural size. 
 VII. Gelatin plate, eight days at 22°. Natural size. 
 View of the colonies against a white back- 
 ground. 
 VIII. Gelatin plate, three days at 22°. x60. Above, 
 superficial j below, deep-seated colonies. 
 
 34 
 
Tab. 24. 
 
 \TiT 
 
 Aiisi \ y i;i'irhhol(i.Miinfhi'i 
 
Tab. 25. 
 
 !;. ;.l,!'<v|,| M„...|.cM 
 
Explanation of Plate 25. 
 
 Bacterium prodigiosum. (Ehrenb.) Lehm. and Neum. 
 
 I. Gelatin stick culture, one day at 22". 
 II. Agar streak culture, four days at 22"^. 
 
 III. Agar stick culture, four days at 22°. Puncture 
 
 canal. 
 
 IV. Agar stick culture, four days at 22°. Surface. 
 V. Agar plate, two to four days at 22°. Natural 
 
 size. Colonies with, and without development 
 of coloring matter. 
 VI. Agar plate, eight days at 22°. x 60. Superficial 
 colonies reddish, deep ones yellowish. 
 VII. Gelatin plate, two days at 22°. x60. Superficial 
 colony just beginning to sink. 
 VIII. Gelatin plate, two days at 22°. Natural size. 
 IX. Potato culture, eight days at 22°. Typical, with 
 
 metallic reflex on the surface. 
 X. Potato culture, eight days at 22°. Atypical, white 
 deposit. 
 XI. Microscopical preparation. Pure culture from 
 agar. x800. Fuchsin stain. 
 XII. Bacteria with several flagella. x 1? 000. Stained 
 by Lofiler's method. 
 
 XII. 
 
 25 
 
Explanation of Plate 26. 
 
 Bacterium kiliense. (Breunig and Fischer) Lehin. 
 and Neum. 
 
 (Kiel water bacillus.) 
 
 I. Agar streak culture, four days at 22'^. 
 II. Gelatin stick culture, four days at 22"". Colony 
 without development of coloring matter. 
 
 III. Gelatin plate, five days at l^"". Natural size. 
 
 Colonies with and without development of color- 
 ing matter. 
 
 IV. Gelatin plate, five days at 22"". x 60. Superficial 
 
 colony. 
 V. Gelatin plate, five days at 22°. x 60. Deep-seated 
 colony. 
 
 VI. Agar, plate, five days at 22°. Natural size. Col- 
 ored and uncolored, superficial and deep-seated 
 colonies. 
 
 VII. Agar plate, five days at 22°. x60. Uncolored 
 colonies. On the right side, superficial; on 
 the left, deep seated. 
 VIII. Agar plate, five days at 22"". x 60. Colored colo- 
 nies. On the right, superficial; on the left, 
 deep seated. 
 
 IX. Microscopical preparation, x 1,000. Pure cul- 
 ture from agar plate. Fuchsin stain. 
 X. Potato culture, five days at 22°. 
 
 XI. Bacteria with several flagella. x 1,000. Stained 
 by Loffler's method. 
 
 r 
 
 XI. 
 
 26 
 
Tab. 26, 
 
Tab. 27. 
 
Explanation of Plate 27. 
 
 Bacterium janthinum. Zopf. 
 
 I. Gelatin stick culture, ten days at ordinary tem- 
 perature. 
 II. Agar streak culture, six days at ordinary tempera- 
 ture. The white borders at the sides also 
 become violet after prolonged standing. 
 
 III. Agar stick culture, seven days at ordinary tem- 
 
 perature. Puncture canal. 
 
 IV. Agar stick culture, seven days at ordinary tem- 
 
 perature. Surface. 
 V. Agar plate culture (x60), four days at ordinary 
 temperature. Superficial and deep colony. 
 Within the former the original colony is still 
 visible. 
 VI. Agar plate culture, eight days at ordinary tem 
 perature. Natural size. The colonies often 
 take on a dark violet color. 
 VII. Gelatin plate culture, six days at ordinary tem- 
 perature. Natural size. The blue zones are 
 not always so deeply colored. 
 VIII. Gelatin plate culture, six days at ordinary tem- 
 perature. X 60. The smaller colony is near 
 the surface, the larger one upon the surface. 
 IX. Microscopical preparation, from a five days' agar 
 
 culture. X TOO. 
 X. Potato culture, six days at ordinary temperature. 
 XI. Bacteria with flagella. x 1,000. Loffler's stain. 
 XII. Bacteria with flagella, from a culture obtained 
 from Sweden, xl;000. 
 
 % 
 
 XL XIL 
 
 27 
 
Explanation of Plate 28. 
 
 Bacterium fluorescens. Fltlgge. 
 
 (Bacillus fluorescens liquefaciens. Fltigge.) 
 
 I. Gelatin stick culture, two days at 22°. 
 
 II. Gelatin stick culture, eight days at 22°. 
 
 III. Agar streak culture, three days at 22°. 
 
 IV. Agar stick culture, four days at 22°. 
 
 V. Gelatin plate, two days at 22°, Part of a super- 
 ficial colony. X 90. 
 VI. Agar plate, twenty -four hours at 22°. x60. e, 
 superficial; i, deep-seated colony. 
 VII. Gelatin plate, three days at 22°. Natural size. 
 VIII. Microscopical preparation. Pure culture from 
 agar plate, x 800. 
 IX. Potato culture, four days at 22°. Natural size. 
 Vide PI. 22, V. ; PI. 14, IX. 
 X. Bacteria with flagella, usually one, more rarely 
 two or more, x 1,000. Loffler's stain. 
 
 X 
 
 28 
 
Tab. 28. 
 
Tab. 29. 
 
Explanation of Plate 29. 
 
 Bacterium pyocyaneum. (Fliigge) Lehm. and Neum. 
 
 (Green pus.) 
 
 I. Gelatin stick culture, three days at 22°. 
 II. Agar streak culture, two days at 37°. 
 
 III. Gelatin plate, two days at 2'!''. x60. Deep- 
 
 seated colonies and some immediately beneath 
 the surface, in younger and older stages. 
 
 IV. Gelatin plate, five days at 22"". x 60. Part of a 
 
 superficial colony. 
 V. Gelatin plate, two days at 22°. Natural size. 
 VI. Agar plate, two days at 37°. Natural size. 
 VII. Agar plate, two days at 37°. x60. Below, deep- 
 seated ; above, superficial colonies. 
 VIII. Potato culture, three days at 37°. Natural size. 
 IX. Microscopical preparation. Pure culture from 
 agar plate, x 800. 
 X. Bacteria with one, more rarely two polar flagella. 
 Xl,000. Loffler's stain. 
 
 29 
 
Explanation of Plate 30. 
 
 Bacterium zopfii. Kurth. 
 
 I. Gelatin stick culture, six days at 22°. 
 II. Gelatin streak culture, thirty-six hours at 37°. 
 In reality of a gray transparent color. 
 III. Agar stick culture, six days at 22°. Puncture. 
 IV. Agar stick culture, six days at 22°. Surface. 
 
 V. Gelatin plate, seven days at 22°. Natural size. 
 VI. Gelatin plate, thirty-six hours at 22°. Natural 
 
 size. 
 VII. Gelatin plate, twenty-four hours at 22°: x 90. 
 Thread-like part of the colony. Deeply situ- 
 ated. 
 VIII. Gelatin plate, twenty-four hours at 22°. x60. 
 Superficial colony. Vide PI. 32, VIII. 5 PL 
 33, VII. 
 
 30 
 
Tab. 30. 
 
 vni 
 
 VTl. 
 
Tab 31 
 
Explanation of Plate 31. 
 
 Bacterium zopfii. Kurth. 
 
 I. Gelatin plate, eight days at 22°. x90. Border 
 
 of a colony. 
 II. Microscopical preparation. xl?000. Pure cul- 
 ture from agar plate. Stained with fuchsin. 
 
 III. Agar plate, twenty -four hours at 37". x60. 
 
 Superficial colony surrounded by innumerable 
 bacteria which have wandered away. 
 
 IV. Agar plate, twenty -four hours at 37°. Natural 
 
 size. 
 V. Agar plate, twelve hours at 37°. Deep-seated 
 
 and superficial colony. 
 VI. Agar plate, four days at 22°. Deep-seated colony. 
 VII. Gelatin plate, eight days at 22°. Sausage -shaped 
 
 forms of a deep-seated colony. 
 VII. Bacteria with numerous flagella. xljOOO. Loff- 
 ler's stain. 
 
 81 
 
Explanation of Plate 32. 
 
 Bacterium vulgare /5 mirabilis. (Hauser) Lehm. 
 and Neum. 
 
 (Proteus mirabilis Hauser.) 
 
 I. Agar stick culture, two days at 22°. Puncture 
 
 canal. 
 II. Agar stick culture, two days at 22"". Surface. 
 
 III. Gelatin stick culture, six days at 22°. 
 
 IV. Agar streak culture, two days at 22''. 
 
 V. Agar plate, seven days at 22°. Natural size. 
 VI. Agar plate, seven days at 22^^. x60. Above, 
 superficial; below, deep-seated colony. 
 VII. Gelatin plate, two days at Y,22' 
 seated colonies. 
 VIII. Gelatin plate, two days at 22°. 
 ficial colony. 
 IX. Potato culture, eight days Sit22°. 
 X. Microscopical preparation. Pure 
 two days old. x800. 
 
 . 60. 
 
 Deep- 
 
 X60. 
 
 Super- 
 
 Natural size. 
 
 agar 
 
 culture, 
 
 32 
 
Tab. 32. 
 
 lu 
 
 vni. 
 
 LuluAnst y. Y Rcirhhold . Miinrheii 
 
Tab. 33. 
 
Explanation of Plate 33. 
 
 Bacterium vulgare Lehm. and Neum, 
 
 (Proteus vulgaris Hauser.) 
 
 I. Gelatin stick culture, twenty-four hours at 22°. 
 II. Agar streak culture, thirty -six hours at 22°. 
 
 III. Agar plate, thirty-six hours at 22°. Natural size. 
 
 IV. Agar plate, four days at 22°. x60. Above, 
 
 superficial ; below, deep-seated culture. 
 V. Gelatin plate, thirty -six hours at 22°. Natural 
 size. 
 VI. Gelatin plate, thirty-six hours at 22°. x60. 
 Eight side, superficial j left side, deep-seated 
 colonies. The lower one, emerging on the sur- 
 face, is beginning to liquefy. 
 VII. Gelatin plate, three days at 22°. x60. Deep- 
 seated colony. Zoogloea form, like bacterium 
 Zopfii. 
 VIII. Microscopical preparation, x 800. Pure agar cul- 
 ture. Fuchsin stain. 
 IX, Bacteria with numerous flagella. x 1^000. 
 
 33 
 
Explanation of Plate 34. 
 
 Bacterium erysipelatos suum. Migula. 
 (Hog erysipelas.) 
 I. Gelatin stick culture, five days at 22°. 
 
 Bacterium murisepticum. Migula. 
 (Mouse septicaemia.) 
 
 II. Agar streak culture, four days at 22°. 
 
 III. Gelatin stick culture, four days at 22°. 
 
 IV. Agar stick culture, four days at 22°. Surface. 
 V. Gelatin plate, four days at 22°. Natural size. 
 
 VI. Gelatin plate, four days at 22°. x60. Super- 
 ficial colony. 
 VII. Agar plate, four days at 22°. x60. Eight side, 
 superficial; left side, deep-seated colony. 
 VIII. Microscopical preparation. Pure agar culture, 
 two days, x 800. 
 IX. Microscopical preparation. Smear preparation 
 from blood of a mouse's spleen. x800. 
 
 34 
 
Tab. 34 
 
Tab. 35 
 
 VIII. 
 
Explanation of Plate 35. 
 
 Bacillus megatherium. De Bary. 
 
 I. Gelatin stick culture, twenty -four hours at 22°, 
 II. Agar streak culture, three days at 22°. 
 
 III. Gelatin plate, thirty-six hours at 22°. Natural 
 
 size. 
 
 IV. Gelatin plate, thirty-six hours at 22"". x 60. Deep- 
 
 seated colony. 
 V. Gelatin plate, thirty-six hours at 22°. x60. Su- 
 perficial colony. 
 VI. Agar plate, four days at 22°. Natural size. 
 VII. Agar plate, one day at 22°. x60. Eight side, 
 superficial ; left side, deep-seated colony. 
 VIII. Agar plate, four days at 22°. x60. Eight side, 
 deep-seated* left side, superficial colony. 
 IX. Potato culture, five days at 22°. Natural size. 
 X. Microscopical preparation. Pure agar culture. 
 X800. 
 XI. Bacilli with numerous flagella. x 1^000. Loff- 
 ler^s stain. 
 
 35 
 
Explanation of Plate 36. 
 
 Bacillus subtilis. F. Cohn. 
 
 (Hay bacillus.) 
 
 I. Gelatin stick culture, thirty-six hours at 22**. 
 
 II. Gelatin stick culture, eight days at 22°. 
 
 III. Agar streak culture, two days at 37°. 
 
 IV. Agar stick culture, two days at 37°. Puncture 
 
 canal. 
 V. Agar stick culture, two days at 37°. Surface. 
 VI. Agar plate, twelve hours at 37°. x60. Super- 
 ficial colony. 
 VII. Agar plate, twelve hours at 37°, x60. Deep- 
 seated colony. 
 VIII. Agar plate, twelve hours at 37°. Natural size. 
 
 36 
 
Tab. 36. 
 
Tab. 37 
 
Explanation of Plate 37. 
 
 Bacillus subtilis. F. Cohn. 
 
 (Hay bacillus.) 
 
 I. Potato culture, seven days at 22°. 
 II. Gelatin plate, two days at 22". x 60. Above, on 
 the right side, a deep-seated colony. Below 
 it, a colony lies directly at the surface. On 
 the left a superficial colony. 
 III. Gelatin plate, two days at 22°. Natural size. 
 IV. Gelatin plate, two days at 22°. xlO. 
 V. Microscopical preparation ( x 1? 000) from an agar 
 colony three hours old at 37°. Stained with 
 fuchsin. 
 VI. Microscopical preparation. Bacilli with flagella. 
 (After Fischer.) Very highly magnified. 
 VII. Microscopical preparation (x 1,000) from an agar 
 colony ten days old at 22°. Contains spores. 
 Unstained. 
 VIII. Microscopical preparation (x700) from an agar 
 colony ten days old at 22°. Double stain with 
 carbolized fuchsin and methyl blue. 
 IX. Bacilli with numerous flagella. x 1? 000. Loff- 
 ler's stain. 
 
 37 
 
Explanation of Plate 38. 
 
 Bacillus anthracis. F. Cohn and E. Koch. 
 
 (Anthrax. ) 
 
 I.-V. Gelatin stick cultures, three days at ^^1°, 
 Figs. I. and II. typical, the others atypical. 
 VI. Agar streak culture, two days at 22*^. 
 VII. Agar stick culture, five days at 22°. Puncture 
 
 canal. 
 VIII. Agar stick culture, five days at 22°. Surface. 
 Atypical. 
 IX. Agar stick culture, five days at 22°. Surface. 
 Typical \ often has a homogeneous whitish-gray 
 color. 
 
Tab. 38. 
 
Tab. 39. 
 
 
Explanation of Plate 39. 
 Bacillus anthracis. F. Colin and E. Koch. 
 
 I. 
 
 (Anthrax.) 
 
 Agar plate, four days at 22°. x 60. On left side, 
 superficial colony; on right side, one lying 
 directly below the surface. Below, a deep- 
 seated colony. 
 II. Agar plate, four days at 22°. Natural size. 
 III. Agar plate, thirty-six hours at 37°. xl50. Bor- 
 der of a streak culture. Superficial colony. 
 IV. Agar plate, thirty-six hours at 37°. xl50. Deep- 
 seated colony. 
 V. Gelatin plate, three days at 22°. Natural size. 
 VI. Gelatin plate, three days at 22°. x60. Super- 
 ficial colony, about to sink. 
 VII. Potato culture, six days at 22°. Natural size. 
 
 39 
 
Explanation of Plate 40. 
 
 Bacillus anthbacis. F. Cohn and E. Koch. 
 
 (Anthrax.) 
 
 I. Smear preparation from the blood of a mouse's 
 
 spleen, x 1,000. 
 II. Impression preparation from agar plate culture, 
 one day at 22°. x 1,000. 
 
 III. Unstained preparation in hanging drop from bouil- 
 
 lon culture, thirty-six hours at 37°. Spores 
 beginning to drop out. x 1,000. 
 
 IV. Anthrax threads from agar, thirty-six hours at 
 
 37°. Stained with Ziehl's solution. Spores 
 red, bacilli blue, x 1,000. 
 V. Involution forms, five weeks old, from agar. 
 
 Stick culture stained with fuchsiuc x 1,000. 
 VI. Unstained preparation in hanging drop from 
 bouillon culture, eight hours at 37°. Begin- 
 ning of sporulation. x 1,000. 
 
 40 
 
Tab. 40. 
 
 1. 
 
Tab. 41, 
 
 FReJchhoW.Muncberi 
 
Explanation of Plate 41. 
 
 Bacillus mycoides. Fltlgge. 
 
 (Eoot bacillus.) 
 
 I. Gelatin stick culture, four days at 22". 
 II. Gelatin stick culture, fourteen days at 22°. 
 III. Agar streak culture, two days at 22°. 
 IV. Agar stick culture, eight days at 22°. Puncture 
 
 canal. 
 V. Agar stick culture, eight days at 22°. Surface. 
 VI. Gelatin plate, one day at 22°. Natural size. 
 VII. Agar plate, one day at 22°. Natural size. 
 VIII. Agar plate, four days at 22°. Natural size. 
 IX. Gelatin plate, four days at 22°. Natural size. 
 The colony is about to sink. 
 
 41 
 
Explanation of Plate 42. 
 
 Bacillus mycoides. Flligge. 
 
 (Boot bacillus.) 
 
 I. Agar plate, one day at 22"^. x20. Superficial 
 and deep colony. 
 II. Potato culture, seven days at 22°. Natural size. 
 III. Microscopical preparation. Pure agar culture, 
 twenty -four hours. Puchsin stain, x 1,000. A 
 few bacilli show spores. 
 ly. Agar plate, one day at 22°. x 100. Superficial 
 and deep colony. 
 
 Bacillus butykicus. Htippe. 
 
 (fiutyric-acid bacillus.) 
 
 V. Potato culture, three days at 22°. 
 VI. Gelatin plate, one day at 22°. x60. Above, su- 
 perficial ; below, deep colony. 
 VII. Gelatin plate, one and a half days at 22°. x60. 
 
 Part of a superficial colony. 
 VII. a. Flagella preparation. xl?000. Loftier' s stain. 
 
 Bacillus vulgatus. Migula. 
 
 (Bacillus mesentericus vulgatus Pltigge. Potato 
 bacillus.) 
 
 VIII. Potato culture, five days at 22°. 
 IX. Potato culture, five days at 22'^. Natural size. 
 Both forms of growth occur. 
 
 VII. a. 
 
 42 
 
Tab. 42. 
 
Tab. 43. 
 
Explanation of Plate 43. 
 
 Bacillus vulgatus. Migula. 
 
 (Bacillus mesentericus vulgatus Mtigge. Potato 
 bacillus.) 
 
 I. Gelatin stick culture, ten days at 22°. 
 
 II. Agar streak culture, ten days at 22°. 
 
 III. Agar stick culture, six days at 22°. Surface. 
 
 IV. Agar plate, six days at 22°. Natural size. 
 
 V. Agar plate, six days at 22°. x60. Deep colony. 
 VI. Agar plate, six days at 22°. x 60. Superficial 
 colonies. 
 VII. Gelatin plate, eight days at 22°. Natural size. 
 VIII. Gelatin plate eight days at 22°. x60. Part of a 
 superficial colony. 
 IX. Gelatin plate, eight days at 22°. xl50. Part of 
 
 a superficial colony. 
 X. Potato culture, five days at 22°. Natural size. 
 XI. Microscopical preparation. Pure culture from 
 agar, one day. x800. Fuchsin stain. 
 XII. Bacilli with numerous flagella. x 1,000. Loffler's 
 stain. 
 
 XIL 
 
 43 
 
Explanation of Plate 44. 
 
 Bacillus mesentericus. Lehm. and Neum. 
 
 (Bacillus mesentericus fuscus Fltigge.) 
 
 I. Gelatin stick culture, two days at 22°. 
 II. Agar streak culture, three days at 22°. 
 III. Potato culture, one day at 22°. Natural size. 
 IV. Potato culture, five days at 22°. Natural size. 
 V. Agar plate, two days at 22°. Natural size. 
 VI. Agar stick culture, four days at 22°. Surface. 
 VII. Agar plate, two days at 22°. x60. Above, super- 
 ficial colony ; below, deep colony. 
 VIII. Gelatin plate, thirty-six hours at 22°. x60. 
 Deep colony. 
 IX. Gelatin plate, thirty-six hours at 22°. x60. 
 Superficial colony. 
 X. Gelatin plate, two days at 22°. Natural size. 
 XI. Gelatin plate, one day at 22°. x60. Eight side, 
 deep colony ; left side, superficial. 
 XII. Microscopical preparation. Pure culture from 
 agar, two days. x800. Fuchsin stain. A few 
 bacilli with spores. 
 XIII. Bacilli with numerous flagella. x 1,000. Loif- 
 ler's stain. 
 
 f 
 
 XIIL 
 
 44 
 
Tab. 44. 
 
 LithJnst.v. F. Reichhold, Miiro ln-i 
 
Tab. 45. 
 
Explanation of Plate 45. 
 
 Bacillus tetani. Nicolaier. 
 
 (Tetanus bacillus.) 
 
 I. Sugar-agar stick culture, three days at 37°. 
 II. Sugar-gelatin stick culture, six days at 22°. 
 
 III. Sugar-gelatin plate, four days at 22°. Natural 
 
 size. Cultivated anaerobic. 
 
 IV. Sugar-gelatin plate, four days at 22°. x60. Su- 
 
 perficial and deep colony. Cultivated ana- 
 erobic. 
 V. Sugar-agar plate, four days at 37". Natural size. 
 Cultivated anaerobic. 
 VI. Sugar-agar plate, four days at 37°. x60. Super- 
 ficial and deep colony. Cultivated anaerobic. 
 VII. Microscopic preparation. Pure culture from 
 sugar-agar, three days at 37°. x 1,000. 
 Bacilli with spores. ZiehPs double stain. 
 VIII. Microscopic preparation. Pure culture from 
 sugar-agar, two days at 37°. x 1,000. A few 
 bacilli with spores. Euchsin stain. 
 IX. Microscopical preparation. Pure culture from 
 sugar-agar, twenty -four hours at 37°. x 1,000. 
 Extremely long filaments with faintly colored 
 interspaces. 
 X. Microscopical preparation. Pure culture from 
 sugar-agar, six days at 37°. x 1,000. Euchsin 
 stain. Long filaments and spore chains with 
 faintly colored interspaces. 
 
 45 
 
Explanation of Plate 46. 
 
 Bacillus Chauvcei of French Writers. 
 (Symptomatic Anthrax.) 
 
 I. Sugar-gelatin stick culture, six days at 22°. 
 
 II. Sugar-agar stick culture, three days at 37°. 
 
 III. Sugar-agar stick culture, three weeks at 37°. 
 
 IV. Sugar-agar plate, four days at 37°. Natural size. 
 
 Cultivated anaerobic. 
 V. Sugar-agar plate, four days at 37°. x 60. Super- 
 ficial and deep colony. Cultivated anaerobic. 
 VI. Sugar-gelatin plate, four days at 22°. Natural 
 
 size. Cultivated anaerobic. 
 VII. Sugar-gelatin plate, four days at 22°. x60. 
 Deep colony. Cultivated anaerobic. 
 VIII. Sugar-gelatin plate, two days at 22°. xl50. Part 
 of a superficial colony. Cultivated anaerobic. 
 IX. Microscopical preparation. Pure culture from 
 sugar-agar, three days at 37°. Bacilli with 
 spores and spores that have fallen out. Fuchsin 
 stain. X 1,000. 
 
 46 
 
Tab. 46. 
 
Tab. 47. 
 
 VTI 
 
Explanation of Plate 47. 
 
 Bacillus cedematis maligni. Koch. 
 
 (Malignant oedema.) 
 
 I. Sugar-agar stick culture, eight days at 37°. 
 II. Microscopical preparation. Elagella plait, x 1,500. 
 (Copied from G. Novy : Zadi. f. Hygiene, Vol. 
 XVII., PI. L, 2.) 
 
 III. Microscopical preparation. Bacilli with flagella. 
 
 Pure culture from agar, twenty-four hours. 
 Loffler's stain, x 1,000. 
 
 IV. Sugar-agar plate, four days at 22*^. x60. Part 
 
 of a superficial colony. 
 V. Sugar-agar plate, six days at 22°. Natural size. 
 VI. Microscopical preparation. Pure culture from 
 agar, two days at 37°. Eods with spores. X 
 1,000. Puchsin stain. 
 VII. Microscopical preparation. Tissue juice from 
 guinea-pig. Smear preparation. (Copied from 
 Praenkel and Pf eiffer : " Mikrophotog. Atlas, " 
 Pi. XXIII., 46.) 
 
 47 
 
Explanation of Plate 48. 
 
 Mycobacterium Tuberculosis (Koch). Lehm. and 
 
 Neum. 
 
 (Tubercle bacillus.) 
 
 I. Glycerin-agar streak culture, fourteen days at 37". 
 II. Glycerin-agar streak culture, forty days at 37°. 
 III. Potato culture, forty days at 37°. 
 IV. Colonies of tubercle bacilli in a blood-serum cul-- 
 ture. x700. (Copied from Koch: ''Aetiol. d. 
 Tubercul. Mittheil. d. kais. Gesundheitsamt," 
 Vol. II., PI. IX., 44.) 
 V. Culture on blood serum from a piece of freshly 
 extirpated scrofulous gland. (Copied as above. ) 
 VI. Giant cell with radiating arrangement of the 
 bacilli. From the cheesy bronchial gland of a 
 case of miliary tuberculosis. (Copied as above, 
 PI. 11. , 9.) 
 VII. Microscopical preparation. Pure culture. Stained 
 by ZiehPs method, x 1, 000. 
 VIII. Branching of tubercle bacilli. (Copied from Hayo 
 Bruns: C. B., XVII., No. 23.) 
 IX. Microscopical preparation. Sputum. ZiehPs 
 stain. X 1,000. 
 X. Individual bacteria. Highly magnified. 
 
 i''^ 
 
 ^o 
 
 48 
 
Tab. 48. 
 
 •^.^ 
 
 iS^^ 
 
 » 
 
 i*^ s 
 
 j^^V^?^ 
 
 r- ^ 
 
 **- ' 
 
 
 
 ,e' 
 
 ll. J 
 
 ^^ 
 
 * 
 
 iiJ 
 
 )^., 
 
 ,.*- % 
 
 mi^^ 
 
 
 & 
 
 ^^ 
 
 ^^^^ 
 
Tab 49. 
 
 VI 
 
Explanation of Plate 49. 
 
 ViBKio CHOLERA. (Koch) Buchner. 
 
 (Comma bacillus.) 
 
 I. Gelatin stick culture, two days at 22°. 
 IT. Gelatin stick culture, seven days at 22°. 
 
 III. Gelatin stick culture, eight days at 22°. Culture 
 
 from a case of Asiatic cholera in Hanover. 
 
 IV. Gelatin stick culture, eight days at 22°. 
 V. Agar streak culture, eleven days at 22°. 
 
 VI. Agar stick culture, eight days at 22°. Puncture 
 canal. 
 VII. Agar stick culture, eight days at ^'T. Surface. 
 VIII. Agar plate, six days at 22°. Natural size. 
 IX. Agar plate, six days at 22°. Culture from a case 
 of Asiatic cholera in Hanover. 
 
 49 
 
Explanation of Plate 50. 
 
 Vibrio cholera. (Koch) Bucliner. 
 
 (Comma bacillus.) 
 
 T. Agar plate, thirty-six hours at 22°. x60. Left 
 
 side, superficial 5 right side, deep colony. 
 II. Agar plate, two days at 22°. x60. Left side, 
 superficial ; right side, deep colony. 
 
 III. Agar plate, three days at 22°. x60. Left side, 
 
 superficial; right side, deep colony. 
 
 IV. Agar plate, three weeks at 22°. x60. Leftside, 
 
 superficial ; right side, deep colony. 
 V. Agar plate, five days at 22° ( x 60) , from a case 
 of Asiatic cholera in Hanover. Superficial and 
 deep colony. 
 VI. Gelatin plate, four days at 22°. Natural size. 
 
 Much depressed liquefaction funnel. 
 VII. Gelatin plate, fourteen days at 22''. Natural 
 size. Colony with pronounced zonal develop- 
 ment. 
 VIII. Gelatin plate, four days at 22°. Shallow zones 
 of liquefaction. 
 IX. Gelatin plate, six days at 22°. Shallow sunken 
 colonies with concentric zones of liquefaction. 
 
 50 
 
Tab. 50. 
 
 nmi 
 
 m. 
 
 iv: 
 
 @ © 
 
 
 
 vn 
 
 \i, 
 
 \; 
 
 VTU 
 
 DC. 
 
Tab. 51. 
 
Explanation of Plate 51. 
 
 ViBBio CHOLERA. (Koch) Buchnei. 
 
 (Comma bacillus.) 
 
 I. Gelatin plate, thirty-six hours at 22°. x 60. Su- 
 perficial and deep colonies. 
 II. Gelatin plate, forty-eight hours at 22°. x60. 
 Left side, superficial ; right side, deep colonies. 
 
 III. Gelatin plate, three days at 22°. x60. Super- 
 
 ficial colonies with zones of liquefaction. 
 
 IV. Gelatin plate, three days at 22°. x60. Deep 
 
 colony. 
 V. Gelatin plate, four days at 22°. x60. Super- 
 ficial colony with zone of liquefaction. 
 VI. Gelatin plate, four days at 22°. x60. Deep 
 
 colony. 
 VII. Gelatin plate, five days at 22°. x60. Deep col- 
 ony from a culture from a case in Hanover. 
 VIII. Gelatin plate, five days at 22°. x 60. Superficial 
 colony. Has undergone complete liquefaction. 
 IX. Gelatin plate, eight days at 22°. x60. Super- 
 ficial colony with zone of liquefaction. 
 
 51 
 
Explanation of Plate 52. 
 
 ViBRia CHOLERA. (Koch) Buchner. 
 
 (Comma bacillus.) 
 
 I. Gelatin plate, five days at 22°. x60. Abnormal 
 shape of a superficial colony. 
 
 II. Gelatin plate, five days at 22". x90. Abnormal 
 
 shape of a superficial colony. 
 III. Gelatin plate, five days at 22°. x60. Deeply 
 sunken, superficial colony with strongly reflect- 
 ing zone of liquefaction. 
 
 ly. Gelatin plate, six days at 22°. x60. Superficial, 
 abnormal colony with compact nucleus. Shallow 
 sinking in, with zone of liquefaction. 
 
 V. Gelatin plate, six days at 22°. x60. Deep, ab- 
 normal colony, dark, with radiating stripes, 
 from the same plate as IV. 
 
 VI. Potato culture, two days at 22°. Natural size. 
 Soaked in a solution of soda before inoculation. 
 VII. Potato culture, five days at 22°. Upon ordinary 
 potato. 
 
 52 
 
Tab. 52. 
 
Tab. 53. 
 
 m 
 
Explanation of Plate 53. 
 
 Vibrio cholera (Koch) Buchner. 
 
 (Comma bacillus.) 
 
 I. Pure culture from bouillon, twenty-four hours at 
 37°. Fuchsin stain, x 1, 000. 
 II. Pure culture from agar, twenty-four hours, x 
 1,000. Loffler's stain of flagella. 
 
 III. Pure culture on gelatin, forty-eight hours. Per- 
 
 fectly fresh preparation from water. (Copied 
 from Fraenkel and Pfeiffer, Fig. 94.) 
 
 IV. Pure agar culture, four weeks old. Involution 
 
 forms stained with fuchsin. 
 V. Vibrio Metschnikovii Gamaleia. Smear prepara- 
 tion from pigeon' s blood. (Copied from Fraen- 
 kel and Pfeiffer, Fig. 102.) 
 VI. Vibrio proteus Buchner. Pure culture in bouil- 
 lon, twenty- four hours. Stained with fuchsin. 
 
 53 
 
Explanation of Plate 54. 
 
 Vibrio albensis. Lehm. and Neum. 
 
 (Fluorescent Elbe vibrio.) 
 
 I. Gelatin stick culture, twenty-four hours at 22°. 
 II. Gelatin stick culture, four days at 22*^. 
 
 III. Gelatin stick culture, ten days at 22°. 
 
 IV. Indol reaction at end of ten days. Bouillon cul- 
 
 ture treated with dilute sulphuric acid. 
 V. Gelatin plate, three days at 22°. x 60. Super- 
 ficial colony. 
 VI. Gelatin plate, three days at 22°. x 60. Deep 
 colony. 
 VII. Gelatin plate, thirty-six hours at 22°. Natural 
 size. 
 VIII. Microscopical preparation. Pure culture on agar, 
 forty-eight hours. Stained with fuchsin. 
 
 54 
 
Tab. 54. 
 
Tab. 55. 
 
 Vlll. 
 
 IX, 
 
Explanation of Plate 55. 
 
 Vibrio danubicus Heider; Vibrio berolinensis 
 Rubner; Vibrioa quatilis Gtinther. 
 
 I. Vibrio danubicus. Gelatin stick culture, three 
 days at 22°. 
 
 III. Vibrio danubicus. Gelatin plate, three days at 
 
 22°. Right side, superficial; left side, deep 
 colony. 
 
 IV. Vibrio danubicus. Microscopical preparation. 
 
 Pure agar culture, twenty-four hours. Stained 
 with fuchsin. x 800. 
 V. Vibrio berolinensis. Gelatin plate, three days at 
 22"^. x60. Right side, superficial ; left side, 
 deep colony. 
 VI. Vibrio berolinensis. Microscopical preparation. 
 Pure agar culture, twenty -four hours. Fuch- 
 sin stain. X 800. 
 II. Vibrio aquatilis. Gelatin stick culture, three 
 days at 22°. 
 VII. Vibrio aquatilis. Gelatin plate, three days at 
 22°. x60. Deep-seated secondary colonies, 
 sta'rting from one point. 
 VIII. Vibrio aquatilis. Microscopical preparation. 
 Pure agar culture, twenty-four hours. Fuch- 
 sin stain. X 800. 
 IX. Vibrio aquatilis. Gelatin plate, three days at 
 22°. x60. Right side, superficial; leftside, 
 deep colonies, 
 
 56 
 
Explanation of Plate 56. 
 
 Vibrio proteus. Buchner. 
 
 (Vibrio Tinkler.) 
 
 I. Gelatin stick culture, one day at 22°. 
 II. Gelatin stick culture, four days at 22°. 
 
 III. Gelatin plate, one day at 22°. Natural size. 
 
 IV. Gelatin plate, four days at 22°. x 60. Super- 
 
 ficial colony. 
 V. Gelatin plate, four days at 22^^. x60. Deep 
 colony. 
 VI. Agar streak culture, six days at 22°. 
 VII. Agar plate, four days at 22°. x 60. Superficial 
 colony. 
 VIII. Agar plate, four days at 22"^. x 60. Deep colony. 
 IX. Agar plate, four days at 22°. Natural size. 
 
 56 
 
Tab 56. 
 
Tab. 49. 
 
Explanation of Plate 57. 
 
 Spirillum rubrum. v. Esmarch. 
 
 I. Agar stick culture, ten days at 22°, 
 II. Agar streak culture, twenty days at 22°. 
 
 III. Aguv plate, five days at 22°. x 60. e, Super 
 
 ficial; if deep colony. 
 
 IV. Gelatin plate, seven days at 22°. x 60. e, Su- 
 
 perficial ; i, deep colony. 
 
 V. Microscopical preparation. Pure culture in ten- 
 fold diluted bouillon, two days at 37°. x 
 1,000. Stained with fuciisin. 
 
 V. a, Flagella preparation of spirillum rubrum. 
 X 1,000, Loffler's stain. 
 
 ^/i 
 
 V. a 
 
 Spirillum ooncentricum. Kitasato. 
 
 VT. Agar plate, seven days at 22°. x 60. e. Super- 
 ficial; if deep colony. 
 VII. Gelatin plate, three days at 22°. x60. 6, Su- 
 perficial; i, deep colony. 
 VIII. Agar plate, seven days at 22°. Natural size. 
 IX. Microscopical preparation. Pure culture in bouil- 
 lon, two days at 37°. x 1,000. Fuchsin stain. 
 
 57 
 
Explanation of Plate 68. 
 Spirilla. 
 
 I. Spirillum serpens with plasma border which is 
 stained with difficulty. x 1,000. Fuchsin 
 stain. (Copied from Zettnow: C. B., X., PI. 5.) 
 II. Spirilla from nasal mucus. Smear preparation. 
 X 1,000. (Copied from Weibel: C. B., TI., p. 
 468, Fig. 1.) 
 
 III. Spirilla from nasal mucus. Agar plate, pure cul- 
 
 ture. X 1,000. (Copied, as above, p. 468, 
 Fig. 2.) 
 
 IV. Spirilla from nasal mucus. Gelatin plate, pure 
 
 culture. X 1,000. (Copied, as above, p. 468, 
 Fig. 3.) 
 V. Spirillum uniula with flagella. x 800. (Copied 
 from Loffler: C. B., VI., PI. I., Fig. 2.) 
 VI. Vibrio spermr^tozoides Loffler. x 1,000. (Cop- 
 ied from Loffler: C. B., VII., PI. III., Fig. 7.) 
 VII. Spirochsetes from mucus of the gums. (Copied 
 
 from Loffler: "Bakterien," PI. I., Fig. 4.) 
 VIII. Recurrens spirilla. Human blood, smear prepara- 
 tion. (Copied from Fraenkel and Pfeiffer: 
 "Atlas," No. 134.) 
 IX. Eecurrens spirilla. Human blood, spirilla ar- 
 ranged in a stellate shape. (Copied from M. 
 J. Soudakewitsch : Annates de V Inst. Pasteur, 
 Vol. v., 1891, p. 514, PI. 14, Fig. 1.) 
 
 58 
 
Tab. 50. 
 
 #. 
 
 1 
 
 11. 
 
 □ CO] 
 
 ffl. 
 
 iv; 
 
 VTl 
 
 VI. 
 
 * t € 
 
 vm 
 
 DC. 
 
THE PROPERTY OF 
 
 llmeDiaBD Medical Coliegeef tie ra< 
 
Tab. 51. 
 
Explanation of Plate 59. 
 
 Leptothrix epidermidis. Biz. 
 
 I. Gelatin stick culture, two days at 22°. 
 II. Agar streak culture, two days at 22°. 
 
 III. Agar stick culture, two days at 22°. Puncture 
 
 canal. 
 
 IV. Agar stick culture, two days at 22°. Surface. 
 V. Agar plate, two days at 22°. Natural size. 
 
 VI. Agar plate, two days at 22°. x90. Part of 
 superficial colony. 
 VII. Agar plate, two days at 22°. x 90. Deep colony. 
 VIII. Gelatin plate, two days u.t 22°. Natural size. 
 IX. Gelatin _ plate, one day at 22°. e, Superficial; 
 if deep colony. 
 X. Potato culture, three days at 22°. Natural size. 
 XI. Microscopical preparation. Pure agar culture, 
 two days at 22°. x 1,000. Fuchsin stain. 
 XII. Microscopical preparation. Bouillon culture in 
 hanging drop, two days at 22°. x 1,000. 
 
 59 
 
Explanation of Plate 60. 
 
 OospoRA FARciNicA (Noccaid) Sauv. and Rad. 
 
 I. Agar streak culture, eight days at 22°. 
 II. Gelatin stick culture, twelve days at 22°. 
 
 III. Agar stick cultutre, eight days at 22°. Puncture 
 
 canal. 
 
 IV. Agar stick culture, eight days at 22°. Surface. 
 V. Gelatin plate, ten days at 22°. Natural size. 
 
 VI. Gelatin plate, ten days at 22°. x60. Superficial 
 (e) and doep-seated (1) colonies. 
 VII. Agar plate, six days at 22°. Natural size. 
 VIII. Agar plate, aight days at 22°. Upper colony sur 
 perficial ; lower one deep. 
 IX. Potato culture, soven days at 22°. Natural size. 
 X. Microscopical preparation. Bouillon pure cul- 
 ture, two days. x800. Stained with fuchsin. 
 
 60 
 
Tab. 52. 
 
 
 
 ^^^^^^^^^^^ 
 
 r* 
 
 ' 
 
 ■^ 
 
 
 / 
 
 
 e 
 
Tab. 61. 
 
Explanation of Plate 61. 
 
 OospoRA CHROMOGENES. Lehm. and Neum. 
 
 (Cladothrix dichotoma Autorum non Colin. ) 
 
 I. Gelatin stick culture, six days at 22°. 
 II. Agar streak culture, six days at 22°. 
 
 III. Agar stick culture, six days at 22°. Puncture 
 
 canal. 
 
 IV. Agar stick culture, six days at 22°. Surface. 
 
 V. Gelatin plate, eight days at 22°. Natural size. 
 View upon a white background. 
 VI. Gelatin plate, eight days at 22°. Natural size. 
 
 View upon a black background. 
 VII. Gelatin plate, eight days at 22°. x 60. Part of 
 a superficial colony. 
 VIII. Agar plate, four days at 22°. x60. Superficial 
 and deep colony. 
 IX. Potato culture, three days at 22°. Natural size. 
 X. Microscopical preparation. Bouillon pure cul- 
 ture, three days at 22°. x 1, 000. Stained with 
 fuchsin. 
 
 61 
 
Explanation of Plate 62. 
 
 OospoRA BO VIS. (Harz.) Sauv. and Ead. 
 
 (Actinomyces.) 
 
 I. Agar streak culture, six days at 37°. 
 II. Agar streak culture, thirty days at 37°. 
 III. Gelatin stick culture, fourteen days at 22°. 
 IV. Gelatin plate, six days at 22°. Natural size. 
 V. Agar plate, six days at 37°. Natural size. 
 VI. Agar plate, six days at 37°. x60. Superficial 
 
 and deep colony. 
 VII. Gelatin plate, six days at 22°. xOO. Superficial 
 and deep colony. 
 VIII. Potato culture, ten days at 37°. Natural size. 
 IX. Microscopical preparation. Bouillon pure culture, 
 three days at 37°. x 1,000. Fuchsin stain. 
 
Tab 62. 
 
 
 VI 
 
 vn. 
 
 IX. 
 
Tab 63. 
 
 
Explanation of Plate 63. 
 
 Mycobacterium lepr^ (Arm. Hansen) Lehm. and 
 Neum. 
 
 Bacterium influenza, E.. Pfeiffer. 
 
 Bacterium pestis, Lehm. and Neum. 
 
 I. Mycobacterium leprae. Giant cell from leprous 
 ulcer of epiglottis. xljOOO. Stained by Kus- 
 elP s method. (Copied from Seif ert and Kahn : 
 "Atlas d. Histopath. d. Nase," 1875, PI. 38, 
 Fig. 75 b.) 
 
 II. Mycobacterium leprae. Transverse section of 
 blood-vessel in a leprous testicle; bacilli in 
 endothelium and in a white blood globule. 
 Stained according to Gram and with Bismarck 
 brown, eosin, bergamot oil. x 1,000. 
 
 III. Mycobacterium leprae. Longitudinal section of 
 ulnar nerve. Staining as above. (Copied 
 from Lie : " Path. Anatomic d. Lepra, " Arch, 
 f. Dermatol, und Syi^h., Vol. XXIX., 1895, 
 PI. VI.) 
 
 IV. Streptobacilli in soft chancre. Section of an un- 
 treated chancre, twelve days old. Stained by 
 Unna's method. (Copied from Petersen : " Ue- 
 ber Bacillenfund bei Ulcus molle," C. B., 
 XIIL, PI. 4.) 
 V. Bacterium influenzae. Smear preparation from 
 the nasal secretion, x 1,000. Stained with 
 fuchsin. 
 
 VI. Bacterium pestis. Smear preparation from lym- 
 phatic gland of a rat which died suddenly. 
 X 1,000. (Copied from Yersin, semi-schematic 
 on account of imperfect photogram, Annates de 
 VInstitut Pasteur, 1894, PI. XII., Vol. 8, 
 Fig. 2.) 
 VII. Bacterium pestis. Bouillon pure culture. (Cop- 
 ied, as r.bove. Fig. 3.) x 1,000 
 
 63 
 
A. Introduction to the Morphology of 
 Bacteria. 
 
 By the term bacteria (schizomycetes of Naegeli) is 
 meant a very large group of the lowest vegetable or- 
 ganisms, which are morphologically very simple and 
 uniform, but biologically are extremely differentiated. 
 They are related to the lowest algae (pliycochroma- 
 cea) and the lowest fungi by so many intermediate 
 forms that a strict separation by a rigid definition 
 appears difficult. Various bacteria also exhibit great 
 similarity* to the simplest flagellates, which are usu- 
 ally regarded as animals. 
 
 A definition is rendered more difficult by the fact 
 that botanical investigations of bacteria are compara- 
 tively rare, and that we still possess very imperfect 
 knowledge concerning various details in the struc- 
 ture of bacteria (ramifications, separately stained 
 parts t). 
 
 * Vide Biltschli in Bronn's " Klassen des Tierreiches, " vol. i., 
 part ii., Mastigophora. 
 
 f It is to be noted, moreover, that according to Brefeld's my- 
 cological investigations (vol. viii., p. 274), forms develop dur- 
 ing the process of development of higher fungi which possess a 
 striking resemblance to bacteria during many successive genera- 
 tions. We must therefore concede the possibility that among 
 the varieties of bacteria a number do not deserve the term 
 " species, " but belong to the category of other fungi. 
 5 
 
66 
 
 ATLAS OF BACTERIOLOGY. 
 
 The following definition will suffice, perhaps, for 
 the practical requirements of bacteriology : 
 
 Small unbranched * cells (almost f) always free from 
 chlorophyll, with a thickness which is hardly ever 
 more than 2 p., and extremely rarely 3-5 jj- ; they have 
 a globular, rod, thread, or screw shape, without any 
 
 a ^6/ 
 
 \c/ 
 
 \d y 
 
 m 
 
 ••%#^**^ 
 
 i 
 
 Fig. 1. — The Forms of Bacteria according to Buchner. 
 
 other organs than flagella which serve for movement. 
 Yegetative proliferation takes place by transverse 
 
 * Concerning our knowledge of branched bacteria, mde pp. 67 
 and 68. 
 
 f Practically important bacteria containing chlorophyll are un- 
 known. But J. Frenzel's green tadpole bacillus must probably 
 be classed among the schizomycetes. The position of Dangeard's 
 eubacillus multisporus among the bacteria seems more doubtful. 
 L. Klein described colorless forms with bluish-green spores. 
 
MORPHOLOGY OF BACTERIA. 67 
 
 division, very rarely by longitudinal division. One 
 series of forms develop endogenous, round, permanent 
 spores; in others conidia-like constrictions (arthro- 
 spores) have been observed or claimed. Other modes 
 of proliferation are unknown at the present time. 
 
 So far as we know, the schizomycetes occur only in 
 the forms here delineated, and which were first com- 
 pletely named by H. Buchner. 
 
 Forms of Solitary Growth. 
 
 Spherical form (a), not coccus. 
 
 Oval form (6), the long diameter at the most twice 
 as large as the transverse diameter. 
 
 Short rods (c), the long diameter is two to four 
 times the transverse diameter. 
 
 Long rods (d) , the long diameter is four to eight 
 times the transverse diameter. 
 
 Filamentous shape (e). 
 
 Half-screw or comma (/), a very short section of a 
 screw, at the most a half winding. 
 
 Short screw (g) , a short screw winding. 
 
 Long screw or spiral form {h). All screw forms 
 may occur either with steep or flat threads. 
 
 Spindle form {i). 
 
 Oval rods (/?) are distinguished from the spindle 
 form by the lesser attenuation of the extremities; 
 from the oval form by their greater length (two to 
 four times the transverse diameter) . 
 
 Club form {l). 
 
 Growth in Groups. 
 
 Double spheres (m), with the separation merely 
 indicated; roll shape or biscuit shape (n). 
 
68 ATLAS OF BACTERIOLOGY. 
 
 Spherical series (o), up to eight spheres, with the 
 separation merely indicated; torula shape (p). 
 
 Spherical threads (q), or, if curved, rosary shape 
 (s) ; with the separation merely indicated ; filaments 
 free from torula (r) . 
 
 Grape shape (^). Double rods (u). Filaments of 
 links (v). 
 
 Tetrads (w), a combination upon one plane of four, 
 eight, sixteen, or more cells. 
 
 Dice shape (x), a combination of eight, thirty -two, 
 etc., cells. 
 
 The formation of branches (dichotomy), i.e., the 
 production of a lateral sprout in bacteria, was un- 
 known until recently, and is at all events rare. It has 
 been demonstrated positively in the tubercle, diphthe- 
 ria, and glanders bacilli (vide Plate 48, Fig. YIII.), 
 so that for the present these varieties occupy a posi- 
 tion between the bacteriaceae proper and the hy phomy- 
 cetes or filamentous fungi. 
 
 A different interpretation attaches to pseudodichot- 
 omy, which, according to Babes (Z. H., XX., 412), 
 occurs not very rarely in the most typical bacteria. 
 Either the lower link of a filament grows past the 
 upper one and to one side, or, in a coccus series, a 
 division of a coccus parallel to the direction of the 
 filament suddenly initiates the beginning of a sec- 
 ond filament. 
 
 Much has been written recently concerning the 
 structure of the bacterium cell. We must confine our- 
 selves to a mere abstract. 
 
 According to Biitschli ("Ueber den Bau der Bak- 
 terien," etc., Heidelberg, Winter, 1890) (Fig. 3), the 
 bacterium cell consists of a membrane; a layer of 
 
MORPHOLOGY OF BACTERIA. 
 
 69 
 
 plasma, wliich takes haematoxylin stain with diffi- 
 culty, is often very thin, and indeed often present 
 only at the extremities; and a large central body 
 
 B 1 P 
 
 r/ 
 
 / 
 
 ^ 
 
 
 
 CS 
 
 cs 
 
 .0 
 
 ©, 
 
 
 
 
 
 Q 
 
 Ck 
 
 
 
 r\ _. 
 
 ^ 
 % 
 
 
 
 
 
 88 
 
 80^ 
 
 8 
 
 8 
 
 9 
 
 8 
 
 
 
 /-* 
 
 
 
 
 
 c/ 
 
 8 
 
 
 
 O 
 
 a 6 
 
 Fig. 8.— Pseudodichotomy. a. In bacilli; 6, in streptococci. 
 
 (nucleus), which stains better with hsematoxylin. 
 The latter shows a distinct, the former not always a 
 distinct honey-combed structure. Among the meshes 
 of the comb, which stain blue with hsematoxylin, are 
 situated in the central body numerous granules which 
 are stained red by haematoxylin. 
 At an earlier period Schottelius (C. B., IV., 705) 
 
 Fig. 3.— Chromatium Okenii 
 Ehrbg. (After Butschli.) 
 
 Fig. 4.— Bacillus oxalaticus 
 Migula. (After Migula.) 
 
 expressed a similar opinion of the structure of bacte- 
 ria. According to him the bacillus anthracis consists 
 of a narrow nuclear filament, which stains a blackish- 
 red with a very dilute watery solution of fuchsin, and 
 
70 ATLAS OF BACTERIOLOGY. 
 
 a protoplasmic body, which does not stain so readily. 
 These two structures together constitute the bacillus 
 as ordinarily conceived; they are surrounded by a 
 membrane which stains with difficulty {vide page 72). 
 According to Alfred Fischer * (Fig. 4), the condi- 
 tions are very simple and essentially different from 
 those just described. The bacterium consists of a 
 cell membrane, a protoplasmic tube, and a central 
 fluid; nothing is yet known concerning a nucleus. 
 
 Fio. 4 a.— Plasmolysis, according to A. Fischer, a, Spirillum undula; 
 6, bacterium Solmsii ; c, vibrio cholerae. 
 
 In solutions of salts (sodium chloride, potassium 
 nitrate, etc.) — and the more rapidly the more con- 
 centrated the solution — the abstraction of water pro- 
 duces "plasmolysis," i.e., a retraction of the proto- 
 plasmic tube with partial detachment from the cell 
 wall, t This explains numerous bright vacuoles which 
 develop in many bacilli on making an ordinary cover- 
 glass preparation, and which were formerly often 
 regarded as spores. 
 
 At the same time and independently of A. Fischer, 
 * " Untersuchungen iiber Bakterien, " 1894, Berlin. Reprint 
 from the Jahrb. f. wiss. Botauik, xxvii., vol. 1, 
 
 f Desiccation on the cover-glass often suffices to produce pic- 
 tures of plasmolysis. 
 
MORPHOLOGY OF BACTERIA. 71 
 
 Migula arrived at the same conclusions from a study 
 of the very large bacillus oxalaticus, a sporulating 
 variety related to the hay bacillus. He emphasizes 
 particularly the fact that he has never succeeded in 
 staining the "central body" darker than the proto- 
 plasm. In the protoplasmic tube which has been 
 squeezed out of the cell membrane, the central space 
 for the fluid can be made esi)ecially distinct by the 
 fact that in media which abstract water it becomes 
 smaller ; in water it becomes larger. 
 
 In very many varieties the interior of the bacterium 
 cells is found, after suitable staining, to contain pecu- 
 liar granules. Babes, their discoverer, applied to 
 them the non-committal term metachromatic gran- 
 ules (i.e.y staining differently than the body of 
 the bacterium), while Ernst, their first thorough 
 investigator, terms them nuclei or sporogenous 
 granules. 
 
 For the literature, which is rich in controversy, 
 I refer to Babes (Z. H., XX., 412), and here will 
 merely give the very plausible and clear views of K. 
 Bunge, the most recent student of the subject. Bunge 
 {Fort. d. Med., XIII., 1895) distinguishes: 
 
 1. Ernst's granules. They are stained by warm 
 Loffler's methyl blue, and are differentiated black- 
 ish blue by a solution of Bismarck brown^ but they 
 disappear on boiling. These granules are entirely 
 absent in some sporulating varieties (anthrax, mega- 
 therium) ; in others it can be proven that they have 
 no relation to spores — hence they are cell granules of 
 unknown rank. 
 
 2. Sporule preliminary stages (Bunge 's granules). 
 Small granules, the majority of which are usually 
 
72 
 
 ATLAS OF BACTERIOLOGY. 
 
 fouDd in tlie sporulating cells ; they are not stained 
 by Ernst's method, but stain in boiling Loffler's 
 solution. After preliminary treatment of the dried 
 preparation with chromic acid, sodium hyperoxide, 
 or hydrogen hyperoxide, they are best shown by the 
 
 Bacterium pneumoniae Bacillus anthracis 
 (Friediander). (Cohn). 
 
 Fig. 5.— Formation of a Capsule. (Schematic.) 
 
 streptococcus lanceolatus 
 (Gamal.). 
 
 ordinary spore staining (vide Technical Appendix). 
 The mature spore is produced by the union of several 
 small preliminary stages. 
 
 Bunge explains the controversies by the frequent 
 confusion of the two different varieties of granules. 
 
 Concerning the cell membrane, it is to be noted that 
 often it is not sharply defined on the outside and ap- 
 pears somewhat swollen. In some varieties of bacte- 
 ria (" capsule bacteria" of writers) the thickening of 
 the membrane or of the outer layer of the membrane 
 is so great that the bacterium appears to be surrounded 
 by a veritable mucous envelope or capsule, which is 
 characterized by its slight response to staining with 
 aniline colors. It is an interesting fact that these bac- 
 teria form capsules only when they grow in the ani- 
 mal body or upon special nutrient media, such as fluid 
 blood serum, bronchial mucus, and, according to 
 
MORPHOLOGY OF BACTERIA. 73 
 
 Paulsen, milk.* The capsules do not form when the 
 cultures are made on gelatin, agar, and potatoes. 
 
 Peculiar unilateral thickenings or swellings of the 
 membrane are found in bacterium pediculatum, which 
 is described as a rare cause of the 
 "frog-spawn disease" of sugar fac- 
 tories (Fig. 6). 
 
 In the spherical forms the outer fiq. 6. -b a c t e r i u m 
 surface of the bacteria is' almost al- pediculatum. (After 
 
 , , . , 1 1 , -I ', ' Koch and Hosfius. ) 
 
 ways smooth ; m the short rods it is 
 often smooth and without appendages, but the larger 
 rod and screw forms are usually provided with single 
 or numerous thin flagella. These are sometimes dis- 
 tributed over the entire body of the bacterium, some- 
 times they form a bundle at one pole, sometimes there 
 is only a single polar flagellum. Shortly before divi- 
 sion bacteria with polar position of the flagella show 
 one flagellum or a bundle of flagella at each pole. 
 As A. Fischer clearly proved, the flagella are not 
 structures similar to the retractile and extensible 
 pseudopodia, but are true hair-like formations which 
 develop from outgrowth. In order to color the fla- 
 gella it is necessary to treat the bacteria with unusu- 
 ally powerful staining reagents. Then the mem- 
 
 *It is not certain that pronounced capsule formation alwa^'S 
 takes place in these nutrient fluids. Recently various authors 
 have called attention to the fact that capsule-like formations are 
 observed extensively among bacteria. Johne describes a method 
 by which they are easily made visible in anthrax, and distinct 
 capsules are also seen in this way in bacillus megatherium, bacillus 
 oxalaticus, etc. Bab6s has depicted capsules in streptococcus 
 pyogenes, and we have occasionally seen similar appearances in 
 many bacteria. Masses of bacteria which are united into mucous 
 clumps by swelling of the capsules are called " zooglcea. " 
 
74 ATLAS OF BACTERIOLOGY. 
 
 brane, which usually remains colorless with ordinary- 
 stains, is also stained and the bacteria appear to be 
 much thicker. Occasionally broad layers of the 
 membrane remain unstained, and the flagella are then 
 situated upon a narrow annular areola, separated from 
 the bacillus by a colorless zone (Zettnow, von Stock- 
 
 5E/ 
 
 r\^ 
 
 -4 ^ 
 
 a h c 
 
 Fig. 7.— Types of Flagella. a, Vibrio oholerse, a flagellum at one ex- 
 tremity; 6, bacterium syncyaneum, a bundle of flagella at one extrem- 
 ity, rarely on the side; c, bacterium vulgare, flagella arranged round 
 about. 
 
 lin, A. Fischer). Unfortunately many of the meth- 
 ods used in staining lead forthwith to exfoliation and 
 degeneration of the flagella, so that their perfect ex- 
 hibition is often difficult. The above figure gives 
 a schematic representation of the three modes in 
 which bacteria are provided with flagella. 
 
 In the cultures of bacteria which are rich in flagella, 
 Loffler first observed the occasional production of 
 peculiar, switch-like bodies, composed of flagella 
 which had fallen off or had been cast off and were 
 plaited into one another (vide Plate 47, Fig. II.). 
 
 The power to produce flagella may be lost entire- 
 ly for generations ; whether permanently is ^ill un- 
 known. Vide Micrococcus agilis, sarcina mobilis 
 (Lehmann and Neumann) . 
 
 Ordinary vegetative increase of bacteria is effected 
 
MORPHOLOGY OF BACTERIA. 75 
 
 by a transverse constriction in the middle of the bac- 
 terial cell, which has either been very little (spherical 
 bacteria) or considerably elongated. As a rule, the 
 micro-organisms separate soon after fission, but the 
 opposite event may occur in all groups of bacteria, 
 so that, for example, chains of spheres or rods de- 
 velop. Under certain nutritive conditions the bacte- 
 riacese, vibriones, and higher bacteria give rise to the 
 production of long threads, but later these may again 
 be resolved into links. According to all recent inves- 
 tigations, division of the cell starts in the protoplas- 
 mic layer upon the wall, the central "nucleus" or 
 "cavity" is divided passively, and the cell membrane 
 takes part secondarily. This is evidently opposed to 
 the interpretation of the central body as a nucleus, 
 because division of the nucleus always precedes divi- 
 sion of the cell. 
 
 Longitudinal growth with transverse fission is the 
 rule for the mass of bacteria, but in certain forms — 
 for example, sarcina — there is a regular alternation of 
 the fission in the three principal planes. At least 
 occasional division along two planes at right angles 
 to one another has been observed in very different 
 bacteria — for example, in streptococci — and thus cells 
 in four parts may develop, with bifurcation of the 
 chain {vide Fig. 2). 
 
 Longitudinal division of rod forms has been ob- 
 served rarely but undoubtedly (Babes: Z. H., XX.). 
 Metschnikoff observed stellate division in a sporu- 
 lating organism known as "Pasteuria," but this can 
 hardly be classed among the bacteria in the narrower 
 sense. 
 
 Ordinary vegetative proliferation must be distin- 
 
76 ATLAS OF BACTERIOLOGY. 
 
 guished from that due to the formation of spores. 
 We are acquainted to-day with: (1) Endospores, 
 strongly refracting oval or round bodies developing in 
 the interior of the cell, and which as a rule possess 
 considerable resistance to injurious influences (heat, 
 dryness, chemicals) ; and (2) arthrospores (De Bary, 
 
 Hiippe), i.e.y sprout-like 
 constriction of one end of 
 the cell. These spores 
 (Fig. 8) are also said to 
 Fig. 8. — Arthrospores of Vibrio exhibit increased resist- 
 
 choleraB, according to Huppe. ^^^^^ ^^^ ^^ ^-^^ ^^^^^^ 
 
 investigations have furnished no absolutely positive 
 proof of the formation of arthrospores which ex- 
 hibit increased resistance, the difficult question of 
 arthrospores, important as it is, must be regarded 
 as still open. 
 
 In the following pages the term spores refers only 
 to endogenous permanent forms. 
 
 In the different varieties the development of the 
 endospores runs a similar but not identical course. 
 In examining any definite variety for the development 
 of spores, we resort, as a rule, to agar streak or po- 
 tato cultures, which are kept at a temperature best 
 adapted to the variety in question. At the end of 
 twelve, eighteen, twenty-four, thirty, thirty-six hours, 
 we examine small tests of the streak culture, first un- 
 stained in water and with a narrow angle of aperture. 
 If it is thought that round or oval, strongly refracting 
 spores have been found, the spores are stained ac- 
 cording to Neisser's or Hauser's method (vide Tech- 
 nical Appendix) . For the careful study of the devel- 
 opment of spores, it is best to place a few bacilli in a 
 
MORPHOLOGY OF BACTERIA. 77 
 
 hanging drop of gelatin or agar, and (if necessary, 
 with the aid of warming apparatus or in a well-heated 
 room) to observe and draw definite individual cells 
 uninterruptedly. 
 
 Motile varieties always come to a standstill (ac- 
 cording to A. Fischer) before the formation of spores, 
 but they do not cast off their flagella. Certain varie- 
 ties first grow into long filaments, which at the be- 
 ginning are unsegmented. This variety includes the 
 bacillus anthracis, whose sporulation will be selected 
 as an example {vide Plate 40, Figs. VI. and III.). 
 
 The previously homogeneous bacteria first exhibit 
 a delicate, cloudy opacity ; then, according to Bunge, 
 the very finest granules are replaced by a smaller 
 number of somewhat coarser granules, which coalesce 
 until small, rounded spores are situated at regular 
 intervals (Plate 40, Fig. VI.), and are converted grad- 
 ually into the oval, strongly refracting, mature spores 
 (Plate 40, Fig. III.). 
 
 When sporulation is complete, we find in the bac- 
 terial filament a delicate septum between two spores 
 (Plate 40, Fig. IV.). Not all segments which have 
 begun the development of spores by the formation of 
 spherical preliminary stages mature these spores. 
 Indeed, certain varieties, as the result of various con- 
 ditions of culture, gradually suffer a permanent loss 
 of the power of producing mature spores and form 
 only preliminary stages, which are physiologically 
 valueless (Eoux, K. B. Lehmann). 
 
 According to Lud. Klein (C. B., VII., 440), spor- 
 ulation is entirely different in five usually motile, an- 
 aerobic forms of bacilli (bacillus De Baryanus, Solm- 
 sii, Peromelia, macrosporus, limosus), which were 
 
78 ATLAS OF BACTERIOLOGY. 
 
 discovered and studied by him (but unfortunately not 
 in pure cultures) . In these the process ran the fol- 
 lowing course : Without any cessation in the motion 
 of the bacillus, one extremity becomes somewhat en- 
 larged and acquires a slightly greenish tinge. The 
 
 ^ 
 
 ^ 
 
 /? 
 
 <^ 
 
 Fig. 9.— Types of Spores. 
 
 entire contents of the distended part now contract 
 into a spore of bluish-green color and striking bril- 
 liance. 
 
 In the most important varieties the mature spores 
 appear as follows (Fig. 9) : 
 
 1. The spore lies in the interior of a non-dis- 
 tended, short bacterium cell (a). 
 
 2. The spore lies in the interior of a non-distended, 
 short bacterium cell, which forms merely a link of a 
 long filament (b). 
 
 3. The spore lies in the interior of a bacterium cell, 
 which has been distended in the middle and has be- 
 come spindle shaped (d). 
 
 4. The spore lies at the extremity of a non-distended 
 short bacterium cell, apparently projecting far beyond 
 it (c). 
 
 The germination of spores has been little studied. 
 They are generally set free before germination by rup- 
 ture of the filament. An outgrowth of the spores in 
 
MORPHOLOGY OF BACTERIA. 
 
 79 
 
 the bacillus at right angles to the direction of the fila- 
 ment is rarely observed {vide Sorokin, C. B., I., 465). 
 
 The following cut shows the germination of a few 
 closely allied varieties which were studied by L. 
 Klein. 
 
 The examination is made in a hanging drop of gel- 
 atin or agar. This may furnish very valuable mate- 
 
 Z 3 
 
 
 3^5 
 
 °os/ 
 
 Fig, 10.— Development of Spores, according to L. Klein, a, Bacillus 
 leptosporus L. Klein; 1-3, the enlarging spore; 4, the spore is con- 
 verted into the bacillus without any sharp demarcation ; 5-10, further 
 growth ; 11-16, development of the spores. 6, Bacillus sessilis L, Klein ; 
 1-4, the spore swells; 5, the spore sends out a little rod at one pole, 
 and remains behind as an empty envelope; 6-8, further growth; 9-13, 
 development of the spores. 
 
 rial in differential diagnosis, as it seems to differ 
 greatly in details. 
 
 1. Bacillus anthracis. The spore swells, its refrac- 
 tive power diminishes, its sharply defined membrane 
 becomes indistinct, and without any sharp demarca- 
 tion the spore becomes a young bacterium cell, which 
 grows further and divides again. 
 
 A similar condition obtains in the bacillus lepto- 
 sporus Klein, described by L. Klein (C. B., VI., 377), 
 which is characterized by narrow, almost quadrangu- 
 lar spores (Fig. 10, a). 
 
80 ATLAS OF BACTERIOLOGY. 
 
 2. Bacillus subtilis Cohn. The membrane of the 
 growing spore bursts at the equator, the firm-walled 
 membrane of the spore adheres not infrequently to 
 the emerging young rod, even after it has grown into 
 a long filament. 
 
 3. Bacillus sessilis Klein. The spore enlarges to a 
 marked degree, then ruptures at one pole, and from 
 the envelope of the spore grows a motionless filament, 
 to which the yellowish-green, contracted membrane 
 of the spore remains adherent for a very long time 
 (Fig. 10, h). 
 
 In old cultures of bacteria we almost always find 
 dead, often very queerly shaped bacterial cells (invo- 
 lution, degeneration forms), which are shown in Plate 
 40, Fig. Y., and Plate 53, Fig. YI. These swollen, 
 bent, often unrecognizable forms stain poorly with the 
 ordinary reagents. The beginner will often regard in- 
 volution forms as the result of fouling ; the resort to 
 plate cultures will soon show whether we have to deal 
 with one or more forms of bacteria. 
 
 B. The Chemical Composition of Bacteria. 
 
 The body of bacteria consists in great imrt of water, 
 salts, and albuminoids ; * extractive matters which 
 are soluble in alcohol, and other bodies (triolein, 
 tripalmitin, tristearin, lecithin, cholesterin) which 
 are soluble in ether, are present in smaller amounts. 
 
 * Albumin and salts may amount to ninety -eight per cent of 
 the dried bacteria (cholera vibrio), and on the other hand as 
 much as twelve per cent of carbohydrates may be present in the 
 membranes. Hellmich found a globulin in the bacterial albu- 
 min (Arch. f. exp. Path. u. Pharm., xxvi., 345). 
 
THE CHEMICAL COMPOSITION OF BACTERIA. 81 
 
 In no variety of bacteria could E. Cramer discover 
 grape sugar; some varieties (bacillus butyricus, lep- 
 tothrix forms) contain starch-like masses which are 
 stained blue b}^ iodine. True cellulose was discovered 
 by Drey fuss in bacillus subtilis and in an organism 
 closely related to bacterium coli, and the bacillus tuber- 
 culosis also forms cellulose in the animal body. But 
 no cellulose could be obtained from cultures of bacil- 
 lus tuberculosis and a "capsule bacillus from water," 
 closely related to bacillus pneumoniae Friedlander, 
 while they contained a large amount of a gelatinous 
 carbohydrate (CeHj^OJ, which is closely allied to 
 hemicellulose (concerning the literature vide Nishi- 
 mura: A. H., XYIII., 318 and XXI., 52). The mu- 
 cus of leuconostoc mesenterioides was shown by 
 Scheibler {Ghem. Centralhl.j XI., 181) to be a carbo- 
 hydrate, C^Hj^Oa dextro-rotatory. Kramer obtained 
 a similar substance from the membranes of bacillus 
 viscosus sacchari. Nuclein has not been extracted, 
 but among the nuclein bases xanthin, guauin, and 
 adenin have been found in considerable amounts. 
 One group of bacteria deposits suljjhur granules, 
 which are derived from sulphuretted hydrogen (beg- 
 giatoa, thiothrix) ; another variety, which is classed 
 among bacteria by many authors, secretes ferric oxide 
 into its membrane from ferruginous waters (cladothrix, 
 crenothrix) . 
 
 The methodical investigations of E. Cramer have 
 shed some light upon the quantitative relations, al- 
 though accurate statements have been obtained hith- 
 erto only concerning bacterium prodigiosum, bacillus 
 pneumoniae, and a few related varieties, and a series 
 of forms of vibrio cholerae {vide E. Cramer: A. H., 
 6 
 
82 ATLAS OF BACTEEIOLOGY. 
 
 XIII., 70; XVI., 150; and XXII., 167). The fol- 
 lowing statements and figures must suffice for the 
 limits of this work. 
 
 The amount of water in a culture which has grown 
 upon a solid nutrient medium, and likewise the 
 amount of ash, depend in a very great measure upon 
 the composition of the nutrient medium. 
 
 Eor example, bacterium prodigiosum contains, 
 when cultivated on potato, 21.49 per cent dry sub- 
 stance, 2.70 per cent ash in the fresh substance; 
 when cultivated on carrots, 12.58 per cent dry sub- 
 stance, 1.31 per cent ash in the fresh substance. 
 
 Apart from the concentration of the nutrient me- 
 dium, higher temperatures and youth of the cultures 
 increase the amount of dry substance and ash. 
 
 The amount of dry substance of bacteria also varies 
 in its composition in the same variety, under the in- 
 fluence of the nutrient medium. 
 
 For example, the bacterium pneumoniae Fried. , upon 
 a nutrient medium of meat-infusion agar, contained : 
 
 With 1 per With 5 per 
 cent peptone, cent peptone. 
 
 Albumin 71.7 79.8 
 
 Ether and alcoholic extract 10. 3 11. 28 
 
 Ash 13.94 10.36 
 
 « 
 
 and with one per cent peptone and five per cent grape 
 sugar : 
 
 Per cent. 
 
 Albumin 63.6 
 
 Ether and alcoholic extract 22. 7 
 
 Ash 7. 88 
 
 It is evident that an increase in the amount of pep- 
 tone in the nutrient medium causes an increased 
 
THE CHEMICAL COMPOSITION OF BACTERIA. 83 
 
 amount of albumin in the bacillus, while an increased 
 quantity of grape sugar diminishes the amount of 
 albumin. 
 
 The differences are much greater as regards the 
 dry substances of cholera vibriones when cultivated 
 upon albuminous soda bouillon and upon the non- 
 albuminous Uschinsky nutrient medium. Cramer 
 found (the figures represent the averages from experi- 
 ments with five cholera species) that : 
 
 Albumin. Ash. 
 
 Percent. Percent. 
 
 Cholera vibriones on soda bouillon contained 65 31 
 
 Cholera vibriones on Uschinsky solution contained .45 11 
 
 In the latter case there were evidently very large 
 amounts of non-nitrogenous bodies, which may be re- 
 garded in part as hydrocarbons (or fats). 
 
 A very important point in classification — although 
 more in a critical negative sense — is the fact discov- 
 ered by Cramer that closely allied varieties which ex- 
 hibit analogous, slightly varying composition upon 
 several nutrient media, suddenly act differently upon 
 a new medium. The most interesting illustration was 
 the conduct of five cholera varieties, which in soda 
 bouillon produced vibriones of almost exactly the 
 same constitution, while they differed greatly in 
 Uschinsky solution: 
 
 Soda Bouillon. 
 
 Albumin. Ash. Total. 
 
 Cholera, old 65.12 31.55 96.67 
 
 Cholera, Hamburg, wmter of 1893. 69.25 25.87 95.12 
 
 Cholera, Paris 62.25 32.80 95.05 
 
 Cholera, Shanghai 64. 25 33. 87 98. 12 
 
 Cholera, Hamburg, autumn of 
 
 1893 63.94 29.81 93.75 
 
84 ATLAS OF BACTERIOLOGY. 
 
 UscHiNSKY Solution. 
 
 Albumin. Ash. Total. 
 
 Cholera, old 48. 13 7. 14 55.27 
 
 Cholera, Hamburg, winter of 1892. 35.75 13.70 49.45 
 
 Cholera, Paris 65.63 9.37 70.00 
 
 Cholera, Shanghai 47. 50 1 1. 64 59. 14 
 
 Cholera, Hamburg, autumn of 
 
 1893 34.37 14.74 49.11 
 
 This result again shows how dangerous it is to dis- 
 tinguish two varieties by relying upon a single chemi- 
 cal or biological reaction. Some of these varieties 
 need merely acquire the power of forming thick cell 
 membranes in Uschinsky solution in order to explain 
 these remarkable differences. How easily, for exam- 
 ple, could a writer be led, from these figures, to re- 
 gard the bacilli of the Paris cholera as a distinct spe- 
 cies, because they contain almost twice the amount 
 of albumin in Uschinsky solution as those of the 
 Hamburg cholera. 
 
 So far as I know the spores of bacteria have not 
 been closely analyzed, but from the analogy to the 
 spores of mould fungi we may expect them to contain 
 a diminished amount of water. 
 
 C. The Vital Conditions of Bacteria. 
 
 1. NUTRIENT MEDIA. 
 
 While a number of schizomycetes have been found 
 hitherto only in the human or animal organism, and 
 therefore appear to be strict parasites (for example, 
 spirillum Obermeieri), the majority of parasitic vari- 
 eties can also be cultivated, either readily (for exam- 
 
THE VITAL COITDITIONS OF BACTERIA. 85 
 
 pie, bacterium typhi) or with difficulty (for example, 
 micrococcus gonorrhoeae) in artificial nutrient media. 
 Among the inhabitants of the inanimate surroundings 
 of man, the so-called saprophjtes^ the majority can be 
 easily cultivated in the same artificial media as para- 
 sites; while in others, for example certain salivary 
 and water bacteria, such cultivation meets with in- 
 surmountable obstacles. 
 
 All nutrient media must be rich in water, and the 
 presence of salts and of a supply of carbon and nitro- 
 gen is also indispensable. The majority of the prac- 
 tically important and all the pathological varieties 
 have a predilection for albuminous and feebly alka- 
 line nutrient media. 
 
 The demands of the bacteria upon the composition 
 of the nutrient media vary extremely. As Mead Bol- 
 ton showed, a number of water bacteria (bacillus 
 aquatilis Fliigge and bacillus erythrosporus Fliigge) 
 are satisfied with water which has been distilled twice 
 in glass vessels. Here the proliferation of the bacte- 
 ria must have taken place either at the cost of traces 
 of impurities or of the ammonia and carbonic acid of 
 the atmosphere. 
 
 In water which contained ammonium carbonate as 
 the sole source of carbon and nitrogen, and was ac- 
 cordingly freei from all organic nutritive material, 
 Heraeus observed abundant proliferation of a variety 
 of fungus — that is, a development of cell substance 
 from the simplest material, such as occurs otherwise 
 only in the higher plants which work with chlorophyll 
 in combination with sunlight. Hiippe and particu- 
 larly Winogradsky have shown the correctness and 
 importance of this obsers^ation as the result of care- 
 
86 
 
 ATLAS OF BACTERIOLOGY. 
 
 ful studies. The energy necessary to the albumin 
 synthesis seems to be gained by oxidation of ammo- 
 nia into nitric acid. 
 
 Very few practically important bacteria exhibit 
 such simplicity, but very many can dispense at least 
 with albumin in the nutrient and thrive in solutions 
 of very simple composition. Formerly cultures in 
 such fluids were employed very often, and recently 
 Uschinsky has again resorted to simple nutrients. 
 But instead of Uschinsky 's somewhat complicated so- 
 lution : 
 
 Water 1,000 
 
 Glycerin 30-40 
 
 Sodium chloride 5-7 
 
 Calcium chloride 0.1 
 
 Magnesium sulphate ., . 0.2-0.4 
 
 Dikalium phosphate . . . 3-2. 5 
 
 Ammonium lacticum . . . 6-7 
 
 Sodium asparaginicum . 3-4 
 
 we may choose much simpler solutions ; for example, 
 on the recommendation of Voges and C. Fraenkel 
 {Hyg. Rundschau, 1894, No. 17) for one litre: 
 
 Sodium chloride 5 gm, 
 
 Neutral commercial sodium phosphate 2 gm. 
 
 Ammonium lactate 6 gm. 
 
 Asparagin 4 gm. 
 
 In this fluid (although it contains no sulphur) there 
 grow: 
 
 Very Well. 
 Bacillus subtilis and mycoides. 
 
 Bacterium syncyaneum, pyocyaneum, coli, acidi lactici, 
 pneumoniaj, mallei, vulgare. All vibriones. 
 
 Feebly. 
 
 Micrococcus pyogenes a 
 
 aureus. 
 Streptococcus pyogenes. 
 
 Bacterium typhi. 
 Bacillus anthracis. 
 
THE VITAL CONDITIONS OF BACTERIA. 87 
 
 Not at All. 
 Bacillus tetani I Bacterium eryaipelatos sunm. 
 
 Bacterium murisepticum. I Bacterium cuniculicida. 
 
 The addition of the other substances recommended 
 by Uschinsky did not cause vigorous growth of other 
 varieties (such as diphtheria and tetanus), while on 
 the addition of three to four per cent glycerin the 
 medium becomes very serviceable even for the tubercle 
 bacillus. 
 
 Although cultures in the simple nutrient media just 
 described possess great theoretical interest, they are 
 used very little for purposes of differential diagnosis. 
 
 Much more frequent use is made of flesh-water pep- 
 tone gelatin, flesh-water peptone agar, bouillon (with 
 or without the addition of grape sugar or milk sugar), 
 glycerin agar, milk, potato discs. 
 
 We must always have these on hand, because no 
 differential diagnosis is possible without them, and 
 no variety can be properly described which has not 
 been tested in regard to its condition in all these nu- 
 trient media (with the exception of glycerin agar). 
 
 Less use is made of the following nutrient media : 
 potato water, lamb bouillon, blood serum (fluid and 
 firm), serum agar, agar smeared with blood, meat, 
 pieces of bread, mashed potatoes, rice, boiled or raw 
 
 2. REACTION OF THE NUTRIENT MEDIA. 
 
 As has been remarked above, the large majority of 
 bacteria, especially the pathogenic forms, have a 
 predilection for neutral or feebly alkaline nutrient 
 
88 ATLAS OF BACTERIOLOGY. 
 
 media. Formerly it was recommended that a solu- 
 tion of soda should be added gradually to the nutri- 
 ent medium until it turns red litmus paper to a faint 
 blue color. 
 
 Every chemist knows that there is no sharply de- 
 fined final reaction for the titration of phosphatic nu- 
 trient media with litmus ; furthermore, that different 
 litmus papers influence the result, and that, finally, 
 titration is quite impossible by gaslight. For this 
 reason W. K. Schultz, in 1891, recommended phenol- 
 phthalein as an indicator in agar tritration, and 
 advised that 8-10 c.c. less of normal soda lye be 
 added to a litre of the nutrient medium than is 
 necessary for complete neutralization with this indi- 
 cator. In this way a medium is obtained whose 
 reaction is suitable to very many bacteria, but there 
 are some which require complete neutralization (C. 
 B., X., 52). 
 
 Without having noticed this suggestion I conceived 
 the same idea, in 1892, during my investigations on 
 bread acids. Since 1894, the neutral gelatin (or 
 agar) used in my laboratory as a nutrient medium 
 has been treated with as much soda lye as is neces- 
 sary to slight reddening of an addition of phenol- 
 phthalein. All the plates in this Atlas have been 
 made according to such cultures, after experiments 
 on five important bacteria had shown that the addi- 
 tion of alkalies and acids to this neutral medium had 
 not improved their growth. Since then, Mr. Winkler, 
 a student of medicine, has systematically tested the 
 power of growth of the large majority of bacteria 
 described in our Atlas. This has been done upon the 
 following nutrient media : 
 
THE VITAL CONDITIONS OF BACTERIA. 89 
 
 1. Upon agar which, after the use of phenol- 
 phthalein, was neutralized with normal soda. 
 
 2. On "acid" agar, i.e., neutral agar which has 
 been treated with 10 c.c. of normal sulphuric acid to 
 1 litre. 
 
 3. On three varieties of alkaline agar, ^.e., on neu- 
 tral agar which has received 10, 20, and 30 c.c. of 
 normal alkali to 1 litre. 
 
 The results laid down in Table I. show, in brief, 
 that almost all bacteria thrive well upon three of 
 these nutrient media. 
 
 At all events the medium made neutral by phenol- 
 phthalein may be recommended unreservedly as a 
 universal nutrient; the virulence of the varieties ex- 
 amined by us (anthrax, bacterium coli, mouse sep- 
 ticaemia, chicken cholera) was also well maintained 
 thereon. 
 
 This reaction possesses the advantage that it is 
 easily prepared and represents a sharply defined 
 point, viz., that in which all the free acids and the 
 acid salts are converted into neutral salts (mono- 
 sodium phosphate into disodium phosphate) . 
 
 If acid media are to be employed, it is best to start 
 with one which has been neutralized with phenol- 
 phthalein, to which 10, 20, or 30 c.c. of normal acid 
 per litre may be added. According to Winkler the 
 first degree of acidity is well tolerated by almost all 
 bacteria. According to Schliiter's statements (C. B., 
 XI., 589), which are confirmed by recent publications, 
 many tolerate a much higher degree of acidity ; even 
 as much at 100 c.c. of normal acid per litre, according 
 to experiments made in our laboratory. 
 
 Apart from yeast and mould fungi, acid nutrient 
 
90 ATLAS OF BACTERIOLOGY. 
 
 media should always be employed as auxiliaries 
 when we have to deal with the isolation of a bacteri- 
 um from an acid medium. In counting the germs in 
 the air, earth, water, milk, etc., the neutral medium 
 should always be used. 
 
 3. INJURY TO BACTERIA BY CHEMICAL SUB- 
 STANCES. 
 
 In the presence of an excess of acids or alkalies we 
 have just recognized a factor which exerts an inhibi- 
 tory influence on development, and, in still greater 
 intensity produces death. The most varied chemi- 
 cals act in a similar manner after a certain degree of 
 concentration. The most effective substances are 
 known as antiseptics or disinfectants. 
 
 With Hiippe, we usually distinguish the following 
 degrees of action : 
 
 1. The growth is not disturbed but the pathogenic, 
 zymogenic functions are weakened — attenuation, miti- 
 gation. 
 
 2. The organisms can no longer proliferate but are 
 not killed — asepsis, kolysepsis. 
 
 3. The vegetative conditions of the micro-organisms 
 are destroyed but not the permanent forms — anti- 
 
 4. The vegetative and spore forms are killed — 
 sterilization or disinfection. 
 
 Inasmuch as the test of the resisting power to 
 chemicals plays a minor part for diagnostic purposes, 
 this section will be treated very briefly. 
 
 The following plan should be adopted in order to 
 determine the minimum concentration of the chemical 
 
THE VITAL CONDITION'S OF BACTERIA. 91" 
 
 poison which will just produce asepsis, i.e., inhibi- 
 tion of development. 
 
 For example, a ten-per-cent solution of the disin- 
 fectant is prepared, and 1, 0.5, 0.3, 0.1 c.c, etc., are 
 added to 10 c.c. of liquefied gelatin. The tubes then 
 contain 1 per cent, 0.5 per cent, 0.^ per cent, 0.1 
 per cent of the disinfectant; stick, streak, or plate 
 cultures are then made with the bacterium which is 
 to be tested. We may also inoculate with material 
 which contains only spores (material which has been 
 freed from all bacilli by heating for half an hour to 
 70° C.) in order to note whether these spores grow 
 into cultures. 
 
 Behring makes this test in the following practical 
 form : From the fluid, infected nutrient medium (for 
 example, serum) which is to be tested a drop is 
 taken before the addition of the antiseptic, and, after 
 being placed on the lower surface of a cover-glass, is 
 enclosed, by means of some vaseline, in a hollowed 
 glass slide (vide Technical Appendix) . Then larger 
 and larger quantities of the disinfectant are added 
 gradually to the serum tube, and after each addition 
 a drop culture is again made. After remaining from 
 twenty-four to forty-eight hours in the incubator, 
 we can convince ourselves with the microscope of the 
 growth in the different drops. 
 
 In case of a degree of concentration necessary to 
 antisepsis, the fungus is cultivated in bouillon and 
 10 c.c. of the bouillon (which is still free from spores 
 and which has been filtered through asbestos in order 
 to get rid of any clumps of bacilli which may be 
 present) are replaced with a corresponding amount 
 of the disinfectant solution. From these tubes we 
 
92 ATLAS OF BACTERIOLOGY. 
 
 take, at the end of one minute, five minutes, ten 
 minutes, fifteen minutes, thirty minutes, one hour, 
 etc., a small platinum loop full of material, place the 
 latter in 10 c.c. of lukewarm liquefied gelatin, and form 
 plates. We then obtain statements like the following : 
 X per cent of the disinfectant proves fatal in twenty 
 minutes, y per cent in one minute, etc. If we sus- 
 pect that the trace of disinfectant, which is conveyed 
 by the loop, may have made the gelatin aseptic and 
 may thus have simulated destruction of the bacteria, 
 we should make a control inoculation of fresh material 
 in gelatin to which a similar trace of the disinfecting 
 fluid has been added. 
 
 The disinfectant to be tested should always be dis- 
 solved in water. If, on account of the slight solubil- 
 ity in water, the use of alcohol in the production of 
 the original solution is indispensable, special control 
 experiments are necessary in order to show that the 
 action of the alcohol was not injurious. 
 
 When using nutrient media which are rich in 
 albumin, we require much larger amounts of the 
 disinfectant, both for the production of asepsis as 
 well as for that of antisepsis, than when using media 
 which are poor in albumin.* Thus in bouillon 
 creolin produces asepsis when present in the propor- 
 tion of 1 : 15000-1 : 5000 ; in beef serum only in the 
 proportion of 1 : 150. In bouillon free from peptone 
 or containing one per cent peptone, cholera vibriones 
 are killed in one-half hour on the addition of 0.01 per 
 cent HCl ; on the addition of two per cent peptone, 
 only when 0.04 per cent HCl is added. For diag- 
 nostic purposes we will usually make the tests in one 
 * Phenol is said to be an exception. 
 
THE VITAL CONDITIONS OF BACTERIA. 93 
 
 per cent peptone solutions if we do not wish to use 
 one of the non-albuminous media described on page 
 86. At all events, the bacteria which are to be com- 
 pared must be treated in exactly the same way, and, 
 if the results are to be published, the various condi- 
 tions of the experiment must be described in detail. 
 Of bacteria which are free from spores not much is 
 known concerning their varying resistance according 
 to variety and nutrient medium, but a few statements 
 in this regard have been made concerning staphylo- 
 cocci (Esmarch: Z. H., V., 1889, p. 72). 
 
 A combination of disinfectants increases their 
 action. In particular, the addition of acid (hydro- 
 chloric or tartaric acid) intensifies the effect of subli- 
 mate, and also of solutions of phenol and cresol. 
 Moreover, the effect is more certain upon a few than 
 upon many germs, and greater at a higher than at a 
 lower temperature. 
 
 4 DEFICIENCY OF FOOD AND WATER. 
 
 If bacteria which require nutritious substances in 
 order to thrive are placed in distilled water, they 
 usually die rapidly (within a few days) . In spring 
 water (even when sterilized), the duration of life is 
 usually not more than eight to fourteen days, and 
 proliferation is rare. In a series of cases, however, 
 a much longer duration of life had been observed 
 {vide Loffler : " Das Wasser u. d. Mikroorg.," Fischer, 
 1896). The sensitiveness of bacteria to a deficiency 
 of water varies greatly. Upon drying nutrient media 
 growth soon ceases. On the other hand, the dura- 
 tion of life upon nutrient media (agar, gelatin, 
 
94 
 
 ATLAS OF BACTERIOLOGY. 
 
 potato) which are drying slowly at the temperature 
 of the room, is often astonishingly long, even when 
 this cannot be attributed to the development of endo- 
 spores. Even after the lapse of a year it is found 
 occasionally that such a shrivelled remnant of a cul- 
 ture furnishes the most beautiful cultures in bouillon. 
 
 The question has often been investigated — and with 
 very contradictory results — as to the length of the time 
 during which bacteria free from spores, which have 
 dried upon pieces of glass or threads of silk, may 
 remain alive. We know now that this is influenced 
 by numerous factors. An idea of their viability is 
 furnished by the following table of Sirena and Alessi 
 (C. B., XL, 484). 
 
 In bouillon cultures free from spores or in watery 
 deposits of bacteria silk threads were dipped, and 
 part of them were placed in test tubes one-third full 
 of sulphuric acid or calcium chloride, part were al- 
 lowed to dry in the open air under various conditions. 
 
 
 Period at which Death Occurred 
 (in Days). 
 
 On drying. 
 
 3 03 
 
 ^1 
 
 a . 
 II 
 
 5* 03 CO 
 
 S3 O 0) 
 
 5 o * 
 
 0. 
 
 03 O 
 O 
 
 t-H 
 
 Vibrio cholerce asiaticae 
 
 Bacterium cholerae gallinarum 
 Bacterium typhi 
 
 1 
 
 2 
 
 41 
 
 35 
 
 63 
 
 114 
 
 1 
 1 
 1 
 
 44 
 53 
 31 
 
 1 
 
 1 
 
 18 
 
 31 
 
 31 
 
 131 
 
 1 
 
 5 
 
 64 
 
 5 
 
 164 
 
 12 
 59 
 
 68 
 
 Bacterium mallei 
 
 Bacterium erysipelatos suum 
 Streptococcus lanceolatus 
 
 59 
 
 192 
 
 Cholera vibriones are especially well known for 
 their slight power of resistance to desiccation. Ex- 
 
THE VITAL CONDITIONS OF BACTERIA. 95 
 
 tended experiments of tlie authors just mentioned 
 put their duration of life at from one to five hours 
 according to the mode of desiccation (the results are 
 similar to those of E. Koch in his first experiments). 
 But it is evident from the results obtained by all 
 writers that desiccation experiments must be espe- 
 cially varied and many-sided, if they are to be con- 
 vincing. The surprising result has recently been 
 obtained in regard to very many varieties which are 
 sensitive to desiccation (this is true particularly of 
 the cholera vibriones) that, under certain circum- 
 stances, they may remain alive, when dried, for a 
 much longer time. Thus Koch found the duration 
 of life to be a few hours; Kitasato (Z. H., Y., 135) 
 fourteen days; French writers and Berckholz (A. G. 
 A., Y., 1) found it one hundred and fifty to two hun- 
 dred days under specially favorable conditions. Ac- 
 cording to most writers these favorable conditions in- 
 clude: stay in the desiccator, removal from agar or 
 potato cultures instead of bouillon cultures, the use 
 of silk threads instead of pieces of glass. Special 
 mention must also be made of the fact that in none 
 of these experiments could anything of the nature of 
 spores (arthrospores) be positively recognized. 
 
 5. RELATIONS TO OXYGEN AND SOME OTEHR GASES. 
 
 In their relations to oxygen bacteria are divided 
 usually into three classes (Fliigge and Liborius) : 
 
 /. Strict Aerobics. — Growth occurs only when the 
 air finds access; any obstruction to the latter inter- 
 feres with the growth. Free oxygen is particularly 
 necessary to the development of spores. 
 
96 ATLAS OF BACTERIOLOGY. 
 
 //. Strict Anaerobics. — Growth and sporulation 
 take place onl^^ during complete exclusion of oxygen. 
 This class includes bacillus oedematis maligni, bacil- 
 lus tetani, bacillus Chauvoei, and a large number of 
 inhabitants of slime and earth. When exposed to the 
 free oxygen of the atmosi)here the vegetative forms of 
 these bacteria perish very readily, but the spores ex- 
 hibit great resistance to oxygen. As the anaerobics 
 are excluded from the main supply of energy which 
 is at the command of aerobic bacteria (oxidation of 
 the absorbed nutritive material by means of free oxy- 
 gen) , they must rely upon nutritive substances which 
 possess great potential energy and which, like grape 
 sugar, for example, set free energy (heat) by sei)ara- 
 tion into two smaller molecules (for exami:>le, alcohol 
 and carbonic acid ; or acetic and lactic acids) . Hence 
 anaerobics are almost always cultivated upon gelatin 
 or agar which contains one to two per cent of grape 
 sugar. 
 
 III. Facultative Aerobics and Facultative Anae- 
 robics. — The large majority of the bacteria which, as 
 a rule, are cultivated aerobic (including almost all 
 the pathogenic forms) tolerate a restriction in the 
 supply of oxygen without suffering injury or ex- 
 hibiting diminished growth. In many cases life 
 in the animal body, for example in the intestinal 
 canal, decidedly involves a diminution or aboli- 
 tion of the supply of oxygen. When oxygen is 
 excluded the formation of pigment is almost al- 
 ways abolished, while virulent products of dis- 
 assimilation are produced in greater abundance 
 (Hiippe). 
 
 It is a very important fact that recent investigations 
 
THE VITAL CONDITIONS OF BACTEBIA. 97 
 
 have shown that aerobic races exist among the anae- 
 robic varieties. 
 
 It is observed not very rarely that varieties which 
 on isolation exhibited more or less anaerobic growth 
 (for example, grew chiefly into the depth of the agar 
 stick canal), in time manifest a purely aerobic con- 
 dition, i.e., distinct growth upon the surface and 
 dwarfed growth in the canal. 
 
 These observations show that two varieties cannot 
 be distinguished from one another by simply calling 
 one aerobic, the other anaerobic. 
 
 In addition to the strict anaerobics all the faculta- 
 tive anaerobic varieties thrive well in nitrogen and 
 hydrogen, but they tolerate carbonic acid in various 
 ways. 
 
 A large number do not flourish at all, but their 
 development is entirely checked until oxygen is again 
 supplied— for example, bacillus anthracis, bacillus 
 subtilis, and allied forms. Of several varieties (an- 
 thrax, cholera) it has been ascertained that the major- 
 ity of individuals are killed very quickly by carbonic 
 acid, while certain ones oif er a very vigorous resistance 
 and render complete sterilization by CO^ impossible. 
 A second group exhibits — especially when the ex- 
 periment is made at incubating temperature — feeble 
 growth (staphylococci, streptococci), while a third 
 group is not at all injured (bacterium prodigiosum, 
 bacterium acidi lactici, bacterium typhi). These 
 grow as well as they do in the air, and the liquefac- 
 tion of the gelatin is not interfered with. As a matter 
 of course, pigment is not formed on account of the 
 absence of oxygen. A mixture of twenty -five per cent 
 air with seventy-five per cent carbonic acid exerts no 
 7 
 
98 ATLAS OF BACTERIOLOGY. 
 
 demonstrable injurious influence upon bacteria which 
 remain absolutely undeveloped in pure carbonic acid 
 (C. Fraenkel: Z. H., Y.) 
 
 Sulphuretted hydrogen in large amounts is al- 
 ways an active bacterial poison; small amounts kill 
 the bacterium Pfliigeri very rapidly (Lehmann and 
 Tollhausen: C. B., V., 785). 
 
 6. INFLUENCE OF TEMPERATURE ON THE LIFE OF 
 BACTERIA. 
 
 Each variety of bacteria makes certain demands 
 upon the temperature of its nutritive medium. Vege- 
 tative bacterial life is possible from 0° to about 70°, 
 but there are some varieties which flourish at the 
 lower range, others at the upper range. In each 
 variety the minimum and maximum of temperature 
 are separated by about 30°, and the following com- 
 prehensive classification may be made according to 
 the temperature requirements : 
 
 PsychropMlic bacteria : minimum at 0°, best at 15°- 
 20°, maximum at about 30°. These varieties usually 
 live in the water. They include, for example, many 
 phosphorescent bacteria of the ocean (vide Forster : 
 C. B., XII., 431). 
 
 Mesophilic bacteria: minimum at 10°-15°, best at 
 37°, maximum at about 45°. These include all the 
 pathogenic varieties, because acclimatization to the 
 bodily temperature is a necessary condition of their 
 pathogenic action. 
 
 Bacillus vulgatus, * which still thrives at 50°, fur- 
 nishes a transition to the following group. 
 
 * Bacillus vulgatus thrives at from 15°-50°, and odg variety of 
 Globig's ranges from 5r-68°, but such cases are very rare. Glo- 
 
THE VITAL CONDITIONS OF BACTERIA. 99 
 
 Thermophilic bacteria: minimum at 40°-49°, best 
 at 50°-55°, maximum at GO'^-TO". These include many 
 bacteria of the soil, and almost all the sporulating 
 bacilli related to bacillus mesentericus. According to 
 Globig about thirty varieties are still capable of de- 
 velopment at 60°, and a few at 70° (Z. H., III., 294). 
 Miquel {Ann. de Micrograph., I., 4; C. B., V., 281) 
 has described a bacillus thermophilus Miqu. , which 
 thrives at from 42°-72°, best at 65°-70°, and has its 
 habitat in i)rivies, the intestinal contents, and dirty 
 water. The description is insufficient to distinguish 
 the bacillus. 
 
 Kecently Lydia Rabinowitsch has described eight 
 thermophilic facultative anaerobic varieties; they 
 were all non-motile sporulating rods*, which throve 
 best at 60°-70°, but proliferated slowly even at 34°-44°, 
 best in an anaerobic agar culture (Z. H., XX., 163). 
 These varieties are widely diffused, particularly in 
 the faeces, but Rabinowitsch did not make any com- 
 parison with the forms described by previous writers. 
 
 By gradually increasing and lowering the temper- 
 ature Dieudonne (C. B., XVI., 965) succeeded in in- 
 creasing the temperature interval within which the 
 bacillus anthracis is capable of proliferating. The 
 bacillus could be adapted gradually to a temperature 
 of 42°. According to the assumption of some writers 
 pigeons are tolerably immune to ordinary anthrax on 
 account of their high temperature (42°), but when 
 the bacilli had been adapted to high temperatures 
 the pigeons died more frequently after inoculation. 
 
 Still more striking;; Tfcre , the results Vh^n DiQudonne 
 
 big found unusuaDy narrow ranges for many thermopuile varie- 
 ties; for example, oueiorm §rew pirly at;'bet\^?('ji^r'\65*.\ ' 
 
100 ATLAS OF BACTERIOLOGY. 
 
 gradually acclimatized the bacilli to a temperature of 
 12° and showed that they could then kill frogs which 
 are kept at 12°. 
 
 Temperatures somewhat below the minimum for 
 the variety in question inhibit the development but 
 are not otherwise injurious. Petruschky has recently 
 recommended keeping them in an ice-box (about 
 4°-6°). He claims that in this way varieties which 
 perish easily can be kept not only alive and capable 
 of proliferation but also virulent, after they have 
 been allowed to grow for two days at a temperature 
 of 20° (streptococci, etc.). 
 
 Temperatures below 0° also act very slowly and 
 injure the different varieties with varying rapidity. 
 
 If temperatures 5° -10° above the best act upon the 
 culture, the latter is injured in various ways. Eaces 
 of diminished intensity of growth develop, the viru- 
 lence and fermentative power diminish, and the capa- 
 bility of sporulation is gradually lost. The injurious 
 influence sometimes predominates in one direction, 
 sometimes in another. If the maximum temperature 
 is exceeded, the culture dies. For the psychrophilic 
 forms about 37°, for the mesophilic forms about 60°, 
 for the thermophilic forms 75°, are quite rapidly 
 fatal temperatures. No bacterium free from spores 
 can tolerate a temperature of 100° even for a few 
 minutes. 
 
 7. MECHANICAL AND ELECTRICAL EFFECTS. 
 
 OiIj: xuttur^ea- are. jr^a^e, almgst exiclusively upon 
 nutri^nffc. j^iedi^.^hicJi.aiTe k^eptrquje;; (it is only to 
 secjaEOr abundant sporulation in ^uid media, in the 
 
THE VITAL CONDITION'S OF BACTERIA. 101 
 
 case of aerobic varieties, that a slight movement of 
 the fluid is usually secured). Hence a theoretical in- 
 terest alone attaches to the fact that, according to 
 Meltzer's recent investigations, brief or feeble shak- 
 ing of bacteria cultures in vessels one-third full acts 
 favorably on the development of the bacteria, while 
 constant and vigorous shaking for a number of hours, 
 especially when balls, of glass are placed in the fluid, 
 scatters the bacteria into a fine dust and kills them. 
 The various bacteria act in different ways (Ztschr. f. 
 Biolog., XXX., p. 454). 
 
 Meltzer makes the very remarkable statement that 
 the feeble tremor which a steam engine running day 
 and night communicated to the floor of a brewery 
 was sufficient to kill, in four days, all the germs of 
 bacillus mycoides and subtilis kept in a bottle of 
 nutrient fluid. 
 
 Concerning our scanty knowledge of the influence 
 of the electrical current upon bacteria, vide Frieden- 
 thal: C. B., Part L, XIX., 319. 
 
 The majority of the effects of the electrical currents 
 hitherto observed are readily explained by the action 
 of heat and electrolysis. 
 
 8. EFFECT OF LIGHT. 
 
 The development of many bacteria, perhaps of the 
 majority, is impeded by the action of diffuse daylight 
 upon the cultures, and still more by the action of 
 direct sunlight. After a time the bacteria lose the 
 power of proliferating freely in the dark and we ob- 
 tain a generation of feeble organisms ; for example, 
 they liquefy imperfectly, form pigment imperfectly, 
 
102 ATLAS OF BACTERIOLOGY. 
 
 are less pathogenic, etc. It is only after repeated 
 transference to fresh nutrient media in the dark that 
 they regain their old power. When the action of 
 light is still more prolonged the micro-organisms die. 
 In order to test the sensitiveness to light it is best, 
 according to H. Buchner, to expose to diffuse light 
 or to sunlight densely crowded plates of gelatin or 
 agar, a black paper cross being pasted on the light 
 side. In order to exclude the action of heat the light 
 may first be passed through a layer of water or 
 alum a few centimeters in thickness. After exposure 
 to the light for one-half, one, one and a half, two 
 hours, etc. , the plates are placed in the dark and it is 
 noted whether the bacteria develop only at the loca- 
 tion of the cross. When all the colonies which were 
 illuminated have perished, we find a sharply defined 
 cross, formed of cultures in a light field. 
 
 During March, July, and August bacteria putidum 
 and prodigiosum are killed in one-half hour by direct 
 sunlight. In November, at the end of one and a half 
 hours, their power of producing pigment and tri- 
 methylamin is interfered with materially, they grow 
 slowly, and bacterium prodigiosum liquefies poorly. 
 The organisms died in one and a half and two and a 
 half hours. 
 
 In diffuse daylight, inhibition of development oc- 
 curs in the spring and summer in three and a half 
 hours, in winter in four and a half hours; death 
 occurs in from five to six hours. The electric arc 
 light, of 900 candle power, inhibited development in 
 ^ye hours, and killed the germs in eight hours. 
 Bacterium coli, bacterium typhi, and bacillus anthra- 
 cis reacted in a similar manner. 
 
THE VITAL CONDITIONS OF BACTERIA. 103 
 
 The ultra violet, violet and blue light have a power- 
 ful injurious effect, green light has a feeble effect, 
 and red and yellow have none at all. 
 
 The action of light seems to be dependent in part 
 on the oxygen of the air. Strict anaerobic (tetanus) 
 and facultative anaerobic varieties (bacterium coli) 
 tolerate sunlight very well if there is complete exclu- 
 sion of oxygen. 
 
 Richardson and recently Dieudonne have discov- 
 ered a fact which possesses great importance in re- 
 gai-d to the mechanism of the action of light, al- 
 though it does not explain everything. They found 
 that hydrogen hyperoxide (H^OJ develops in a short 
 time (in ten minutes in direct sunlight) upon illumi- 
 nated agar plates, but only in blue to ultra violet 
 light. * An agar plate, half covered with black paper, 
 is exposed to the light, then a paste containing a 
 small amount of potassium iodide is poured over it 
 and this followed by a weak solution of sulphate of 
 ferric oxide, the illuminated side turns a bluish-black. 
 In gases which contain no oxygen H.,02 does not form 
 and light does not give rise to any injury. This 
 also explains the fact that slight " attenuation" of the 
 bacilli is also observed frequently when agar plates 
 which have been standing in the sun f are inocu- 
 lated. Bacteria which have been previously ex- 
 posed to the light develop with special difficulty on 
 an illuminated nutrient medium. 
 
 * Hours elapse before H2O2 can be demonstrated upon gelatin. 
 
 f Other decompositions of the nutrient media by sunlight may 
 interfere occasionally with the subsequent growth of bacteria, 
 for example, the development of formic acid from tartaric acid 
 (Duclaux) . 
 
104 ATLAS OF BACTERIOLOGY. 
 
 9. EFFECT OF OTHER BACTERIA UPON BACTERIAL 
 GROWTH. 
 
 Althougli it is the object of every bacteriologist to 
 obtain only pure cultures, it must not be forgotten 
 that in nature bacteria often appear in mixed cul- 
 tures. When water, milk, the intestinal contents of 
 sick or healthy individuals, etc., are examined, sev- 
 eral varieties will always be found at the same time. 
 Although this admixture usually appears to be 
 purely accidental, it is found on closer investigation 
 that, in the domain of bacteriology, there are syn- 
 ergetic (favoring the growth of one another) and 
 antagonistic (injuring one another) varieties. Nencki 
 speaks of symbiosis and enantobiosis. 
 
 Garre demonstrated the antagonism experimentally 
 by making streak cultures of various bacteria upon 
 gelatin plates, in the shape of parallel or intersect- 
 ing lines. It was then found that certain varieties 
 thrive very poorly or not at all when another variety 
 is growing in their immediate neighborhood. In 
 very many cases the antagonism is one-sided. For 
 example, bacterium putidum grows very well when 
 inoculated between closely approximated, well-devel- 
 oped streaks of staphylococci. On the other hand, 
 micrococcus pyogenes does not grow when inoculated 
 between luxuriantly developing cultures of bacterium 
 putidum, and the former remains very meagre when 
 both varieties are applied in streak cultures at the same 
 time (Garre: Corresp. f. Schweizer Aerzte, 1887). 
 
 Or we make plates of gelatin or agar (for liquefy- 
 ing varieties) which have been infected, in the melted 
 
THE VITAL CONDITIOl^S OF BACTERIA. 105 
 
 condition, with an equal number of individuals of two 
 different varieties of bacteria. In many cases only 
 one variety will undergo development (Lewek: C. B., 
 VII., 107). 
 
 The following is the third method of making the 
 experiment. The same fluid nutrient medium is in- 
 oculated with two varieties and later we ascertain the 
 victor in the struggle, either with the microscope or 
 macroscopically upon thin plates. To this category 
 belongs the frequent experience that fermentation- 
 producers, when present in large numbers in a suit- 
 able medium, prevail over contaminating bacteria. 
 The latter sometimes disappear entirely. 
 
 The following practical inference may be drawn 
 from these experiences. In counting bacteria very 
 dense plates may not be regarded as decisive, and in 
 the isolation of certain varieties thin plates may also 
 be necessary. For example, in isolating bacterium 
 Pfliigeri from an abundance of bacterium putidum; 
 no bacteria Pfliigeri grow within a circle of several 
 millimetres around each culture of bacterium putidum 
 (K. B. Lehmann). 
 
 Finally, bacteria may antagonize one another with- 
 in the animal body. As Emmerich showed, animals 
 infected with anthrax may be saved by subsequent 
 inoculation with streptococcus pyogenes. It is im- 
 possible to enter into the mechanism of this process 
 within the limits of this work. 
 
 Greater practical importance attaches to the sym- 
 biosis of bacteria, as is shown by the following 
 examples. 
 
 1. A series ol bacteria thrive better in company 
 with others than alone. Certain anaerobics even 
 
106 ATLAS OF BACTEKIOLOGT. 
 
 thrive on the admission of air, if other aerobic varie- 
 ties are present {vide bacillus tetani). 
 
 2. Certain chemical actions, for example, the de- 
 composition of nitrate into gaseous nitrogen cannot 
 be effected by some bacteria alone, while it can be 
 done by two forms in combination. This experience 
 is to be remembered in looking for the xjrodncers of 
 certain decompositions. When the isolated varieties 
 do not act singly or act incompletely, combinations 
 must be examined. 
 
 3. In a similar way it has been observed, for ex- 
 ample, that among a series of soil bacteria each 
 single variety is not pathogenic, while certain com- 
 binations, when introduced into the animal, make 
 the latter sick. This experience also merits special 
 attention in the search for the producers of a new or 
 obscure disease. 
 
 Some writers also assume the production of cholera 
 by two germs (diblastic theory). 
 
 4. Feeble pathogenic varieties (for example, atten- 
 uated tetanus bacilli) are said to gain in virulence 
 when cultivated with bacterium vulgare. 
 
 D. The Conditions of Formation and Germi- 
 nation of Spores. 
 
 Biological Characters of Spores. 
 
 The extent of the formation of endogenous spores 
 appears to be imperfectly known at the present time. 
 Apart from a large group of bacilli which are re- 
 lated to bacillus anthracis and bacillus tetani, un- 
 doubted endogenous spores are known only in sarcina 
 
FORMATION AND GERMINATION OF SPORES. 107 
 
 pulmonum and the peculiar spirillum endoparagoci- 
 cum. 
 
 As H. Buchner (C. B., YIII., 1) showed, the for- 
 mation of spores takes i^lace in suitable varieties 
 when the nutrient medium is beginning to be ex- 
 hausted, i.e., it is most rapid in very poor media. 
 
 On the other hand, a good nutrient medium not 
 alone facilitates the development of the bacilli but 
 also that of the spores, in so far as the vigorously 
 growing bacilli also sporulate luxuriantly and con- 
 stantly. The crop of spores is disproportionately 
 large. Whether the quality (power of resistance) of 
 the spores, which grow upon different nutrient media, 
 also differs, does not seem to have been investigated 
 methodically. 
 
 The temperature must sometimes (always ?) be 
 higher for sporulation than for vegetative growth. 
 For example, the bacillus anthracis flourishes at 
 13°-14°, but does not form spores under 18°. 
 
 All aerobic bacteria require the entrance of oxygen 
 particularly for sporulation. The mode in which 
 facultative anaerobic varieties act has not been as- 
 certained. 
 
 Strict anaerobics produce spores only on the exclu- 
 sion of oxygen or on the admission of oxygen in 
 mixed cultures or when synergetic bacteria have 
 perished. 
 
 Spores never germinate in the exhausted nutrient 
 medium in which they have been formed, or which 
 has been affected injuriously by the products of dis- 
 assimilation. It is only after removal to a new nu- 
 trient medium that germination takes place (the mor- 
 phological details have been described on page 79) . 
 
108 ATLAS OF BACTERIOLOGY. 
 
 Spores are much more resistant than vegetative 
 forms to all injurious influences. They require no 
 nourishment or water in order to remain capable of 
 germination for years and decades,* they are much 
 more indifferent to gases than bacilli, and the spores 
 of anaerobic varieties usually tolerate free oxygen 
 welLt 
 
 The power of resistance of the spores to dry and 
 moist heat is very considerable. Dr^^ heat is toler- 
 ated relatively very well, and many spores resist a 
 temperature of 100°. In the moist condition a tem- 
 perature of 70° kills the anthrax bacillus in one 
 minute, while the spores resist this temperature for 
 hours, and in water or steam at 100° they live from two 
 to ^Ye minutes, occasionally even from seven to twelve 
 minutes. The varying resistance of different anthrax 
 spores (v. Esmarch: Z. H., Y., p. 67) seems to be 
 partly a race peculiarity. It is very probable, more- 
 over, that the nutrient medium, the temperature at 
 the formation of the spores, the degree of maturity, 
 etc., also exert an influence upon the resistance. 
 Careful investigations on this subject are almost en- 
 tirely lacking, but Percy Frankland has shown that 
 spores formed at 20° are more resistant to light than 
 those formed at incubation temperature (C. B., XV., 
 p. 110). 
 
 * According to an observation of v. Esmarch the virulence of 
 anthrax spores seems to be lost, in the course of time, before 
 their power of germination. 
 
 f Dry garden earth containing the spores of malignant oedema 
 preserved the latter excellently in my laboratory for four y^ars. 
 On the other hand, tetanus spores which were dried on threads 
 and kept in the room had perished at the end of three days ; 
 they were still alive on the second day. 
 
FORMATION- AND GERMINATION" OF SPORES. 109 
 
 The resistance is tested by simply hanging in the 
 steam chamber little tulle bags containing fragments 
 or bits of glass upon which anthrax spores have been 
 dried. From minute to minute a bag is removed and 
 the bits of glass placed upon an agar plate which is 
 kept at incubating temperature. Anthrax spores are 
 obtained by careful removal of sporulating agar streak 
 cultures, and warming the emulsion, prepared with 
 little water, to 70° for five minutes. 
 
 The varying resistance of apparently identical an- 
 thrax spores possesses great practical importance: 
 (1) in disinfection tests which may be made only 
 with spores of known resistance ; (2) in differential 
 diagnosis, because it shows that we must be on our 
 guard against creating two species based on a differ- 
 ence in resistance. 
 
 Various forms which occur in hay and soil possess 
 remarkable resistance. 
 
 Christen found (C. B., XVII., p. 498), for example, 
 that in steam under pressure the resisting spores of 
 the soil required for their destruction : At 100°, more 
 than sixteen hours; 105°-110°, two to four hours; 
 115°, thirty to sixty minutes; 125°-130°, five minutes 
 or more; 135°, one to five minutes ; 140°, one minute. 
 The apparatus raised objects very rapidly to the de- 
 sired temperature. 
 
 Spores are also very resistant to chemical agents. 
 Thus, anthrax spores require, according to their origin 
 (v. Esmarch : I. c.) a five-per-cent solution of carbolic 
 acid at least two days, in some cases even forty days. 
 A one-per-cent aqueous solution of corrosive subli- 
 mate is withstood by very resistant anthrax spores as 
 much as three days, although their virulence was lost 
 
110 ATLAS OF BACTERIOLOGY. 
 
 in twenty hours. These tests are made best with thin 
 deposits of the spores in water, to which the disin- 
 fectant is added, as we have indicated above in regard 
 to the tests of antiseptic action against bacilli. 
 
 In order to test the resistance of spores to gases it 
 is best to dry them upon pieces of glass ; the gases 
 are allowed to act first in a dry chamber, then in one 
 saturated with water. 
 
 Spores are also less damaged by light than bacilli 
 are; as in the case of bacilli an oxygenated atmos- 
 phere is necessary in order to produce injury by 
 light. Anthrax spores on agar plates were found by 
 Dieudonne to be killed by direct sunlight in three 
 and a half hours (bacilli in one and a half hours) ; 
 when oxygen was excluded they were not injured by 
 exposure for nine hours. 
 
 E. The Effects of Bacteria, Especially in Re- 
 gard to Their Employment for Diagnostic 
 Purposes. 
 
 The effects of bacteria* in vitro may be classified 
 as (1) mechanical; (2) thermal; (3) optic; and (4) 
 chemical. They will be discussed in this order and 
 a fifth section will deal with the effects of bacteria 
 upon the living animal body and will explain the 
 guiding principles necessary to the comprehension of 
 
 * It goes without saying that a classification of bacteria into 
 zymogenous, saprogenous, chromogenous, and pathogenic, is no 
 longer admissible. For example, bacterium coli produces fer- 
 mentation in solutions of sugar, indol and sulphuretted hydrogen 
 in albuminous media, brownish -yellow foci upon potatoes, and is 
 pathogenic to guinea-pigs, i.e., it combines the characteristics 
 of all four groups. 
 
THE EFFECTS OF BACTERIA. Ill 
 
 their pathogenic iDfluence, the struggle between the 
 bacteria and the tissue cells. 
 
 All the effects of bacteria depend: (1) upon the 
 present condition of the bacteria; (2) upon the nu- 
 trient medium ; (3) upon the entrance of air ; (4) upon 
 the temperature; and (5) upon the illumination. A 
 large number of other circumstances — in part less im- 
 portant, in part imperfectly known — also appear to 
 play a part. 
 
 As the most important points in reference to tem- 
 perature and illumination have already been given, 
 I will discuss chiefly the influence of the nutrient 
 medium and the entrance of air on the one hand, 
 and the composition of the terminal culture on the 
 other hand. Emphasis must be constantly laid upon 
 the latter point in order to show as clearly as pos- 
 sible how much the effects of bacteria vary according 
 as they are examined in a fully virulent zymogenic, 
 chromogenic, or pathogenic condition, or in an attenu- 
 ated condition. 
 
 1. MECHANICAL EFFECTS. 
 
 Under the microscope it is readily seen that many 
 bacteria exhibit a pronounced active movement, and 
 the study of flagella proves that almost all the mo- 
 tile varieties* present flagella and move by means of 
 these appendages. The movement varies greatly in 
 character; for example, creeping (bacillus megathe- 
 rium), waddling (bacillus subtilis), sinuous (vibri- 
 
 * In the actively motile spirochsBte Obermeieri and the slowly 
 creeping beggiatoa flagella have not been demonstrated, so that 
 the motion is supposed to be due to an undulating narrow mem- 
 brane which encloses the organism. 
 
112 ATLAS OF BACTERIOLOGY. 
 
 ones). It is sometimes very slow, sometimes so 
 rapid that observations in detail are hardly possible 
 (bacterium typhi) . 
 
 In some cases it is difficult to decide whether there 
 is a real active movement or whether the micro-organ- 
 isms do not exhibit an unusual degree of the so- 
 called Brownian molecular movement — i.e., the danc- 
 ing and trembling which are also found in finely 
 divided, non-organized particles. In such cases, 
 apart from the attempt to render the flagella visible, 
 it is well to examine the organism in a drop of five- 
 per-cent carbolic acid or one-per-cent corrosive subli- 
 mate. If the movements then continue, we have had 
 to deal only with molecular movements. Some 
 varieties do not always exhibit movements of their 
 own, but they may be absent in certain nutrient 
 media. According to A. Fischer the vital movements 
 may be lacking, although the flagella are perfectly 
 developed — for example, in bacillus subtilis upon a 
 nutrient medium containing two to four per cent 
 ammonium chloride. In two different cultures of 
 micrococcus agilis Ali-Cohen, drawn from a good 
 source, we saw neither vital movements nor flagella, 
 and reached the conclusion that the same variety 
 may occur with or without flagella. 
 
 Certain chemical substances attract bacteria (posi- 
 tive chemotaxis), others repel them (negative chemo- 
 taxis). Oxygen in particular attracts aerobic, and 
 repels anaerobic bacteria. As Beyerinck showed, 
 very beautiful chemotaxic or aerotaxic figures can be 
 obtained in the following way : In a test tube filled 
 three-quarters full with sterilized water is placed an 
 unsterilized bean, pea, or the like. By diffusion the 
 
THE EFFECTS OF BACTERIA. 113 
 
 bean gives off nutritive substances, which slowly ex- 
 tend ui^ward. In this feeble nutrient solution cer- 
 tain bacteria which have been introduced with the 
 bean develop in sharply defined horizontal planes, 
 which slowly ascend. Certain varieties form several 
 planes above one another. I have had these interest- 
 ing statements investigated by Mr. Miodowski, who 
 corroborated them in great measure. But instead of 
 the non-sporulating bacillus perlibratus Bey., which 
 usually formed the planes in Beyerinck's experi- 
 ments, we found chiefly an organism allied to ba- 
 cillus mesentericus and bacillus subtilis {vide Bey- 
 erinck: C. B., XIV., 827, and Miodowski: Diss., 
 Wiirzburg, 1896). 
 
 Schenk has observed a positive thermotropism. 
 If a hanging drop containing bacteria is warmed at 
 one point with a warm wire (temperature difference 
 8°-10°) the bacteria congregate in that spot (C. 
 B., XIY.). 
 
 2. OPTICAL EFFECTS. 
 
 Phosphorescent bacteria are distributed quite 
 widely, especially in and near salty media (the ocean, 
 rivers, salted fish) , and a considerable number — main- 
 ly bacilli and vibriones — have been carefully studied. 
 Phosphorescence is a vital symptom and does not 
 depend upon the oxidation of a photogenic substance 
 secreted by the bacteria (K. B. Lehmann and ToU- 
 hausen : C. B. , V. , 785) . It is destroyed by all factors 
 which injure the life of the bacteria; cold produces 
 rigidity of the organisms and interrupts the phos- 
 phorescence as long as it lasts. High temperatures, 
 acids, chloroform, etc., interfere temporarily with the 
 8 
 
114 ATLAS OF BACTERIOLOGY. 
 
 phosphorescence. Living bacteria can always be ob- 
 tained from phosphorescent cultures, and a filtered 
 culture free from germs is always devoid of phos- 
 phorescence. But although the organism cannot give 
 light without life, it may live without giving light — 
 for example, in an atmosphere of carbonic acid. In 
 like manner the muscles cannot contract without life, 
 but they may be alive without contracting. 
 
 According to Beyerinck (C. B., YIII., pp. 716 and 
 651), who includes all phosphorescent bacteria in one 
 (physiological) genus, photobacterium, they require 
 peptone and oxygen in order to produce light. Four 
 of his six varieties also require, in addition to pep- 
 tone, a supply of carbon which may also contain 
 nitrogen. Small amounts of sugar (dextrose, levu- 
 lose, galactose, maltose), glycerin, and asparagin act 
 in this way. In some varieties a higher percentage 
 of sugar causes cessation of the phosphorescence, 
 after the formation of acids and marked fermentation. 
 
 When the phosphorescence is to be maintained, we 
 would recommend a gelatin nutrient medium, made 
 by cooking fish in sea water, to which one per cent 
 peptone, one per cent glycerin, and one-half per 
 cent asparagin have been added. But even in this 
 medium phosphorescence is soon lost if inocula- 
 tions are infrequent, so that in the majority of labor- 
 atories the phosphorescent bacilli do not emit light. 
 By repeated rapid transfers to a suitable nutrient 
 medium we can often succeed in restoring the photo- 
 genic power. I recommend that two salt herrings be 
 cooked in one litre of water, and ten per cent gelatin 
 added to the filtrate without neutralization. 
 
THE EFFECTS OF BACTERIA. 115 
 
 3. THERMIC EFFECTS. 
 
 The development of heat during the metabolism 
 of bacteria is not noticeable in our ordinary cultures 
 on account of its slight amount. Even luxuriantly 
 growing, fermenting fluid cultures do not reveal to the 
 hand any noticeable production of heat. 
 
 But there is no doubt, on the other hand, that the 
 heat given out by moist decomposing organic matters, 
 such as beds of tobacco, hay, manure, etc., depends, 
 at least in part, on bacterial activity. In view of the 
 high temperature produced, it is very probable, ac- 
 cording to Lydia Kabinowitsch, that the thermophilic 
 bacteria take part in the process. Careful investiga- 
 tions concerning the producers of these high temper- 
 atures are still wanting (vide Eabinowitsch : Z. H., 
 XX., 163). 
 
 4. CHEMICAL EFFECTS. 
 
 The chemical actions of bacteria, which are accom- 
 panied in part by the production of light, and always 
 by the production of heat, are known only in their 
 main outlines, despite the extremely numerous and 
 successful investigations of the last twenty-five years. 
 In many cases we know only the final products, and 
 have no accurate information concerning the mechan- 
 ism of their development, the intermediate pro- 
 ducts, and the substances which appear in small 
 quantities. 
 
 We may distinguish the following three principal 
 varieties of chemical efl'ects : 
 
 1. The bacteria store up their cell substance. 
 
116 ATLAS OF BACTERIOLOGY. 
 
 2. The bacteria excrete ferments, designed to make 
 the surrounding nutrient medium more suitable for 
 assimilation. Tho products which develoj^ at this 
 time in the vicinity of the bacteria may be called 
 transformation products. 
 
 3. The bacteria assimilate some substances and 
 excrete others — true products of disassimilation. A 
 separation of fermentative products and disassimila- 
 tive products, such as is still attempted at times, is 
 incorrect because the substances are only fermented 
 when thej^ have previously entered the bacterium 
 cell. Hence fermentation products are products of 
 disassimilation under the influence of special nutri- 
 tion (vide page 124). 
 
 I. Bacterial Ferments and the Changes Produced 
 BY Them. 
 
 Under the term ferments in the narrower sense 
 (enzymes) we refer to chemical bodies which, in mini- 
 mum amounts and without being used up, are able to 
 separate large amounts of complicated organic mole- 
 cules into simple, smaller, more soluble and diffusible 
 molecules.* 
 
 Ferments may be regarded as chemical only when 
 we can prove : 
 
 1. That the fermentation continues in the presence 
 of substances (for example phenol, three per cent; 
 thymol, .01 per cent; chloroform, ether) which kill 
 bacteria but do not endanger ferments ; or 
 
 2. That the germless filtrate of the bacterial culture 
 
 * This definition does not hold good for a single ferment, the 
 milk ferment, which coagulates the milk {nde page 123) . 
 
THE EFFECTS OF BACTBBIA. 117 
 
 through a cIslj or porcelain cylinder possesses the 
 power of fermentation ; or 
 
 3. That this power inheres in a sterile preparation 
 of the ferment, made in the shape of a powder. 
 
 Of the extremely numerous details which we have 
 learned from Fermi's methodical and thorough inves- 
 tigations, we can here give only the most important. 
 All ferments dialyze as little as ordinary albuminoids 
 through good parchment paper. 
 
 Proteolytic — i.e., albumin-dissolving enzymes — are 
 widely distributed. The liquefaction of the gelatin, 
 which is chemically allied to albumin, in our nutrient 
 media is sure evidence of the presence of a proteolytic 
 ferment. As the reaction at which the gelatin is dis- 
 solved is always or may be alkaline, the bacteria 
 cultures do not contain pepsin (which is effective only 
 with acid reaction) but trypsin. The different bac- 
 terio-trypsins vary greatly in their resistance to heat 
 (in a moist condition they tolerate a temperature of 
 from 55°-70° for one hour), their sensitiveness to dif- 
 ferent acids, etc. Some are efficient even when a con- 
 siderable amount of acid has been added, but they 
 never act better than in an alkaline reaction. 
 
 The action on fibrin is much weaker than that on 
 gelatin, and hence Fermi has recommended the fol- 
 lowing method as the most convenient and certain 
 demonstration of the presence of even a trace of pro- 
 teolytic ferment. A non-neutralized solution is made 
 of about seven per cent gelatin in one per cent car- 
 bolic acid and equal amounts are placed in test tubes 
 of the same size. The solution to be tested for the 
 ferment is then placed on the solid gelatin, after re- 
 ceiving two per cent carbolic acid. We can then read 
 
118 ATLAS OF BACTEKIOLOGY. 
 
 off on a millimetre scale, at the temperature of the 
 room, the rate at which the liquefaction of the gelatin 
 proceeds for days and weeks. Qualitative tests may 
 be simply made by using 1 c.c. of a liquefied gelatin 
 culture which has been sterilized with carbolic acid. * 
 This material also suffices in testing the influence of 
 the nutrient medium upon the formation of the fer- 
 ment. By this method we may also compare the 
 action of different degrees of concentration of differ- 
 ent bacterio-trypsins. The less the percentage of 
 gelatin and the nearer the temperature to incubating 
 temperature, the more certainly do we obtain the 
 action of even traces of ferment. In such critical 
 cases the experiment is continued for two weeks and 
 we then note whether the test tubes in the refrigera- 
 tor, provided with the ferment, remain fluid, while 
 the control tubes remain rigid. 
 
 In order to demonstrate the production of a true 
 peptone, we proceed in the following way : 
 
 The variety of bacteria in question is cultivated 
 upon a fluid albuminous nutrient medium free from 
 peptone (blood serum, milk serum, milk). If the 
 culture grows well, all the albuminoids, with the 
 exception of the peptone, are precipitated by the ad- 
 dition of solid ammonium sulphate (about 30 gm. to 
 20 c.c). Milk and milk serum may be warmed to 
 60°-80°, blood serum to about 40°. The precipitate 
 is then filtered, the filtrate cooled; a part is made 
 strongly alkaline by the addition of potash, and one- 
 per-cent solution of copper sulphate is then added 
 
 * As a matter of course we must never fail to make a control 
 test with two-per-cent solution of carbolic acid in water (free 
 from germs) . 
 
THE EFFECTS OF BACTERIA. 119 
 
 drop by drop. The appearance of a rose color indi- 
 cates the presence of peptone.* 
 
 The formation of proteolytic ferments varies in 
 many, perhaps in all, species to a much greater extent 
 than we would imagine from the ordinary descrip- 
 tions. In the case of two phosphorescent vibriones 
 Beyerinck found that one which at first liquefied 
 gelatin very slowly, did so more rapidly after longer 
 culture, while the other variety acted in the opposite 
 way. Katz made a similar observation in experi- 
 ments on Australian phosphorescent bacteria. Max 
 Gruber and Firtsch have watched very closely the 
 development of feebly liquefying races in vibrio pro- 
 teus (A. H., VIII., 369), and similar statements have 
 been made concerning cholera vibrio, bacterium 
 vulgare, and micrococcus pyogenes. Indeed, some 
 observers have even seen a liquefying streptococcus 
 pyogenes. 
 
 We have also observed in many varieties that on 
 thin plates the individual distinctly visible, super- 
 ficial colonies exhibit very different degrees of lique- 
 faction. In fact a beginner would be convinced that 
 he had to deal with several varieties. 
 
 It is to be regretted that, as a result of these obser- 
 vations, one of the most convenient diagnostic aids, 
 viz., the liquefaction of gelatin, has lost consider- 
 ably in value. 
 
 The causes of the increase and decrease of liquefac- 
 tion with prolonged culture are looked for in our 
 artificial nutrient media, or in the influence of the 
 
 * Recent investigations have shown, however, that in addition 
 to peptone a few albumoses remain unprecipitated in part by 
 ammonium sulphate. 
 
120 ATLAS OF BACTEEIOLOGY. 
 
 products of disassimilation of tlie micro-organism, 
 but we are unable to give any positive data. 
 
 Concerning the influence of the nutrient media upon 
 the formation of trypsin in a culture or the liquefac- 
 tion of the gelatin, the following facts are known: 
 
 1. The majority of circumstances which impair the 
 growth of a variety of bacteria upon a nutrient 
 medium also interfere with liquefaction — for exam- 
 ple, the addition of phenol, or a large percentage of 
 glycerin. Wood found that the impaired power of 
 liquefying gelatin, which was produced by phenol, 
 was transmitted during several generations upon a 
 good nutrient medium (C. B., YIII., 266). 
 
 2. The liquefying facultative anaerobics do not 
 liquefy gelatin* in hydrogen and nitrogen, but they 
 do in carbonic acid, if they are able to grow in the 
 latter medium. As the gases, according to Fermi, 
 have no effect upon the action of the ferment, they 
 must influence the formation of the ferment. Strict 
 anaerobics, on the other hand, produce the most pro- 
 nounced liquefaction of gelatin. 
 
 3. In many bacteria the addition of sugar inter- 
 feres not with their growth, but with the liquefaction 
 of gelatin — for example, in bacterium vulgare (proteus 
 vulgaris) but not in bacillus subtilis (Kuhn: A. H., 
 Xin., 70). This is explained, perhaps, by the fact 
 that bacterium vulgare produces an acid from sugar, 
 and the vulgare trypsin is very sensitive to acids. 
 Upon 10 c.c. of a one-per-cent grape-sugar gelatin, in 
 five days bacterium vulgare produced 3.7 c.c. of one- 
 tenth normal acid, vibrio proteus 2.1 c.c, bacillus 
 
 *With the single exception of bacterium prodigiosum, but 
 this also ceases to liquefy on the addition of grape sugar. 
 
THE EFFECTS OF BACTERIA. 121 
 
 subtilis 1.7 c.c, bacillus anthracis 0.9 c.c. ; bacte- 
 rium vulgare was the only one which did not produce 
 liquefaction. 
 
 4. In fluid, non-albuminous, glycerin-containing 
 (free from sugar) nutrient media, very few bacteria 
 produce proteolytic ferments — for example, bacterium 
 prodigiosum and bacterium pyocyaneum. The pro- 
 duction of ferment also apj^ears to be less on pep- 
 tone bouillon than on peptone - bouillon gelatin 
 (Fermi). 
 
 Upon albuminous nutrient media the liquefying 
 bacteria produce bitter products of disassimilation 
 —for example, this is done in milk by very many 
 varieties (Hiippe) An enumeration of the trypsin- 
 forming varieties is unnecessary because they are 
 characterized as trypsin-producers by their liquefac- 
 tion of gelatin. 
 
 The other ferments have been studied less care- 
 fully. 
 
 Diastatic ferments convert starch into sugar. They 
 are demonstrated in the following manner: A thin 
 starch paste containing one per cent thymol is com- 
 bined with a culture to which one to two per cent thy- 
 mol has been added, and is kept six to eight hours in 
 the incubating chamber. A little Fehling's solution is 
 then added and sugar is recognized by the reduction 
 of copper (reddish-yellow precipitate) on boiling. 
 We can also make a direct examination of mashed 
 potato cultures of the bacteria by boiling the cultures 
 and testing the extract. 
 
 According to Fermi about one-third of the varie- 
 ties examined — only upon albuminous nutrient media 
 — possess the power of forming such a ferment (A. H., 
 
122 ATLAS OF BACTERIOLOGY. 
 
 X., and C. B., XII., p. 713) viz., the bacilli of the sub- 
 tilis group (anthrax, megatherium, Fitzianus, etc.), 
 the vibriones related to the cholera vibrio, also micro- 
 coccus tetragenus, micrococcus mastitidis, bacterium 
 violaceum, bacterium mallei, bacterium pyogenes 
 foetidum, bacterium phosphorescens, bacterium pneu- 
 moniae, bacterium synxanthum, bacterium aceticum; 
 the others are not active or are doubtful. In addition 
 all the actinomyces and oospora varieties (with the ex- 
 ception of oospora carnea). The majority of the 
 varieties mentioned subsequently convert the sugar 
 into acid but some do not, for example, bacillus 
 subtilis. 
 
 Inverting ferments (i.e., those which convert cane 
 sugar into grape sugar) are rare, according to Fermi 
 and Montesano. They are demonstrated in the fol- 
 lowing way : A one to two per cent solution of cane 
 sugar containing one per cent of carbolic acid is 
 added to a culture containing one per cent of carbolic 
 acid. After a few hours we test whether the fluid 
 reduces Fehling's solution; as is well known, cane 
 sugar does not produce this reaction. Control tests 
 with a solution of cane sugar alone are always neces- 
 sary. Bacteria invertin tolerates (always?) a tem- 
 perature of 100° for more than an hour, and also de- 
 velops upon a non-albuminous nutrient medium if 
 glycerin is present. The above-named writers men- 
 tion only the following forms as producers of invert- 
 ing ferments ; bacillus megatherium, bacillus kiliense, 
 bacillus fluorescens liquefaciens, bacterium vulgare, 
 vibrio cholerse and Metschnikovii. 
 
 The attempts to find a ferment similar to emulsin 
 have been unsuccessful. Micrococcus pyogenes 
 
THE EFFECTS OF BACTERIA. 123 
 
 tenuis transfori][is amygdalin into benzaldehyd, but 
 this function cannot be separated from cell life. 
 
 Eennet ferments — i.e., bodies which coagulate milk 
 of a neutral (or amphoteric) reaction and indepen- 
 dently of the action of acids — are not wanting among 
 the bacteria. It can be demonstrated, for example, 
 in not too old cultures of bacterium prodigiosum 
 which, sterilized at 55°-60°, can easily coagulate 
 sterilized milk solid in one or more days (Gorini : C. 
 B., XIL, 666). 
 
 So far as I know, thorough investigations concern- 
 ing the distribution of this ferment are still lacking. 
 We may suspect it in all varieties which coagulate 
 milk without possessing the power of forming lactic 
 acid out of milk sugar. 
 
 II. The Chemical Actions of Bacterial 
 Metabolism. 
 
 Like the production of ferments, the majority of 
 the other chemical actions of bacteria depend, in 
 great measure, on the nutrient medium. This is 
 most striking when the growth of many forms of 
 bacteria is observed upon an albuminous nutrient 
 medium, which at one time is free from sugar, at 
 another time contains sugar. In the former event, 
 apart from pigment substances and perhaps some 
 badly smelling substances, hardly any perceptible 
 metabolic products are formed , but in the latter event 
 there is often a very striking change, characterized 
 by the development of gas and active production of 
 acid. The organism joroduces fermentation in the 
 sugar-containing medium, in the other it does not. 
 
124 ATLAS OF BACTERIOLOGY. 
 
 On account of the practical (and diagnostic) im- 
 portance of the fermenting power we must here give 
 a precise definition of this process. The term fermen- 
 tation is used in literature in various senses. 
 
 1. Some writers call every typical decomposition 
 produced by bacteria a fermentation, and speak, for 
 example, of the putrid fermentation of albuminoids. 
 
 2. Others confine the term fermentation to proc- 
 esses which are attended with the visible develop- 
 ment of bubbles of gas. According to this definition 
 the conversion of nitric acid into nitrogen is a fer- 
 mentation as well as the fermentation of milk sugar 
 by bacterium acidi lactici. 
 
 3. Still others use the term only in cases of decom- 
 position of hydrocarbons with or without the forma- 
 tion of gas. 
 
 It seems to me that the term fermentation is always 
 in place when it can be shown that an organism, in 
 addition to or instead of its other metabolic products, 
 forms one or a few special metabolic products in an 
 unusual amount — metabolic products which are al- 
 most always derived from the merely superficial 
 splitting up of a bacterial nutrient which is easily split 
 up. Oxidative fermentation is rarer. A necessary 
 condition of fermentation is the presence of a definite 
 nutrient matter which the bacteria attack with special 
 ease, often discarding substances which are less acces- 
 sible but which they decompose in the absence of 
 the fermenting substance. 
 
 Every fermentation is intended to carry a supply 
 of energy to the fermenting organism. In the fer- 
 mentation which splits up organic material, this is 
 due to the fact that the complicated, fermentible 
 
THE EFFECTS OF BACTERIA. 125 
 
 molecule in the bacterial cell is decomposed into 
 smaller particles, during which process heat is given 
 off. I will illustrate this by the ordinary form of 
 fermentation of sugar in which the process is very 
 simple. 
 
 CeHiaOe = SCsHeO + 2C0, 
 
 1 grape sugar = 3 alcohol + 2 carbonic acid. 
 
 Or, 
 Or, 
 
 CeHisOs = 2C3H6O3 
 
 1 grape sugar = 2 lactic acid. 
 
 CeHiaOe = 3C2H4O2 
 
 1 grape sugar = 3 acetic acid. 
 
 The organism requires such a source of energy, 
 particularly when it grows in the absence of oxygen, 
 and there is a failure of the source of energy at the 
 command of the aerobic varieties and which consists 
 in the oxidation of absorbed substances by the oxy- 
 gen which has been taken up. Hence all anaerobic 
 varieties are provided with great power of fermenta- 
 tion of sugar, and some facultative anaerobics only 
 give rise to fermentation of a saccharine nutrient 
 when oxygen is excluded. 
 
 In contradistinction to fermentation by the split- 
 ting-up process is the much rarer oxidative fermen- 
 tation, the best example of which is the production 
 of acetic acid from alcohol. Here we find a one-sided 
 metabolic activity of the acetic acid bacteria. These 
 obtain a considerable supply of energy, not by split- 
 ting up, but by oxidation of the absorbed alcohol. 
 The gain in energy occurs simply from a one-sided 
 intensification of the ordinary nutritive processes of 
 bacteria. 
 
 It is evident from these remarks that products of 
 
126 ATLAS OF BACTERIOLOGY. 
 
 fermentation are products of metabolism like all the 
 other products of the bacterial cell, and hence a di- 
 vision of fermentations in principle is not warranted. 
 But it will be advisable to discuss the individual bac- 
 terial products according to their development upon 
 a saccharine or non-saccharine nutrient medium, and 
 then to add some functions of the bacteria which are 
 manifested by decomposition of salts of the fatty 
 acids, alcohols, etc. 
 
 A. Functions upon which the Amount of 
 Sugar in the Nutrient Medium Exerts 
 no Great Influence. 
 
 1. Formation of Pigment. 
 
 The chemistry of the pigment matters has been 
 very little studied, but in recent times a preliminary 
 survey has been made by some of Migula's pupils. 
 In regard to the fluorescent pigments I follow the 
 statements of K. Thumm ( " Arbeiten d. bact. Instituts 
 Karlsruhe," published by Klein and Migula, Vol. I., 
 Pt. 3, p. 291) and those of Paul Schneider (eod. loco, 
 Yol. I., Pt. 2, p. 201) in regard to the other pigments. 
 
 1. Ked and Yellow Pigments. According to 
 Schneider the twenty -seven yellow and red bacte- 
 ria furnish, in almost all cases,* pigments which are 
 soluble in alcohol, insoluble in water, f and are also 
 
 * The coloring matter of micrococcus cereus flavus Passet was 
 soluble only in dilute caustic potash. 
 
 f A striking contrast to these results is furnished by M. Freund 
 (C. f. B., xvi., 640). In examining four newly discovered red 
 and yellow bacteria he found the pigment always soluble in 
 water, and insoluble in alcohol and ether. 
 
SUGAR IN" THE NUTRIENT MEDIUM. 127 
 
 soluble in ether, carbon bisulphide, benzol, and chlo- 
 roform. 
 
 The large majority,* in the dry condition, are 
 colored bluish-green by concentrated sulphuric acid 
 and red or orange by caustic potash, or they retain 
 these colors when so treated. But the various pig- 
 ments show various chemical differences and quite a 
 different reaction in the spectrum. The majority 
 may be placed unhesitatingly in the large group of 
 lipochromata which are widely distributed in the 
 animal and vegetable kingdoms, and to which belong 
 the coloring matter of fat, yolk of the egg, the carotin 
 of carrots, and many others. 
 
 Entirely different from these substances are the 
 pigments of bacterium prodigiosum and bacterium 
 kiliense. These take a brownish-red color with con- 
 centrated sulphuric acid, and a yellowish-brown 
 and yellowish-red color with caustic potash. They 
 are allied to one another but still quite distinct, f It 
 has often been assumed, especially on account of the 
 golden shimmer of the prodigiosum culture, that we 
 have to deal here with a coloring matter resembling 
 fuchsin, but on careful examination the resemblance 
 is found to be very superficial. 
 
 Violet Pigments. Bacterium violaceum and bac- 
 
 * Thirteen red and fourteen yellow bacteria were examined, 
 and the only exceptions were bacterium prodigiosum and bacte- 
 rium kiliense. Schneider furnishes full tabulated statements 
 concerning the reactions of the alcoholic solution and of the dry 
 coloring matter with various agents, and also concerning the 
 spectrum reactions. 
 
 f The fact that this coloring matter or one of its derivatives is 
 not entirely insoluble in water is evident from the fact that in 
 old agar cultures garnet-red pigment is diffused in the agar. 
 
128 ATLAS OF BACTERIOLOGY. 
 
 terium janthinum were found to contain a violet 
 coloring matter, which was insoluble in water, readily 
 soluble in alcohol, but insoluble in ether, benzol, and 
 chloroform. In the dry state it is turned yellow hj 
 concentrated sulphuric acid and emerald green by 
 caustic potash. In alcoholic solution it assumes a 
 greenish to bluish-green color on the addition of 
 strong acids and ammonia. The pigment loses its 
 color on the addition of zinc and sulphuric acid. 
 
 Claessen and Schneider examined, in a very imper- 
 fect manner, the beautiful blue coloring matter of 
 bacterium indigonaceum Claessen. This pigment is 
 insoluble in the ordinary solvents; in hydrochloric 
 acid it gives at first a blue, then a yellowish-brown 
 solution. Other acids dissolve it but cause decom- 
 position. Caustic potash gives a bluish-green color. 
 
 Distinct from these blue coloring matters is the 
 blue pigment formed by bacterium syncyaneum (blue 
 milk) in addition to bacterio-fluorescein (vide below) 
 and for which I propose the term syncyanin. Thumm 
 describes the pigment as very unstable. Acids color 
 it steel blue ; in slight acidity it is bluish-black, neu- 
 tral black, and alkaline brownish-black. 
 
 According to the recent investigations of Thumm 
 the fluorescent pigments, which are found in numer- 
 ous cultures, are all identical. The coloring matter, 
 for which I propose the term bacterio-fluorescein, is 
 lemon yellow and amorphous in the dry state, soluble 
 in water and dilute alcohol, and insoluble in strong 
 alcohol, ether, and carbon bisulphide. The watery 
 solution, when concentrated, has an orange color, 
 when diluted, a pale yellow color; with acid reaction 
 it shows no fluorescence, with neutral reaction a 
 
SUGAR IN THE NUTRIENT MEDIUM. 129 
 
 bluish, with alkaline a green fluorescence. The 
 fluorescence of the cultures is at first blue, later 
 green, on account of the increase of the ammonia 
 formed by the bacteria. The pigment is not sensi- 
 tive to oxidizing substances. Colorless preliminary 
 stages have not been observed. Phosphoric acid and 
 magnesium are necessary to the development of bac- 
 terio-fluorescein. 
 
 The variations in the chromogenic functions have 
 been the subject of numerous investigations. All 
 possible factors which have an unfavorable influence 
 on the growth of the bacteria also diminish the de- 
 velopment of pigment. After continued culture upon 
 unsuitable nutrient media or at improper tempera- 
 tures, etc. , the formation of pigment by later genera- 
 tions may remain permanently diminished. 
 
 For example, there are races of bacterium syncy- 
 aneum which form no trace of coloring matter in agar 
 or milk, but on potato give a dark color even to the 
 parts around the culture. The development of pig- 
 ment appears to have been lost here simply on account 
 of the rare inoculation of the agar cultures. 
 
 At 37° bacterium prodigiosum forms no pigment, 
 and if the cultures are kept up at this temperature for 
 a long time, the production of pigment will be lost 
 for many generations even under favorable conditions 
 (Schottelius). 
 
 Very interesting communications are scattered 
 throughout the literature on pigment-forming races 
 among otherwise colorless varieties. For example, 
 Fawitzky reports yellow to rusty red colonies of 
 streptococcus lanceolatus; Kruse and Pasquale ob- 
 served colored races of streptococcus pyogenes 
 9 
 
130 ATLAS OF BACTERIOLOGY. 
 
 (Ziegler's "Beitrage," XII.). Kutscher lias recently 
 published the experience that a pseudo-glanders 
 bacillus, taken from the animal, had a bright orange- 
 red color only in the first culture upon serum, but 
 this color changed to white after a few inoculations. 
 Perhaps still greater importance attaches to the 
 often made observation that, as the result of in- 
 ternal causes, colored and uncolored colonies of one 
 variety, for example, bacterium kiliense, occasion- 
 ally develop upon plate cultures. 
 
 2. The Formation of Alkaline Metabolic Products and 
 Urea Fermentation. 
 
 According to v. Sommaruga (Z. H., XII., 273) 
 aerobic bacteria, when growing in a non-saccharine 
 nutrient medium, always produce an alkali from the 
 albuminoids. 
 
 When sugar is present the majority of varieties 
 form acid out of the sugar, in addition to the produc- 
 tion of alkali, and the originally neutral or feebly 
 acid reaction of many young bacterial cultures is ex- 
 plained simply by a slight percentage of sugar in the 
 bouillon (derived from the meat). When the sugar 
 is used up, the production of alkali becomes more 
 pronounced (Th. Smith). 
 
 So far as we know at present, the alkaline bodies 
 produced are ammonia (occasionally perceptible to 
 the sense of smell), amine and ammonia bases. In 
 order to determine the degree of production of the 
 alkali, we titrate tubes which contain 10 c.c. peptone 
 bouillon, uninoculated, and one to eight days after 
 inoculation with one-tenth normal acid and phenol- 
 
SUGAR IlSr THE NUTRIENT MEDIUM. 131 
 
 plithalein as indicator. The difference in the titra- 
 tions gives the increase of alkali. 
 
 The following will serve as an illustration of the 
 production of alkali by bacteria which in the pres- 
 ence of sugar form acid actively (5-7 c.c. normal acid 
 to 100 c.c). One hundred cubic centimetres of a 
 nutrient medium containing traces of meat sugar and 
 rendered neutral by phenolphthalein used up : 
 
 When Inoculated with Bacterium Coli. 
 
 At the end of five days 0.1 normal sodium. 
 
 At the end of ten days 0.1 normal sodium. 
 
 At the end of fifteen days 0.25 normal acid. 
 
 A special form of alkali production by bacteria is 
 the conversion of urea into ammonium carbonate: 
 CO(NH,), + 2H,0 = C03(NH,),. 
 
 Leube (Yirch. Arch., 100, p. 540) first isolated a 
 few organisms frona decomposing urine which pro- 
 duced ammonia from urea : micrococcus urese Leube, 
 bacillus ure?e Leube. This is also done by sarcina pul- 
 monum and a few other unnamed varieties. Fliigge 
 has described a micrococcus ureae liquefaciens. 
 
 We have cultivated a large number of white lique- 
 fying and non-liquefying cocci and rods from decom- 
 posing urine. None of them possessed in any strik- 
 ing degree the power of setting free ammonia from 
 diluted urine or a nutrient medium treated with urea, 
 although they flourished in these solutions. It can- 
 not be denied that natural urea fermentation depends 
 partly on symbiosis. 
 
 The ability to decompose urea does not seem to be 
 very widespread. Among twenty-four organisms ex- 
 amined Warington found that two alone (micrococcus 
 
132 ATLAS OF BACTERIOLOGY. 
 
 ureae and bacillus fluorescens) produced ammoniacal 
 decomposition of urine. 
 
 Among sixty varieties only three (bacterium vul- 
 gare, bacterium prodigiosum, and bacterium kiliense) 
 developed a distinct ammoniacal odor in sterilized 
 human urine. 
 
 Leube employed Jacksch's nutrient solution : In 1 
 litre 0.125 acid potassium phosphate, 0.062 mag- 
 nesium sulphate, 5 gm. Seignette salts, which were 
 sterilized . by boiling. To the sterile solution he 
 added 3 gm. urea which had been sterilized in a dry 
 state at 106^ (boiling of urea solutions is to be 
 avoided because a part of the urea is thus converted 
 into ammonium carbonate) . In order to demonstrate 
 the presence of the ammonia Leube employed Ness- 
 ler's reagent, a very sensitive test. For the study 
 of the quantitative relations vide Brodmeier (C. B., 
 XVIII., p. 380). Urea is not decomposed upon a 
 nutrient medium which contains sugar. Burri, Her- 
 feldt, and Stutzer (C. B., Pt. II., Vol. I., 284) recently 
 described three rods which decompose urea very 
 vigorously. 
 
 In addition to ammonia Brieger's investigations 
 have disclosed a large number of basic crystalline 
 nitrogenous bodies as products of bacterial metab- 
 olism. These bodies are now usually called pto- 
 mains (7rrw//a, putrefaction) or putrefaction alkaloids, 
 when they are not poisonous, and toxins* when they 
 are poisonous. 
 
 * With the growth of our knowledge of bacterial poisons the 
 conception of toxins has been enlarged, so that now the major- 
 ity of writers call all bacterial poisons toxins, irrespective of 
 their chemical constitution. 
 
SUGAR IK THE NUTKIENT MEDIUM. 133 
 
 So far as they have been closely examined, the 
 majority belong to the following groups : 
 
 1. Amins. Methylamin, dimethylamin, and tri- 
 
 /CH3 /CH3 
 
 N-H N— CH3 
 
 \H \H 
 
 methylamin, similar to ethylamin, diethylamin, and 
 
 /CH3 
 
 N— CH3 
 
 xCHs 
 
 triethylamin. Ethylendiamin || „ and its homo- 
 
 logues, dimethylethylendiamin-putrescin, with which 
 sepsin is isomeric; pentamethylendiamin is called 
 cadaverin. The most virulent one is ethylendiamin. 
 
 2. Ammonium Bases. The best known is cholin- 
 
 /CH3 
 
 ^CH3 
 bilineurin = N^- — CH3 
 
 Muscarin (CgHj^NOg) is closely allied, and likewise 
 vinylcholin (C.H.^NO) and neuridin (C,H,,NJ. 
 
 3. Pyridin derivatives. Derived from pyridin 
 CgH.N ; the principal ones that have been found are 
 coUidin C^H^N and i^arvolin CgHjgN. 
 
 4. Indol (C«H,N) and skatol (C,H,N), vide page 142. 
 
 In addition, amido acids (leucin, tyrosin, etc.), sub- 
 stances allied to guanidin (C(NH)(NH2)2) and numer- 
 ous other imperfectly characterized bodies have been 
 discovered. It would be useless to mention them 
 here as the poisonous ones are no longer regarded as 
 the true viruses of the disease {vide page 135) . 
 
134 ATLAS OF BACTERIOLOGY. 
 
 The isolation of these bodies can only be hinted at. 
 According to Brieger's method, which is usually 
 employed, the culture of a feebly acid reaction (hy- 
 drochloric acid) is brought to a boil for a short time, 
 the filtrate then condensed into a syrup, dissolved in 
 ninety-six-per-cent alcohol, and then freed from im- 
 purities (especially traces of albumin) by alcoholic 
 lead acetate. The lead is then removed, the filtrate 
 concentrated, and from this the mercurial binary 
 compound of the ptomains are precipitated with al- 
 coholic solution of corrosive sublimate. When the 
 alcohol has been removed by heat and the mercury 
 by sulphuretted hydrogen, the characteristic gold and 
 platinum binary compounds are produced, or we at- 
 tempt directly to obtain the crystalline chlorhydrates 
 and, by the aid of caustic soda, the free, often fluid. 
 
 Some ptomains, like very many vegetable alka- 
 loids, can be easily obtained with ether in a watery 
 solution as soon as they have been set free by potash 
 lye. But Brieger's method is much better because it 
 secures many substances which do not dissolve in 
 ether. 
 
 3. Formation of Complicated "Albumin-like'' Toxic 
 Metabolic Products, 
 
 (" Toxalbumins, " Toxins.) 
 
 In connection with the discussion of the relatively 
 simple, basic, more or less poisonous metabolic prod- 
 ucts of bacteria, we may make a few brief remarks 
 on other bacterial poisons. In the present state of 
 our knowledge they may be divided into two classes. 
 
SUGAR IK THE NUTRIENT MEDIUM. 135 
 
 1. Bacterial Proteins (Buchner). — This term refers 
 to certain albuminoid substances which produce fe- 
 ver (pyogenic) and inflammation (phlogogenic). 
 They are obtained by boiling, for several hours, po- 
 tato cultures together with one-half-per-cent potash 
 lye (about fifty volumes potash to one volume bac- 
 terial substance). The clear fluid, filtered through 
 paper, allows the precipitation of the proteins after 
 careful feeble acidulation. The proteins may then 
 be washed, dried, and, before using, dissolved in a 
 weak solution of soda. 
 
 The best-known protein is Koch's tuberculin. Mal- 
 lein also belongs to this category. According to 
 Buchner and Roemer all bacterial proteins have es- 
 sentially the same action. According to Mathes deu- 
 teroalbumose, which is obtained by the action of 
 pepsin on albumin and has no connection whatever 
 with bacteria, produces the same effects on tubercu- 
 lous guinea-pigs. 
 
 2. '^ Toxalbumins.'' — C. Fraenkel and Brieger 
 (Deut. med. Wschr., 1890, 4 and 5) confirmed in great 
 measure the statements of earlier observers (Christ- 
 mas, Roux and Yersin, Hankin) that measures which 
 precipitate albumin will also precipitate from the 
 bouillon cultures of many bacteria amorphous poisons 
 which exert an intense and usually specific (similar 
 to the living culture) toxic action. They called these 
 poisons toxalbumins and considered them analogous 
 to the toxic albuminoid bodies in many plants (ricin 
 in ricinus communis, abrin in abrus precatorius, 
 etc.). The majority of investigators regarded — and 
 some still regard — these poisons as " labile" albumi- 
 noids, which are derived from the bacterial cell. 
 
136 ATLAS OF BACTERIOLOGY. 
 
 They are also regarded as analogous to snake poisons 
 and to the enzymes. With these bodies they share a 
 great sensitiveness to heat, reagents, light, etc. 
 
 The toxalbumins are obtained as a raw product by 
 precipitating, with absolute alcohol or ammonium 
 sulphate, old bouillon cultures of the bacteria which 
 have been concentrated in a vacuum, and which have 
 been freed from living germs by passing through a 
 porcelain filter. If the ammonia salt has been used, 
 this is removed from the filtered precipitate by dialy- 
 sis with flowing water in a parchment coil and, after 
 renewed concentration in a vacuum, precipitation of 
 the bodies with absolute alcohol. It has recently 
 been discovered that zinc chloride precipitates these 
 bodies quantitatively, and the toxins can be separated 
 from the precipitate by the aid of sodium phos- 
 phate (Brieger and Boer: Z. H., XXI., 268). 
 
 From the beginning, however, doubts were ex- 
 pressed whether these toxalbumins were not merely 
 carried down by the precipitated albumin and perhaps 
 had no connection with the albumin. 
 
 In the case of tetanus poison, Brieger and Cohn 
 (Z. H., XY., 1) succeeded in obtaining from the raw 
 product, by means of lead acetate and ammonia, a 
 pure virus which showed a faint violet color with 
 copper sulphate and soda lye but gave no albumin 
 reaction ; it is free from phosphorus and almost en- 
 tirely from sulphur. It thus seems to be proven that 
 the tetanus virus is not an albuminoid. 
 
 The statements of Uschinsky that he obtained an 
 albuminoid tetanus virus and diphtheria virus upon 
 a non-albuminous nutrient medium have not been 
 tested hitherto because German observers did not 
 
SUGAR INT THE NUTRIENT MEDIUM. 137 
 
 succeed in securing a sufficient growth of these organ- 
 isms upon a non-albuminous medium. Brieger and 
 Cohn found that cholera vibriones formed a non- 
 albuminous virus upon the Uschinsky nutrient me- 
 dium. The diphtheria virus is also recognized now 
 as free from- albumin (Brieger and Boer: L c). 
 
 It is becoming more and more customary to call 
 the bacterial poisons simply toxins and to ignore 
 entirely the existence of the above-described crystal- 
 lizable toxins of simple constitution. 
 
 Concerning the other characteristics of these tox- 
 ins I will make a few remarks, taking the tetanus 
 virus as an illustration (Brieger and Cohn: I. c). 
 Waterjt solutions are not coagulated by heat but lose 
 their poisonous properties in time. The addition of 
 small amounts of acid or alkali to produce solution, 
 and prolonged transmission of carbonic acid and 
 sulphuretted hydrogen impair the toxicity very ma- 
 terially. In the dry state the virus tolerates a tem- 
 perature of 70° very well for a long time, higher 
 temperatures decompose it rapidly. When dried 
 and protected from light, air, and moisture, it is 
 converted slowly into an inert substance. It is bet- 
 ter preserved when covered with absolute alcohol, 
 anhydrous ether, and the like. 
 
 The virulence of the purest tetanus virus is almost 
 inconceivable. A mouse weighing 15 gm. is killed 
 by 0.00005 mgm. ; a man weighing 70 kgm., with the 
 same susceptibility, would be killed by 0.23 mgm. 
 Thirty to one hundred milligrams of strychnine are 
 required to kill a man. 
 
138 ATLAS OF BACTERIOLOGY. 
 
 4. Sulphuretted Hydrogen, 
 
 Sulphuretted liydrogen is a very widely distributed 
 bacterial product. It is easily demonstrated by 
 fastening, by means of the cotton plug, a moist strip 
 of lead acetate paper in the neck of the culture tube 
 and closing it with a rubber cap (made of black rubber 
 free from sulphur). Frequent observations of the 
 originally brownish, later black, often very feeble dis- 
 coloration of the paper is necessary, because some- 
 times the color fades away at a later period. Tests 
 which are apparently negative should not be ter- 
 minated too soon. The literature consists mainly of 
 articles by Petri and Maassen (A. G. A., YIII., 318 
 and 490), Eubner, Stagnitta-Balistreri, and Niemann 
 (A. H., XVI.). 
 
 Sulphuretted hydrogen may be formed from : 
 
 1. Albuminoid bodies. (It is well known that 
 mere boiling eliminates H^S from egg albumin). 
 According to Petri and Maassen this power inheres 
 in all the bacteria examined upon a fluid nutrient 
 medium which is rich in peptone (five to ten per 
 cent) and free from sugar; in bouillon free from 
 peptone very few varieties form H^S (for example, 
 bacterium vulgare); in bouillon containing one per 
 cent peptone, about fifty per cent of the bacteria 
 (Stagnitta-Balistreri) . 
 
 2. Powdered sulphur. In nutrient media to which 
 pure powdered sulphur has been added all bacteria 
 produce much larger amounts of sulphuretted hy- 
 drogen than without this addition. Petri and Maas- 
 sen regard this production of sulphuretted hydrogen 
 
SUGAR IN- THE NUTRIENT MEDIUM. 139 
 
 as a function of the nascent liydrogen wliich the bac- 
 teria produce, i.e., they regard the formation of H^S 
 as a proof of the formation of nascent hydrogen. 
 
 3. Thiosulphate and thiosulphite. This has been 
 studied especially in yeast but has also been demon- 
 strated in the case of some bacteria (by Petri and 
 Maassen). 
 
 4. Sulphates. Beyerinck in particular has de- 
 monstrated this practically important function for his 
 (morphologically poorly characterized) motile, strict 
 anaerobic spirillum desulphuricans. It is rarely 
 found developed among other bacteria (C. B., Part 
 11. , Vol. L, 1). 
 
 Rubner has shown that in bacterium vulgare the 
 decomposed organic sulphur always suffices for the 
 production of sulphuretted hydrogen. 
 
 The presence of sugar in the nutrient media rarely 
 prevents or diminishes the production of sulphur- 
 etted hydrogen, even when the bacteria are able to 
 decompose (ferment) sugar vigorously. The decom- 
 position of hydrocarbons does not protect the albumi- 
 noids from decomposition. The presence of saltpetre 
 is a disturbing factor and under these circumstances 
 very little H^S but an abundance of nitrite is formed 
 (Petri and Maassen). The exclusion of oxygen favors 
 the production of sulphuretted hydrogen. On pass- 
 ing air through the cultures of facultative anaerobic 
 producers of sulphuretted hydrogen the amount of 
 H^S produced diminishes considerably, and in its 
 place sulphates are formed. 
 
 Many producers of sulphuretted hydrogen also 
 produce stinking mercaptan (CH^— SH), demonstra- 
 ble by tiie green color which it gives to the yellow- 
 
140 ATLAS OF BACTERIOLOGY. 
 
 ish-red isatin sulphate. Upon the culture glass is 
 placed a tube open on both sides ; this is filled with 
 glass beads which are moistened with a one and a 
 half per cent solution of isatin in concentrated sul- 
 phuric acid. The presence of sugar iu the nutrient 
 media diminishes or prevents the formation of mer- 
 captan. 
 
 5. Reduction Processes. 
 
 (Keduction of Coloring Matters, Nitrates, etc.) 
 
 We have seen that aerobic bacteria in general 
 possess the power of converting powdered sulphur 
 into sulphuretted hydrogen and that nascent hydrogen 
 is necessary thereto. 
 
 Similar processes, and probably also due in part 
 to nascent hydrogen, are the following : 
 
 1. Reduction of blue litmus coloring matter, methyl 
 blue, and indigo when added to colorless leuco-prod- 
 ucts. The upper layer in contact with the air often 
 shows no reduction, only the deeper layers. On 
 shaking in the air the color is restored, but occasion- 
 ally the litmus coloring matter is restored with a red 
 color on account of the coincident production of acid. 
 The mode of experiment goes without saying ; bouil- 
 lon serves as the nutrient medium. According to 
 Cahen the reduction of litmus is effected by all lique- 
 fying bacteria. It is observed very beautifully, for 
 example, in bacillus fluorescens liquefaciens, but 
 there are also non-liquefying varieties (for example, 
 bacterium coli) which exhibit this characteristic. 
 
 2. Reduction of nitrates to nitrites and ammonia. 
 The former power seems to belong to very many bac- 
 
SUGAR IN^ THE NUTRIENT MEDIUM. 141 
 
 teria. At least Petri and Maassen found that, among 
 six varieties cultivated in bouillon containing 2.5-5 
 per cent peptone and 0.5 per cent saltpetre, there was 
 almost always a pronounced production of nitrites; 
 in one case, indeed, only ammonia was found. Rub- 
 ner (A. H., XYI., 62) found the production of nitrites 
 absent only in isolated cases. Among twenty-five 
 varieties Warington found that eighteen produced 
 nitrites. In our experiments with bacterium coli, 
 typhi, vulgare, bacillus anthracis, subtilis, vibrio 
 cholerae, the addition of sugar was not a disturbing 
 factor. At the end of three days the nitrite reaction 
 was equally pronounced, with or without the pres- 
 ence of one per cent grape sugar, in one per cent pep- 
 tone bouillon containing one-half per cent saltpetre. 
 
 Nitrites are demonstrated in the following way: 
 After the tubes have remained for a few days in the 
 incubating chamber, some potassium iodide starch 
 solution (thin starch paste with one-half per cent 
 KI) and a few drops of sulphuric acid are added to 
 the nitrate bouillon (also to two uninoculated control 
 tests). The control tubes remain colorless or at the 
 most gradually acquire a very faint blue color, but 
 if nitrites are present, a dark blue to dark brownish- 
 red color develops. Small amounts of nitrite are de- 
 monstrated by metaphenylendiamin and somewhat 
 diluted sulphuric acid (yellowish-brown color) or 
 (most clearly) by a mixture of sulphanilic acid and 
 naphthylamin (red color) . ( Vide Dieudonne, A. G. 
 A., XL, 508). 
 
 The demonstration of ammonia by the addition of 
 Nessler's reagent is permitted only upon inorganic 
 non-saccharine nutrient media. In bouillon Ness- 
 
142 ATLAS OF BACTERIOLOGY. 
 
 ler's reagent is reduced almost immediately to black 
 mercurial oxide. A strip of paper which has been 
 dipped in the reagent may be hung over bouillon 
 cultures, or the latter may be distilled after addition 
 of MgO and the distillate treated with Nessler's re- 
 agent. A yellow to reddish-brown color indicates the 
 presence of ammonia. Control tests must be made. 
 
 6. Aromatic Metabolic Products. 
 
 It is evident that albumin gives rise, under the in- 
 fluence of very many varieties of bacteria, to aromatic 
 bodies of which indol, skatol, phenol, and tyrosin 
 are the best known. Methodical investigations have 
 been made only in regard to indol and phenol, as 
 these bodies are easily recognized. 
 
 Demonstration of indol : To the bouillon culture — 
 whicli should not be less than a week old and made 
 without any addition of sugar — about half its volume 
 of ten-per-cent sulphuric acid is added. If a rose to 
 bluish-red color appears forthwith on warming to 
 about 80°, then indol and nitrite are both present, as 
 this nitrosoindol reaction requires the presence of 
 both bodies. The test is generally successful in 
 cholera and other vibriones and occasionally in diph- 
 theria (red cholera reaction). But as a general thing 
 the addition of sulphuric acid does not suffice, and it 
 is necessary to add a little nitrite. This may be done 
 later, after the culture has been heated without 
 nitrite, and no reaction or a very doubtful one has 
 been obtained. Of the solution containing about 
 0.05 per cent sodium nitrite we add 1 to 2 c.c. until the 
 maximum of the reaction is secured. The addition 
 of strong nitrite solutions gives the acid fluid a 
 
SUGAR IN^ THE NUTRIEN"T MEDIUM. 143 
 
 brownish-yellow color and prevents entirely the de- 
 monstration of indol. 
 
 Demonstration of phenol: The culture, which is 
 made in non-saccharine bouillon, receives about one- 
 fifth its volume of hydrochloric acid and is then dis- 
 tilled. The distillate deposits flocculi with bromine 
 water, or assumes a violet color on the addition of 
 calcium carbonate and cautiously neutralizing, or of 
 neutral very dilute ferric chloride. 
 
 Among sixty varieties examined we found indol 
 formed twenty-three times, and our findings agree 
 with those of Levandovsky (Deiitsch. med. Wschr., 
 1890, No. 51). The chief indol producers are the 
 coli group in the widest sense — glanders, diphtheria, 
 proteus, and the majority of vibriones. According 
 to Levandovsky the indol producers just mentioned, 
 with the exception of the vibriones, also form phenol. 
 We have tested the production of phenol only in bac- 
 terium coli and vulgare and found mere traces in five- 
 day cultures. 
 
 7. Decomposition of Fats. 
 
 Pure melted butter is not a nutrient medium for bac- 
 teria. The rancidity of butter is due to : (1) a purely 
 chemical decomposition of the butter by the oxygen 
 of the air, aided by sunlight (Duclaux, Ritsert) ; (2) a 
 lactic-acid fermentation of the milk sugar which has 
 been left over in the butter (vide v. Klecki, C. B., 
 XV. , 354) . Finally fat is attacked by bacteria and acid 
 is formed, if it is mixed with gelatin as a nutrient 
 medium (pide v. Sommaruga, Z. H., XVII., 441). 
 
144 ATLAS OF BACTERIOLOGY. 
 
 8. Putrefaction (Appendix to 1-7). 
 
 Putrefaction, in the language of the laity, means 
 every decomposition which is produced by bacteria 
 and is attended by the formation of foul-smelling 
 substances. 
 
 On scientific investigation it is found that the al- 
 buminoids and their allies are the substratum of pu- 
 trefaction; at first they are often peptonized, then 
 they are split up still further. 
 
 Typical putrefaction occurs only when the supply 
 of oxygen is wanting or scanty. The vigorous pas- 
 sage of air through a putrefaction bacteria culture— a 
 process which does not occur in natural putrefaction 
 — modifies the process in the most marked manner. 
 In the first place because the anaerobic putrefac- 
 tion bacteria are killed or their growth is inhibited, 
 and secondly by the action of the oxygen upon the 
 products or intermediate products of the aerobic and 
 facultative anaerobic bacteria. Finally, it seems 
 conceivable that the same bacteria (anaerobic and 
 aerobic) may from the start furnish different products 
 of putrefaction. 
 
 Among the putrefaction products we find the bod- 
 ies * described in preceding chapters : peptone, am- 
 monia and amins, leucin, tyrosin and other amido 
 bodies, oxyfatty acids, indol, skatol, phenol, finally 
 
 *It is often said that in every putrefaction the albuminoid 
 bodies are first peptonized, but inasmuch as bacterium vulgare /3 
 Zcnkeri, and bacterium putidum are generally recognized as pro- 
 ducers of putrefaction, and as they do not even liquefy gela- 
 tin, we cannot always speak of peptonization of albumin as 
 constant in putrefaction. 
 
SUGAR IN THE NUTRIENT MEDIUM. 145 
 
 sulphuretted hydrogen, mercaptan, carbonic acid, 
 hydrogen, marsh gas. 
 
 But inasmuch as, in putrefaction of different nu- 
 trient media by different bacteria, the metabolic 
 products just mentioned are found, as a rule, only in 
 part and in extremely varying combinations, putre- 
 faction can hardly be defined more accurately with 
 chemical aids than is possible with the senses. 
 Hence I believe it is best to employ the term putre- 
 faction only in the general lay signification of every 
 foul-smelling decomposition of albuminoids (vide 
 Kuhn: A. H., XIII., 1). 
 
 9. Nitrification. 
 
 The formation of small amounts of nitrous and 
 nitric acids is widely diffused among bacteria. 
 Heraeus (Z. H., 1, 193), who first investigated the 
 subject with pure cultures, found that in sterilized 
 urine which had been diluted fourfold very many of 
 the well-known bacteria form small amounts of nitrite 
 from urea or ammonium carbonate. These include 
 micrococcus pyogenes citreus, bacterium prodigi- 
 osum, typhi, coli, bacillus mycoides, anthracis, 
 vibrio pyogenes, and vibrio proteus. Various soil 
 bacteria also furnish nitrites. The addition of sugar 
 interferes with the production of nitrite from NHg 
 until it is destroyed. The formation of nitrate was 
 not studied by Heraeus. Warington failed to find 
 nitrates in a study of twenty-four varieties in pure 
 cultures in nutrient solutions which formed nitrate 
 distinctly when infected by means of the soil (C. B., 
 YI., 498). 
 
 According to more recent investigations nitrifica- 
 10 
 
146 ATLAS OF BACTERIOLOGY. 
 
 tion is particularly the function of a small, special 
 group of bacteria which are cultivated with difficulty 
 and do not thrive upon our ordinary nutrient media. 
 
 According to Winogradsky, who has done the most 
 work in this department, the facts of the case are as 
 follows : The soil of Europe contains, widely distrib- 
 uted, two micro-organisms, one of which (nitroso- 
 monas) converts ammonia into nitrite, the other (called 
 nitromonas, later nitrobacter) converts nitrite into 
 nitrate. Both varieties are obtained mixed when bits 
 of earth in flasks are dissolved in boiling water 
 (Winogradsky took the water of a fresh-water lake) 
 containing 1 gm. ammonium sulphate and 1 gm. potas- 
 sium phosphate to 1 litre. About 1.0 gm. basic mag- 
 nesium carbonate is added to each flask containing 
 100 c.c. Considerable development of nitrites takes 
 place, and gradually nitrates are also formed. By 
 inoculation of new flasks the nitrifying organisms 
 are obtained gradually in a purer state, and silicic- 
 acid plates finally i)ermit, with difficulty, a pure cul- 
 ture. Burri and Stutzer have recently cultivated upon 
 the ordinary nutrient media a vigorous nitrate pro- 
 ducer (from nitrite), but it forms nitrates only upon 
 inorganic nutrient solutions (C. B., Vol. I., Part II., 
 731). 
 
 P. F. Kichter (C. B., XYIII., Part I., p. 129) ob- 
 served on several occasions a pronounced nitrite 
 reaction in fresh urine evacuated with the catheter. 
 From one specimen he isolated a coccus of medium 
 size, which in twenty minutes produced a very in- 
 tense nitrite reaction in fresh urine. In addition it 
 reduced nitrate to nitrite. 
 
SUGAR IN THE ITUTRIENT MEDIUM. 147 
 
 10. Conversion of Nitrons and Nitric Acids into Free 
 Nitrogen. 
 
 This process is carried on by an entire series of 
 bacteria. Burri and Stutzer (C. B., Part II., Yol. 
 I., No. 7 et seq.) were the first to describe special ni- 
 trate fermenters in such an accurate manner that they 
 could again be recognized. They first isolated from 
 horse manure two bacteria, of which each alone was 
 unable to produce nitrogen from nitrate, but did this 
 vigorously when combined, and when the supply of 
 oxygen was abundant or scanty but never when it was 
 absent. These two synergetic bacteria are : (1) Bac- 
 terium coli (this may be replaced by bacterium typhi) , 
 and (2) a short rod described as bacillus denitri- 
 ficans I. Later these writers found a bacillus deni- 
 trificans II., which alone effected the entire decom- 
 position of nitrate into nitrogen. We found that 
 bacterium pyocyaneum also converts saltpetre into 
 nitrogen. 
 
 The practical importance of these organisms lies 
 in the fact that through their agency considerable 
 amounts of nitrates in the soil, but particularly in 
 manures, may be lost for the nourishment of plants 
 on account of their conversion into nitrogen. 
 
 11. Assimilation of Nitrogen. 
 
 According to our present knowledge no other vege- 
 table family is able to assimilate the nitrogen of the 
 air, but this power does inhere in one form of bac- 
 teria, the bacillus radicicola Beyerinck. This bac- 
 terium is found in the small root knobs of various 
 
148 ATLAS OF BACTERIOLOGY. 
 
 leguminosse and may be cultivated from them. From 
 the different forms of leguminosse we obtain different 
 races of the bacteria, each one being especially 
 adapted to one form of leguminosse; not every race 
 is able to produce the knobs in every form of the 
 vegetable. There are also "neutral" bacteria, found 
 free in the soil, which are not specially adapted to 
 any form of the leguminosse and which are able to 
 produce knobs in very different forms of the vege- 
 table. 
 
 With the aid of these root knobs, which are due to 
 the immigration of the root bacteria, the leguminosse 
 are able to furnish crops which are rich in nitrogen 
 from a relatively sterile soil which is very poor in ni- 
 trogen. The manner in which the absorption of 
 nitrogen takes place is still entirely unknown. It is 
 claimed that the swollen zoogloea form of bacteria 
 (bacteroids*), almost always observed in the knobs, 
 is alone able to absorb nitrogen. Recently it seems to 
 have been j^roven that even without the aid of legu- 
 minosse knob bacteria living free in the soil are able 
 to absorb elementary nitrogen (for a detailed rhume 
 of the present status of the question, see Stutzer : C. 
 B., PartlL, Yol. I., p. 8). 
 
 12, Production of Acids from Carbohydrates. 
 
 As Theobald Smith showed (C. B., XVIII., No. 1), 
 the formation of free acid is only possible on a 
 saccharine nutrient medium. Its production upon 
 ordinary bouillon takes place only when the latter 
 
 * These bacteroids assume the most bizarre shapes, networks, 
 forks, stars. 
 
SUGAR IN^ THE NUTRIENT MEDIUM. 149 
 
 contains grape sugar (derived from the meat).* Ac- 
 cording to Smith all strict or facultative anaerobics 
 form acids out of sugar, the aerobics either do not 
 or they do it so slowly that the formation of the acid 
 is concealed by the parallel production of alkali. 
 Prior to a knowledge of this work we had found that 
 all tested varieties of bacteria (about sixty), which 
 are shown in the Atlas, formed more or less free 
 fixed acid in one per cent grape sugar peptone 
 bouillon (vide Table I). The formation of acid 
 may or may not be attended with visible develop- 
 ment of gas. Intense production of acid may kill 
 the cultures (for example, bacterium coli, vulgare, 
 etc.). 
 
 In many varieties the formation of acid or decom- 
 position of sugar is intense and rapid, so that this 
 metabolism, which is effected chiefly at the expense 
 of the carbohydrates, is called fermentation. Inas- 
 much as this is attended not infrequently by the de- 
 velopment of gas in large quantity, this term also 
 seems justifiable to the laity. 
 
 If, after the sugar is used up, the amount of acid 
 produced is insufficient to kill the bacteria, the metab- 
 olism which ensues is that common to the non-sac- 
 charine nutrient medium, the acid is gradually neu- 
 tralized, and finally an increasing alkaline reaction 
 sets in. 
 
 Among the acids produced (apart from carbonic 
 acid, which will be considered under the heading of 
 "Production of Gas") the most important and widely 
 distributed is lactic acid. In addition we almost 
 
 * According to Th. Smith, seventy -five per cent of commercial 
 beef contains distinct amounts of sugar (up to 0.3 per cent). 
 
150 ATLAS OF BACTERIOLOGY. 
 
 always find, at least in traces, formic acid, acetic 
 acid, proprionic acid, butyric acid, and not infre- 
 quently some ethyl alcohol, aldehyde, or acetone. In 
 rarer cases the lactic acid is wanting and only the 
 other acids are formed. 
 
 In order to obtain and separate the acids we 
 employ the following method: In 1 litre flasks are 
 placed i litre peptone bouillon with two to ^yq per 
 cent grape sugar or milk sugar and perhaps 
 10 gm. calcium carbonate. The acids formed com- 
 bine with the calcium carbonate into a soluble 
 lime salt and carbonic acid escapes; the reaction 
 of the fluid — and that is the main thing — remains 
 neutral. A strongly acid reaction would inter- 
 fere prematurely with the further growth of the 
 bacteria. 
 
 When the growth has ceased (in eight to fourteen 
 days) the undissolved carbonate is filtered off, and 
 the reaction being neutral, the alcohol, aldehyde, 
 acetone, etc., are distilled; the fluid is boiled down 
 considerably during this process. The three sub- 
 stances just mentioned are detected in common by 
 Lieben's iodoform reaction. To the slightly warmed 
 fluid in a test tube are added five to six drops of 
 pure ten-per-cent potash lye, then a weak iodine- 
 potassium iodide solution is added drop by drop 
 until a brown color is produced, and the latter is 
 made to disappear by a drop of potash. The charac- 
 teristic iodoform odor and the precipitation of micro- 
 scopic small six-angled iodoform plates are convinc- 
 ing evidence. For the differentiation of alcohol, 
 aldehyde, and acetone, vide Yortmann, " Analyse or- 
 gan. Stoffe," 1891. 
 
SUGAR IK THE NUTRIENT MEDIUM. 151 
 
 A strong acid reaction is now secured with phos- 
 phoric acid and the volatile acids are distilled off 
 with the aid of a current of steam. The distillation 
 must be prolonged because the complete removal of 
 the volatile acids is difficult. The lactic acid is left 
 in the distillate, is obtained by shaking repeatedly 
 with pure ether, and the ether is then distilled off. 
 
 The lactic acid obtained is always ethylidenlactic 
 CH3 
 
 acid CHOH, which occurs in two stereoisomeric forms : 
 
 COOH 
 
 (1), dextro-rotatory with laevo-rotatory zinc salt, and 
 (2) Isevo-rotatory with dextro-rotatory zinc salt. If, 
 as happens very frequently, exactly the same number 
 of molecules of left and right lactic acids are present, 
 then the combination is optically inactive and forms 
 the so-called "fermentation lactic acid." 1 assume 
 that both lactic acids often develop from sugar, but 
 that some varieties of bacteria thrive mainly on one, 
 some on the other form, so that sometimes there is a 
 uniform combination, sometimes one form predom- 
 inates or alone remains. 
 
 Since Schardinger {Mitt. f. Chem., XI., 545) dis- 
 covered that the previously unknown left lactic acid 
 was the product of a short rod bacillus in water, the 
 pupils of Nencki and Kubner have made numerous 
 investigations on the lactic acids formed by the dif- 
 ferent varieties, in the hope of utilizing the results 
 for purposes of differential diagnosis. 
 
 For the method of determining the form of lactic 
 acid, vide Nencki (C. B., IX., 305) and Gosio (A. H., 
 XXL, 115). 
 
152 ATLAS OF BACTERIOLOGY. 
 
 The most important results of the investigations 
 
 are: 
 
 Bacterium coli 
 
 Bacterium Bischleri 
 
 Bacterium typhi 
 
 Micrococcus acidi paralactici . 
 
 Vibrio cholerse (Calcutta) 
 
 Vibrio cliolerse (Massaua) . . . . 
 
 Vibrio Metschnikovi 
 
 Vibrio danubicus 
 
 Vibrio "Wernicl^e" L,II.,I.-I. 
 
 Vibrio "Dunbar" 
 
 Vibrio proteus 
 
 Vibrio Weibel 
 
 Vibrio Bonhoff b 
 
 Vibrio berolinensis 
 
 Vibrio aquatilis 
 
 Vibrio tyrogenes 
 
 Vibrio Bonhofl: a 
 
 Inactive 
 lactic acid. 
 
 + 
 
 + 
 
 Right lactic 
 
 acid = 
 
 paralactic 
 
 acid. 
 
 + 
 
 + 
 
 + 
 
 Left lactic 
 acid. 
 
 + 
 
 + 
 
 Although these results are not yet of much impor- 
 tance, a continuance of these theoretically interesting 
 studies is desirable. 
 
 Various bacteria— which have in great part been 
 imperfectly studied morphologically and biologically 
 — are able to produce butyric acid, butyl alcohol, or 
 both from carbohydrates. 
 
 A review of these varieties is found in an article by 
 Baier (C. f. B., Part II., Yol. I., p. 17). Here we 
 will only mention : bacillus butyricus Hiippe (appar- 
 ently also other aUied varieties), the imperfectly 
 described granulobacter poly my xa Beyerinck, and 
 several anaerobic varieties (Clostridium butyricum of 
 the authors). 
 
 In connection with the fermentation of sugar we 
 
SUGAR IN THE NUTRIENT MEDIUM. 153 
 
 may mention tlie splitting up of cellulose by various 
 bacteria, which are found particularly in the gastric 
 and intestinal contents of herbivora, and in muck, 
 and which form marsh gas as a striking product. 
 
 Unfortunately the decomposition of cellulose by 
 bacteria has been imperfectly studied. It appears to 
 be certain, however, that at least one anaerobic variety' 
 decomposes cellulose into marsh gas and carbonic 
 acid. But the most recent investigator of this ques- 
 tion. Van Senus, maintains that the anaerobic bacil- 
 lus amylobacter isolated by him will attack cellulose 
 only in symbiosis with another small bacillus (vide 
 the resumS by Herzfeld: C. B., Part I., Vol. II., p. 
 114). 
 
 13. Formation of Gas from Carbohydrates and other 
 Fermentihle Fatty Bodies. 
 
 The only gas which develops in visible amounts 
 upon a non-saccharine nutrient medium is nitrogen. 
 If sugar is vigorously attacked by bacteria, the devel- 
 opment of gas may be lacking inasmuch as pure lactic 
 or acetic acid is produced (for example, typhus 
 bacillus on grape sugar) ; but very often there is a 
 notable development of gas, especially when the air 
 is excluded. About one-third of the varieties which 
 form acid vigorously also produce an abundance of 
 gas. This consists of carbonic acid which, accord- 
 ing to Smith (C. B., XYIII., 1) is always combined 
 with hydrogen. Marsh gas appears to be formed 
 rarely (apart from the bacteria which decompose cel- 
 lulose). Last year Mr. Conrad isolated in my labor- 
 atory, a bacterium allied to bacterium coli, which 
 gives rise to the fermentation of sauerkraut and 
 
154 
 
 ATLAS OF BACTERIOLOGY. 
 
 always, even when the nutrient medium is free from 
 cellulose, forms some marsh gas in addition to car- 
 bonic acid and hydrogen. 
 
 In order to determine whether gas is formed, we 
 should use the agitation culture on one-per-cent grape 
 
 Fig. 11.— Bacterium Coli upon Sugar Agar at the end oflVelve, Twenty- 
 four, and Forty -eight Hours. 
 
 sugar agar. At the end of twenty -four hours (often 
 at the end of eight to twelve hours when incubating 
 temperature may be employed) the agar is infiltrated 
 with bubbles of gas or even split up by numerous 
 deep rifts and fissures. If the gas is to bo measured 
 or analyzed, it is best, according to Th. Smith, to re- 
 ceive it in the fermentation flask which has been used 
 
SUGAR IN" THE NUTRIENT MEDIUM. 
 
 155 
 
 ^\ 
 
 for such a long time in physiological and pathologi- 
 cal chemistry. 
 
 The tubes, which should have the shajje shown in 
 Fig. 12, are filled with one-per-cent grape sugar pep- 
 tone bouillon and sterilized in the 
 steam chamber. After inoculation 
 with a platinum loop in the incubat- 
 ing chamber the following facts de- 
 velop : 
 
 1. If the opacity is produced only 
 in the open spherical part of the flask, 
 we have to deal with an aerobic va- 
 riety; if produced only in the closed 
 tube and the globe remains clear, we 
 have to deal with an anaerobic variety. 
 
 2. The daily amount of gas pro- 
 duced is marked with ink ; if the cali- 
 bre of the tube is known, we are able 
 
 to state, after the formation of gas has ceased on 
 the fourth to sixth day, what percentage of gas was 
 produced on each day. 
 
 3. A rough analysis of the gas should be made. 
 After the amount of gas has been noted, we fill the 
 open sphere completely with ten-per-cent soda lye, 
 close it firmly with the thumb, and shake it for a 
 while. At the end of two minutes all the gas is al- 
 lowed to pass into the closed tube, and after the 
 thumb is removed the new volume of gas is read off. 
 The part which has disappeared is carbonic acid, the 
 rest is nitrogen, hydrogen, and marsh gas. The quan*- 
 titative analysis of these gases is best done by means 
 of Hempel's gas pipettes [vide Winkler: "Lehrb. 
 d. techn. Gasanalyse," Freiburg, 1892). The method 
 
 Fig. 12.— Fermen- 
 tation Flask. 
 
156 ATLAS OF BACTERIOLOGY. 
 
 is based on the fact that hydrogen, when mixed with 
 oxygen and passed over glowing palladium asbestos, 
 is converted into. water and accordingly disappears; 
 carburetted hydrogen is changed into carbonic acid 
 in a glowing platinum capillary tube, and is measured 
 as such, and the remainder is nitrogen. With some 
 practice the examination is easy and accurate. 
 
 14. Production of Acids from Alcohols and other 
 Organic Acids. 
 
 It has long been known that bacterium aceti or its 
 nearest allies convert weak solutions of ethyl alcohol 
 into acetic acid, at the same time using up a large 
 amount of oxygen : 
 
 JH^OH COOH 
 
 cii 
 
 Higher alcohols, such as glycerin, dulcite, and 
 mannite, are also converted into acids; glycerin as 
 generally as sugar (v. Sommaruga: Z. H., XY., 291). 
 
 Finally, numerous observations have been made on 
 the conversion, b}^ bacteria, of acids of the fatty 
 series (or their salts) into other fatty acids, but unfor- 
 tunately the majority were not made with pure cul- 
 tures which meet the modern requirements. Lactate, 
 mallate, citrate, and gly cerate of lime were usually 
 employed as the material and almost always acid 
 mixtures were obtained as the result of the bacterial ac- 
 tivity. Among these butyric, propionic, valerianic, 
 and acetic acids play the principal part; succinic 
 acid and ethyl alcohol are often found ; formic acid is 
 rarer. Among the gases carbonic acid and hydrogen 
 are especially prominent. 
 
SUGAR IN THE NUTRIEI^^T MEDIUM. 157 
 
 Such experiments were formerly made chiefly by 
 Fitz, and recently have been performed with pure 
 cultures and interesting results by P. Frankland. A 
 couple of illustrations will suffice. Pasteur found 
 that anaerobic bacteria convert lactate of lime into 
 butyrate of lime. 
 
 2(CH3 - CHOH - C00)2Ca = COaCa + 3 CO^ + 4 H^ + 
 Lactate of lime. (CH3- CH2-CH2- COO)Ca 
 
 Butyrate of lime. 
 
 According to P. Frankland the bacillus ethaceticus 
 Fitz converts gly cerate of lime (CH^OH— CHOH— 
 C00)2Ca into ethyl alcohol, acetic acid, carbonic 
 acid, and hydrogen. 
 
 in. The Pathogenic Effects of Bacteria. 
 
 (Pathogenesis, Predisposition, Resistance, Im- 
 munity.) 
 
 Whenever we are able to recognize the nature of the 
 pathogenic action of bacteria, they are found to act 
 by means of the chemical substances which they form 
 in the animal body or which are formed from them. 
 But hitherto we have learned to comprehend only the 
 action of those bacteria which produce toxic sub- 
 stances in cultures, and by means of which we can 
 reproduce the characteristic symptomatology in a 
 more or less accurate manner. 
 
 The bacteria of this category include particularly 
 the bacillus tetani, bacillus diphtheriae, streptococcus 
 pyogenes, micrococcus pyogenes, vibrio cholerae, etc. 
 On page 134 we have given a brief sketch of our 
 chemical knowledge of these toxic substances. 
 
158 ATLAS OF BACTERIOLOGY. 
 
 In an entire series of important infectious dis- 
 eases, on the other hand, we are almost entirely un- 
 able to explain them on a chemical basis. These 
 include anthrax, rabbit septicaemia, hog erysipelas. 
 Filtrates through i)orcelain of the most virulent 
 cultures are inert ; the cultures, which are cautiously 
 killed by briefly warming them or by a short expo- 
 sure to chloroform, produce only the general protein 
 action (fever) when injected. Yet it is probable that 
 even these diseases are toxaemias due to bacterial 
 metabolic products. 
 
 It is to be regarded as an important finding that 
 Petri and Maassen (A. G. A., YIII., 318) were able 
 to demonstrate the sulphmethsemoglobin strii)e in 
 the fresh blood and oedema fluid of erysipelatous hogs 
 — a sign that poisoning with sulphuretted hydrogen 
 at least plays a part in the death of the animals. 
 Similar evidence has also been obtained in malignant 
 oedema. 
 
 Hoffa regards rabbit septicaemia as methylguanidin 
 poisoning (Langenbeck's Arch., 1889, p. 273). Em- 
 merich and Tsuboi {31unch. med. Wschr., 1893, No. 
 25) explain cholera as a nitrite poisoning, but this 
 has been vigorously opposed. 
 
 These explanations are very interesting, but they 
 do not seem to suffice, inasmuch as, apart from the 
 toxic processes just mentioned, there are at least 
 other speciflc processes in the blood and tissues of 
 the animal. This is proven, among other things, by 
 the development of specific protective substances 
 ("anti -bodies"). 
 
 In order that a pathogenic action may be observed 
 the micro-organism must be in a condition of vigorous 
 
SUGAR IN THE NUTRIENT MEDIUM. 159 
 
 virulence, the inoculation must be made upon a sensi- 
 tive animal, and the proper channel of infection must 
 be selected. 
 
 The virulence of bacteria varies like all their other 
 functions (production of coloring matters, fermenta- 
 tion, etc.), and is best retained by constant inocula- 
 tion from one sensitive animal to another. This is 
 also done in many varieties by tolerably frequent 
 transmission (about once a month) from one artificial 
 nutrient medium to another, preferably with an occa- 
 sional intermediate inoculation of an animal. On 
 the other hand, the virulence usually suffers when, on 
 account of rare inoculations, the cultures remain for a 
 long time in contact with their accumulating meta- 
 bolic products. 
 
 Attenuation of the virulence is easily effected : 
 
 (a) By making the cultures at somewhat too high 
 a temperature. For example, at 42.5° anthrax is en- 
 tirely deprived of virulence in three to four weeks, at 
 47° in a few hours, at 50°-53° in a few minutes. By 
 the proper regulation of the action of heat iihe bacil- 
 lus anthracis may be attenuated to such a degree that 
 it will kill only mice, or mice and guinea-pigs, or 
 these animals and rabbits. 
 
 Spores may also be "attenuated" by dry heat or 
 brief, careful disinfection with steam. 
 
 {b) By cultures upon an unsuitable nutrient me- 
 dium. The addition of phenol (1 : 600), potassium 
 bichromate (0.04-0.02 per cent) was emi)loyed suc- 
 cessfully to attenuate bacillus anthracis, iodine tri- 
 chloride to attenuate the bacilli of diphtheria. 
 
 (c) By the action of sunlight, compressed oxy- 
 gen, etc. 
 
160 ATLAS OF BACTERIOLOGY. 
 
 (d) By repeated inoculation of unsuitable animals. 
 The bacilli of hog erysipelas become much less viru- 
 lent from passing repeatedly through the rabbit; the 
 organisms of variola (these are not bacteria) from 
 passing through the body of the cow. 
 
 It is much more difficult to increase the virulence 
 of those bacteria which have been attenuated. On 
 the whole, it may be said that the virulence returns 
 spontaneously so much more readily the more rapidly' 
 the attenuation has been effected. 
 
 Varieties which have slowly and spontaneously lost 
 their virulence may often be restored to increased 
 virulence in the following ways : 
 
 1. Culture in bouillon to which ascites fluid has 
 been added (streptococci, diphtheria). 
 
 2. We first infect especially sensitive animals — par- 
 ticularly very young ones, such as young guinea-pigs 
 —and, when these have succumbed, convey the germs 
 (directly with the blood of the animals) to older and 
 more resistant animals of the most sensitive species, 
 later to more resistant species. Each passage through 
 an animal increases the virulence until finally a certain 
 maximum is reached. 
 
 3. Sensitive animals are infected with large amounts 
 of the fresh bouillon culture of the bacteria. The 
 metabolic products, which are introduced at the same 
 time, then increase the predisposition for the injected 
 organism. 
 
 4. Large amounts of the metabolic products of bac- 
 terium vulgare are injected with the bacteria (this has 
 been especially useful in the case of staphylococci 
 and streptococci). The explanation of the effect is 
 the same as that of 3. 
 
SUGAR IN THE NUTRIENT MEDIUM. 161 
 
 5. We inject — for example, with the attenuated 
 bacillus oedematis maligni or anthracis — another 
 variety which per se is almost entirely harmless — for 
 example, bacterium prodigiosum. 
 
 6. We inject the culture, mixed with an injurious 
 substance of non-bacterial origin — for example, lactic 
 acid. In bacillus oedematis maligni this has pro- 
 duced increased pathogenic power, probably from 
 local impairment of the anti-bacterial activity of the 
 animal at the site of inoculation. 
 
 The susceptibility of different species of animals 
 and of different individuals to different infectious 
 diseases varies from birth in a striking and not easily 
 explained manner. 
 
 Certain species are absolutely immune against spe- 
 cial infection-producers.* For example, man against 
 rinder pest, the cow against glanders, and all ani- 
 mals which have been tested against syphilis, malaria, 
 and gonorrhoea. 
 
 A series of other diseases is conveyed very rarely 
 and with difficulty to certain animals — for example, 
 anthrax to certain varieties of pigeons, rats, and 
 sheep. This constitutes relative immunity. The 
 more vigorous and, as a general thing, the more 
 mature an animal is, the more completely is its rela- 
 tive immunity developed. Noxious influences of all 
 kinds (hunger, cold, excessive exertion, ingestion of 
 * It is especially remarkable that very closely allied varieties 
 often exhibit astonishing differences. For example, the glanders 
 bacillus can be conveyed very readily to the field mouse but not 
 to the house mouse ; the bacillus anthracis kills the house mouse 
 with almost absolute certainty and is hardly pathogenic to the rat. 
 Micrococcus tetragenus is pathogenic to the white variety of the 
 house mouse, but is not virulent to the gray variety. 
 11 
 
162 ATLAS OF BACTERIOLOGY. 
 
 certain poisons) diminish the immunity to a consider- 
 able extent, so that a large number of organisms 
 which are weakened in this manner succumb to a 
 subsequent infection. 
 
 Hence in every newly isolated variety of bacteria 
 whose pathogenic action we desire to prove it is 
 necessary to experiment ui)on various animals if the 
 experiments on those first selected proved negative. 
 The principal animals for experimentation are : the 
 white domestic mouse, white rat, guinea pig, rab- 
 bit, chicken, pigeon, and, for special purposes, the 
 monkey. More rarely we emj^loy the gray domestic 
 mouse and rat, field mice, dogs, cats, cows, sheep, 
 pigs, and horses. The most convenient animal, but 
 one requiring good care, is the guinea-pig, charac- 
 terized by suitable size, mildness, and modest con- 
 sumption of food. Animal plagues are studied and 
 explained much more readily than human diseases, 
 because the animals are at our disposal for experi- 
 mentation. In difiicult cases various experiments in 
 infection have also been made upon man. 
 
 The causes of congenital immunity (resistance) 
 reside in protective arrangements of the organism 
 which I cannot here consider in detail. It may be 
 said, however, that the views formulated by Buchner 
 as a compromise between the various opposing 
 theories are in tolerable accord with all the facts. 
 In an invasion of pathogenic germs into the resisting 
 organism a part is destroyed by substances (alexins) 
 already present in the serum (and derived from leu- 
 cocytes) ; another part is destroyed by substances 
 which are produced from leucocytes (or other tis- 
 sues) under the influence of the bacteria. A part of 
 
SUGAR IK THE NUTRIENT MEDIUM. 163 
 
 the germs which are destroyed by the leucocytes is 
 absorbed by the latter secondarily, but some germs 
 are undoubtedly ingested alive by the leucocytes. 
 Metschnikoff — the most redoubtable antagonist of 
 Buchner — insists upon the view that the latter proc- 
 ess (phagocytosis), followed by subsequent death of 
 the germs within the leucocytes, is the essential fea- 
 ture of natural immunity. 
 
 An increase of the congenital resistance to various 
 infectious diseases has been effected in a number of 
 ways. Thymus extract, spermin, abrin (toxic al- 
 buminoids from the paternoster pea), papayotin 
 (albumin-dissolving ferment from the papaya), cin- 
 namic acid, iodine trichloride, sodium carbonate, etc., 
 when injected into animals have produced favorable 
 effects, sometimes in one, sometimes in several infec- 
 tious diseases. Indeed, an increased resistance has 
 been observed from the injection under the skin, but 
 especially into the peritoneal cavity, of an entire 
 series of ordinary albuminous substances, such as 
 blood serum and bouillon. 
 
 It is generally assumed that this effect depends 
 upon increased stimulation of the leucocytes to the 
 production of substances which are antagonistic to 
 the bacteria. 
 
 According to the majority of writers there is a 
 sharp contrast between this increased resistance and 
 the specific immunity from a definite disease which 
 develops when an individual has spontaneously ac- 
 quired and passed through this infectious disease or 
 when he has been purposely inoculated with : 
 
 (1) Naturally or artificially attenuated infection- 
 producers of the same variety ; or 
 
164 ATLAS OF BACTERIOLOGY. 
 
 (2) Extinct cultures of the micro-organism in ques- 
 tion; or 
 
 (3) The blood serum or tissue juices of an animal 
 immunized by the plans mentioned under (1) and (2). 
 After (1) and (2) there develops an active immunity ^ 
 after (3) a passive immunity. 
 
 According to the most widely entertained opinion 
 specific immunity depends upon the presence of 
 specific " antisubstances" (Behring) in the blood and 
 tissues of the immunized animal. According to 
 Buchner the "antisubstances" are derived from the 
 injected bacteria cultures and are much more resis- 
 tant than alexins to noxious influences. Thus 
 tetanus antitoxin tolerates a temperature of 70°-80° 
 and the action of sunlight and putrefaction without 
 decomposing. Brieger and Ehrlich have extracted 
 diphtheria antitoxin in a solid form from the milk of 
 goats which were rendered immune against diph- 
 theria. Whether it is an albuminoid or adheres to al- 
 buminoids, is not yet known. The antitoxins are best 
 extracted (Brieger and Boer: Z. H., XXI., 266) by 
 means of zinc chloride, but we have not yet succeeded 
 in freeing them from the last traces of zinc. Accord- 
 ing to Emmerich the " antisubstances," which he calls 
 "immune proteidins," are combinations of a sub- 
 stance furnished by the bacteria with body albumin 
 from the immunized animal. 
 
 In some cases the character of immunity, the action 
 of the "antisubstances," is jjurely antitoxic, a true 
 antidote. The notion, first advanced by Behring and 
 Kitasato, that toxin and antitoxin neutralize one an- 
 other chemically (somewhat like an acid and its base) 
 has not been corroborated. We have to deal rather 
 
SUGAR IK THE NUTRIENT MEDIUM. 165 
 
 with an antagonistic action upon the cells of the 
 body analogous to the action of atropine against 
 morphine, except that the antisubstances possess a 
 minimum toxicity or none at all. The proof that an 
 ineffective mixture of toxin and antitoxin still contains 
 a virus is furnished, for example, by the fact that 
 guinea-pigs, upon whom antitoxin has less protective 
 action than upon mice, can be poisoned with mixtures 
 of toxin and antitoxin, which are entirely devoid of 
 effect on mice (Buchner). 
 
 While the "antisubstances" of diphtheria protect 
 very well against the diphtheria virus, they have no 
 injurious effect on the diphtheria bacilli either in 
 vitro or in vivo, i.e., they are not bactericidal. The 
 diphtheria bacilli may grow in the interior of an im- 
 munized organism but they are not harmful. 
 
 Entirely different in principle is the mode of action 
 of the "antisubstances" in cholera. Here they are 
 exquisitely bactericidal, but do not protect against 
 large amounts of the cholera virus (K. Pfeiffer). Ac- 
 cording to Emmerich, this is also true of hog ery- 
 sipelas and pneumonia. 
 
 Much attention has been devoted to the question 
 of the specific action of the "antisubstances." Kich- 
 ard Pfeiffer, the strongest advocate of their absolutely 
 specific action, has defended successfully the follow- 
 ing standpoint in regard to the cholera vibrio and its 
 allies : Every pathogenic organism furnishes, in the 
 body of the actively immunized animal, "antisub- 
 stances" which exert a bactericidal action (often ex- 
 tremely pronounced) only against the organism in 
 question but not against its closest allies. This spe- 
 cific action is so pronounced that Pfeiffer regards 
 
166 ATLAS OF BACTER10L0(^Y. 
 
 it as the most valuable diagnostic measure, for ex- 
 ample, in deciding the question whether an organism 
 is to be regarded as a cholera vibrio or not. Pfeiffer 
 made the same discovery in regard to bacterium typhi 
 and its allies, and this is corroborated by Dunbar, 
 Sobernheim, Loffler, and Abel. 
 
 It must not be forgotten, however, in opposition to 
 these very interesting and surprising findings that a 
 number of investigators (for example, Hiippe) do 
 not recognize a sharp distinction between resistance 
 and specific immunity, but acknowledge only quanti- 
 tative, not qualitative, differences. At all events, we 
 still have much to learn in this difficult field. 
 
 Technical Appendix. 
 
 The following recommendations and brief descrip- 
 tions furnish all the technical directions which are 
 given in a thorough course of bacteriology. We have 
 given only the most necessary data and those which 
 in our experience have proved most practical. 
 
 I. MICROSCOPICAL EXAMINATION OF BACTERIA. 
 
 1. Hints on Microscopical Technique. 
 
 For bacteriological examinations we use almost ex- 
 clusively the modern microscope with Abbe's illu- 
 minating apparatus, iris diaphragm, a low-power 
 lens, and an oil immersion lens. 
 
 A. Low magnifying power (sixty to one hundred 
 
 times) and narrow diaphragm are used for careful 
 
 examination of plate cultures. For this purpose we 
 
 either raise the cover* and examine the colony from 
 
 *Our plate cultures are always poured into cups. 
 
TECHNICAL APPENDIX. 167 
 
 above, or, if we do not wish to soil the plate by open- 
 ing it, place it upon the cover and examine the colony 
 from below. This does not give such characteristic 
 appearances in all cases. 
 
 B. High magnifying power. Oil immersion (seven 
 hundred to twelve hundred times) is used in the ob- 
 servation of individual bacteria. Upon the prepara- 
 tion is placed a drop of oil of cedar, the tube of the 
 microscope pushed down by means of the coarse ad- 
 justment until the lens just touches the surface of the 
 oil, and then adjust it accurately on the preparation 
 with the micrometer screw. 
 
 (a) Unstained Preparations. Narrow diaphragm. 
 They are examined in two ways : 
 
 1. A drop of a fluid pure culture or a drop of water 
 mixed with a trace of pure culture is placed between 
 the slide and cover-glass ; or 
 
 2. In the hanging drop. A platinum loopful of 
 fluid pure culture, or a loopful of bouillon mixed with 
 a trace of pure culture, is placed 
 on a cover-glass, and this laid 
 (reversed) upon a slide which 
 has been hollowed out so that the drop lies in the 
 cavity. The cover-glass is then fixed to the slide 
 by applying a trace of water to the four corners of 
 the cover-glass or by apjilying vaseline, if prolonged 
 observation is required. 
 
 (6) Stained Preparations. Open diaphragm. 
 Abbe's illuminating apparatus. To observe double- 
 stained section preparations we require wide dia- 
 phragm for the bacteria and narrow diaphragm for 
 the tissues. 
 
 C. Cleansing of the preparations and the micro- 
 
168 ATLAS OF BACTERIOLOGY. 
 
 scope. The immersion oil is always brushed off 
 gently, and now and then the lens is rapidly cleansed 
 with xylol and chamois skin; prolonged action of 
 xylol loosens the setting of the lens. Xylol also re- 
 moves dried particles of oil from the cover-glasses of 
 old preparations. 
 
 2. The Most Important Solutions for Making 
 Preparations. 
 
 A. Staining Solutions. 
 
 1. Watery alcoholic solution of fuchsin and methyl 
 blue. A concentrated "stock solution" is made by 
 pouring absolute alcohol over the powdered coloring 
 matters (fuchsin, methyl blue) in bottles, shakiug, 
 letting them stand for a few hours, and then filtering. 
 Of this saturated solution one part is mixed with four 
 parts distilled water and filtered before using. In 
 order to obtain good preparations it is better to stain 
 for a longer time with weak solutions than for a 
 shorter time with strong solutions. 
 
 2. Carbolized fuchsin (Ziehl's solution) : 
 
 Fuchsin 1.0 gm. 
 
 Acid, carbolic, liq 5.0 " 
 
 Alcohol 10.0 " 
 
 Aq. dest 90.0 " 
 
 3. Aniliue fuchsin: 4.0 aniline oil (anilin. pur.) are 
 well shakeu for several minutes with 100 aq. dest., 
 then filtered until all the water runs off clear (then 
 the funnel is removed because otherwise the oil will 
 pass through). In this aniline water are dissolved 4.0 
 gm. fuchsin and it is then again filtered. 
 
TECHNICAL APPENDIX. 169 
 
 4. Aniline gentian (Elirlich's solution): To 100 
 c.c. aniline water add 11 c.c. of an alcoholic con- 
 centrated gentian violet solution (stock solution). 
 This solution does not keep long. 
 
 5. Loffler's methyl blue: To 100 c.c. water, which 
 contains 1 c.c. of a one-per-cent potash lye, add 
 30 c.c. of a concentrated alcoholic solution of methyl 
 blue. The staining power is increased by the addi- 
 tion of the alkali. 
 
 6. Bismarck brown: Prepare like No. 1. (Stains 
 tissues, but bacteria poorly). 
 
 7. Alum carmine: To 100 c.c. of a five-per-cent 
 alum solution add 2 gm. carmine, boil for an hour, 
 and filter. 
 
 B. Differentiation Measures. 
 
 1. Distilled water. 
 
 2. Absolute alcohol. 
 
 3. Iodine-potassium iodide solution (Gram). 
 
 lodin. pur . 1.0 
 
 Potassii iodidi 3.0 
 
 Aq. destil 300.0 
 
 4. Sulphuric acid (twenty-five per cent). 
 
 5. Acetic acid (three per cent). 
 
 6. Acid alcohol. 
 
 Alcohol (ninety per cent) 100 c.c. 
 
 Distilled water. 200 " 
 
 Pure hydrochloric acid 20 gtt 
 
 G. Mordants for the Fhgella. 
 Loffler's mordant: 
 10 c.c. alcoholic solution of fuchsin. 
 50 c.c. cold saturated ferrosulphate solution. 
 100 c.c. twenty -per-cent tannin solution. 
 
170 ATLAS OF BACTERIOLOGY. 
 
 2. Bunge's mordant: 
 
 25 c.c. of a twentyfold diluted officinal ferric chloride 
 
 solution. 
 75 c.c. saturated watery solution of tannin. 
 
 To this solution is added, immediately before using, 
 enough of a tliree-per-cent solution of hydrogen per- 
 oxide to produce a reddish-brown color, and it is 
 then filtered (we have always dispensed with the 
 peroxide). 
 
 D. Substances Used for Clearing Up and Mounting. 
 
 1. Xylol. 
 
 2. Canada balsam. 
 
 3. Dammar varnish. 
 
 3. Preparation of Stained Specimens of Bacteria. 
 A. Smear Preparations. 
 
 1. Ordinary Stain with Fuchsin or Methyl Blue. 
 This may be used for all bacteria with the exception 
 of the tubercle bacillus. 
 
 We place upon the cover-glass or slide a loopful of 
 distilled water, mix with it a trace of pure culture 
 (best from a solid nutrient medium) and then spread 
 the drop in a very thin layer. After the fluid has 
 evaporated the preparation, with the layer turned up, 
 is rapidly drawn three times through the flame in 
 order to fni the bacteria on the glass (not to burn 
 them) and the layer of bacteria is covered with the 
 staining solution. After a brief interval (one minute), 
 perhaps after feebly warming the glass, the prepara- 
 tion is washed with water and allowed to dry (some- 
 
TECHNICAL APPENDIX. 171 
 
 times after cautious warmiug). By means of a drop 
 of Canada balsam the dry cover-glass is finally fixed 
 to the slide with the bacterial layer downward. 
 2. Gram's Stain. 
 
 (1) Making the smear preparation as above. 
 
 (2) Staining with Ehrlich's solution three minutes. 
 
 (3) Washing off with water. 
 
 (4) Differentiation with iodine-potassium iodide so- 
 lution one minute. 
 
 (5) Decolorizing with absolute alcohol up to color- 
 lessness (usually one to two minutes). 
 
 (6) Drying and mounting. 
 
 For the species which are adapted to Gram's stain, 
 vide the table. In our experience the common 
 opinion that every variety of bacteria may be pre- 
 pared invariably either well or not at all according 
 to this method is erroneous. For example, we ob- 
 served among the fluorescents, which are usually 
 described in literature as unstainable, that three 
 varieties out of twelve stained very beautifully after 
 twenty -four hours' culture. Indeed, according to 
 Zimmermann, all fluorescents may be stained in 
 young cultures. 
 
 In like manner we were able to stain the bacillus 
 of symptomatic anthrax which has often been re- 
 garded as incapable of staining. The contradictory 
 statements may be explained in part by the fact that 
 the material emi3loyed has varied greatly in age, and 
 also that the differentiation with alcohol was effected 
 in different ways. But tyrothrix tenuis, which has 
 been regarded as unstainable by Gram's method, was 
 found to stain very well on a subsequent test of the 
 same culture with the same technique. At all events 
 
172 ATLAS OF BACTERIOLOGY. 
 
 at each staining a fresh preparation of anthrax bacil- 
 lus should be stained at the same time and all prepa- 
 rations differentiated with alcohol for an equally long 
 time (one or two minutes). We can then judge very 
 well whether one variety of bacteria retains or gives 
 off the coloring matter. 
 
 3. Capsule Preparation. According to Johne we 
 proceed in the following manner : 
 
 (1) Heating the preparation with two-per-cent so- 
 lution of gentian violet until steam is given off. ^ 
 
 (2) Washing with water. 
 
 (3) Moistening with two-per-cent acetic acid for 
 six to ten seconds. 
 
 (4) Washing with water. 
 
 By this method a very distinct membrane around 
 the intensely colored bacterium cell can often be 
 demonstrated in varieties which are not regarded as 
 "capsular bacteria." The capsules are seen best on 
 examination in water. 
 
 4. Staining of Flagella. The flagella, which are 
 almost always invisible when unstained, are generally 
 prepared according to LofBer's method : 
 
 (1) Rubbing up a trace of young agar streak cul- 
 ture (not bouillon) in a very small drop of water; 
 spread out well, dry rapidly. 
 
 (2) Heating of the preparation with mordant until 
 steam is produced (do not boil) for one-half to one 
 minute. 
 
 (3) Washing off in a vigorous stream of water. 
 
 (4) Washing off in alcohol in order to remove the 
 remains of the mordant adherent at the edges. 
 
 (5) Dropping of the staining fluid (a fev/ crystals 
 are dissolved in 10 c.c. aniline water, and then one 
 
TECHNICAL APPENDIX. 173 
 
 per cent soda lye is added drop by drop until the 
 clear fluid just begins to grow opaque) and beating 
 for one minute until steam is evolved. 
 
 (6) Washing off in water, drying, mounting in 
 Canada balsam. 
 
 The manipulations must be carried out with the 
 most scrupulous cleanliness, and the cover-glasses 
 must be especially well cleaned with acids and alco- 
 hol. The cultures must be young, although it is not 
 necessary, as some authors maintain, to make the 
 staining only in cultures that are twenty-four hours 
 old. We have often obtained very good preparations 
 even at the end of twelve days. The mordants are 
 usually prepared fresh. 
 
 According to Loffler, it is necessary, in the case of 
 the majority of bacteria, to add a definite amount of 
 acid or alkali to the mordant in order to obtain well- 
 stained flagella. Loffler advises that to 16 c.c. of the 
 mordant there be added for : 
 
 Drops. Soda lye. 
 
 Cholera vibrios i to 1 1 per cent 
 
 Spirillum rubrum 9 1 
 
 Bacterium typhi 20 to 22 1 
 
 Bacillus subtilis 28 to 38 1 
 
 Bacillus oedematis maligni. .. 36 to 37 1 
 
 Bacterium pyocyaneum 5 to 6 Equivalent sul- 
 phuric acid. 
 
 Oar results show that in the majority of cases we 
 obtain very useful pictures with the unchanged mor- 
 dant and that the addition of alkali or acid is by no 
 means material. Similar experiences have been had 
 by other writers, for example, Lucksch, Giinther, A. 
 Fischer, Nicolle and Morax, but our investigations 
 have not been concluded. 
 
174 ATLAS OF BACTERIOLOGY. 
 
 Bunge lias recently employed a somewhat different 
 method which also gave us good results, but, like 
 Loffler's method, occasionally left us capriciously in 
 the lurch. 
 
 (1) Preparation of the specimen, according to 
 Loffler. 
 
 (2) Heating with Bunge's mordant for one minute 
 until steam is produced. 
 
 (3) Careful cleaning with water and drying. 
 
 (4) Warming slightly with carbolized gentian violet 
 or carbolized fuchsin. 
 
 (5) Washing in water, drying, and mounting in 
 Canada balsam. 
 
 Most of our specimens are i)repared with Bunge's 
 mordant which is several months old. 
 5. Staining of Endospores.* 
 According to Hauser : 
 
 (1) Preparation of the specimen. (It should be 
 drawn ten times, instead of three times, rapidly 
 through the flame.) 
 
 (2) Staining with watery fuchsin or carbolized 
 fuchsin (Ziehl's solution) ; the preparation, over the 
 flame, is covered freely with the staining fluid, and 
 heated (not boiled) one to two minutes until there is 
 an indication of simmering. The evaporating stain- 
 ing fluid is replaced constantly by fresh fluid. 
 
 (3) Washing with acid alcohol, f until the red color 
 of the preparation is almost gone. 
 
 * Arthrospores possess no undisputed color reactions. For 
 metacJiromatic corpuscles, Ernst's and Bunge's granules, pre- 
 liminary stages of spores, mde page 71. 
 
 f Instead of acid alcohol we may also use thirty per cent 
 nitric acid, five or twenty five per cent sulphuric acid, but 
 these must be allowed to act for a shorter period. 
 
TECHNICAL APPEI^DIX. 175 
 
 (4) After-staining with methyl blue (a few sec- 
 onds). The spores remain red, the bacilli blue. 
 
 6. Staining of Tubercle Bacilli. This is done ac- 
 cording to the same principles as the staining of 
 spores. The preparation is treated in the flame 
 with a deeply staining solution and then everything 
 with the exception of the tubercle bacilli is decolor- 
 ized with some acid solution. 
 
 (a) We may manipulate exactly as in spore staining 
 (according to Ziehl-Neelsen), except that the prepara- 
 tion is drawn only three times through the flame. 
 This method is the only one employed by us. An- 
 other favorite method is the one recommended by A. 
 Fraenkel and Gabbet, in which decolorization and 
 after-staining are effected at the same time. Then 
 the preparation which has been stained with hot car- 
 bolized fuchsin, and washed in water, is placed in the 
 following solution: 
 
 Sulphuric acid 1 
 
 Distilled water 3 
 
 Methyl blue, q.s. uutil the most intense blue color is pro- 
 duced. 
 
 We then wash carefully in water, dry, and mount in 
 Canada balsam. 
 
 However convenient this method may be, it is 
 better, for those who are not very experienced, to 
 stain, differentiate with acids, and after-stain sepa- 
 rately, because in this way success is more assured. 
 
 (h) Ehrlich-Koch's method is also often employed. 
 The dry preparation is drawn through the flame, 
 treated with aniline gentian solution for one to two 
 minutes over the flame and heated with acid (usually 
 thirty per cent nitric acid) for one to four seconds, 
 
176 ATLAS OF BACTERIOLOGY. 
 
 and with sixty per cent alcohol for a few moments. 
 It is then dipped for several minutes in a watery 
 solution of Bismarck brown and washed off in water. 
 The tubercle bacilli then appear violet on a brown 
 background. 
 
 In this form the method is suitable for cover-glass 
 preparations from pure cultures and tuberculous 
 sputum with many tubercle bacilli. If very few or no 
 bacilli are found in the first preparations, we must 
 adopt some method for increasing their numbers. 
 We mention two of the innumerable recommen- 
 dations : 
 
 {a) According to Strohschein : 
 
 Five to ten cubic centimetres of the sputum are 
 mixed with a threefold amount of Wendriner's borax- 
 boracic acid solution,* and after vigorous shaking 
 allowed to settle for four to five days. The mixture 
 becomes fluid and the bacilli settle at the bottom. 
 Such sputum may be used for examination even after 
 the lapse of years. 
 
 {h) According to Dahmen, modified by Heim : 
 
 The entire sputum is cooked from fifteen to twenty 
 minutes in a beaker glass in the steam chamber, then 
 allowed to cool, the opalescent fluid is poured off, and 
 the crumbly sediment is used for smear preparations. 
 
 B. Section Preparations. 
 
 1. Universal method, according to Loffler, adapted 
 to the large majority of bacteria. 
 The section, which lies in alcohol, is conveyed 
 
 * Eight grams borax dissolved in hot water, 12 gin. horacic 
 acid added, and then 4 gr. borax ; after crystallization the solu- 
 tion is filtered. 
 
TECHNICAL APPENDIX. 177 
 
 (spread upon a spatula of German silver or glass) to 
 Loffler's alkaline methyl blue solution for from five to 
 thirty minutes, and is then placed for a few seconds 
 in one-per-cent acetic acid. After the differentiation 
 the section is placed in absolute alcohol, xylol, and 
 Canada balsam. We must try how long the acetic 
 acid may be allowed to act, and must accelerate the 
 dehydration in alcohol as much as possible; the 
 bacilli should be blackish-blue, the nuclei blue, the 
 protoplasm bluish. 
 
 2. Nicolle states that by the following method he 
 has obtained very good section staining of objects 
 which are stained with difficulty — for example, in 
 glanders, typhoid fever, etc. : 
 
 Loffler's blue, one to three minutes. 
 
 Washing in water. 
 
 Treatment with ten-per-cent solution of tannin for 
 a few seconds. 
 
 Washing in water. 
 
 Absolute alcohol, oil of cloves, xylol, Canada 
 balsam. 
 
 3. According to Gram : 
 
 (1) Ehrlich's solution, three minutes. 
 
 (2) Iodine-potassium iodide solution, two minutes. 
 
 (3) Alcohol, one-half minute. 
 
 (4) Alcohol containing three per cent hydrochloric 
 acid, ten seconds. 
 
 (5) Alcohol, several minutes until maximum decol- 
 orization. 
 
 (6) Xylol; finally mounting in Canada balsam. 
 
 If the tissues are to be stained in a contrasting 
 color, the section is placed, after the maximum de- 
 colorization with alcohol, in a watery solution 
 1^ 
 
178 ATLAS OF BACTERIOLOGY. 
 
 (10 : 100) of Bismarck brown for a few minutes, then 
 in absolute alcohol for fifteen to twenty seconds, then 
 in xylol, and finally in Canada balsam. 
 
 4. Botkin maintains that Gram's stain is facilitated 
 by washing in aniline water preparations which have 
 been stained with aniline gentian. The preparations, 
 when taken from the iodine solution, subsequently 
 stand the action of the alcohol very much better. 
 Bacillus oedematis maligni and bacterium pneu- 
 moniae Friedlander can be stained in this way. 
 
 5. Kutscher's modification of Gram's method: 
 
 A concentrated solution of gentian violet is made 
 in a mixture of : 
 
 Aniline water 1 part. 
 
 Alcohol 1 *' 
 
 Five-per-cent carbolized water 1 " 
 
 This concentrated solution is poured drop by drop 
 into a watch-glassful of water until a shimmering 
 layer forms on the surface. The sections are placed 
 in this for ten to fifteen minutes, are then washed off 
 in distilled water, placed one minute in iodine- 
 potassium iodide, then in alcohol, xylol, and bal- 
 sam. Malignant oedema and symptomatic anthrax 
 can also be stained by this method. 
 
 6. If tubercle bacilli are to be stained in sections 
 we use carbolized fuchsin or aniline gentian solution 
 as in cover-glass staining, but we dispense with the 
 heating and instead allow the staining fluid to act for 
 fifteen to thirty minutes, 
 
TECHNICAL APPENDIX. 179 
 
 4. Production of Section Preparations. 
 
 At the autopsy small pieces of the organs are 
 thrown at once into an abundance of absolute alcohol 
 and kept there two to three days, the alcohol being 
 renewed two to three times. In most cases the 
 organs are then ready for cutting. For this pur- 
 pose the firmer part of the kidneys, liver, and muscles 
 are placed on a piece of cork with liquefied commer- 
 cial gelatin * and then again placed, with the cork, in 
 absolute alcohol. At the end of twenty -four hours the 
 organ may be cut with the microtome. More delicate 
 organs must be embedded in celloidin or paraffin ; be- 
 fore staining, the paraffin is removed completely by 
 washing repeatedly in turpentine, or xylol and the 
 prei^aration is placed in absolute alcohol after re- 
 moval from the xylol. 
 
 II. CULTURE OF BACTERIA. 
 
 1. Nutrient Media. 
 
 A. Non-albuminous (according to C. Fraenkel and 
 
 Voges). 
 
 Common salt 5 gm. 
 
 Neutral commercial sodium phosphate 2 " 
 
 Ammonium lactate 6 " 
 
 Asparagin 4 " 
 
 are dissolved in 1,000 gm. of distilled water. We may 
 
 add ten per cent gelatin or one per cent agar, and 
 
 thus obtain a non-saccharine nutrient medium which 
 
 * One part of gelatin is dissolved in two parts of water. 
 
180 ATLAS OF BACTERIOLOGY. 
 
 is suitable to the majority of bacteria. The addition 
 of milk sugar gives a milk-sugar uutrient medium 
 which is free from dextrose (Lehmann and Neumann). 
 B. Albuminous. 
 
 1. Peptone water. In 1 litre of water are dissolved 
 10 gm. dried peptone, and 5 gm. sodium chloride, and 
 sterilized together. 
 
 2. Milk. Fresh milk (best, fresh centrifugal milk) 
 is placed in test tubes and sterilized in the steam 
 chamber for one-half hour on two successive days. 
 Milk which contains the spores of the subtilis group 
 is often incapable of sterilization. 
 
 3. Litmus whey (Petruschky). Casein is cau- 
 tiously precipitated from milk by giving it a very 
 feeble acid reaction with diluted hj-^drochloric acid, 
 the filtrate is boilefl and filtered, and the neutralized 
 fluid mixed with some litmus. This whey is not 
 easily prepared {vide Heim: "Lehrbuch," p. 210). 
 
 4. Hay decoction. About 10 gm. dry hay are boiled 
 in a litre of water. The filtered solution is placed in 
 test tubes, and sterilized for two hours on three suc- 
 cessive days (kept over night in the incubating cham- 
 ber) in order to destroy the very resisting spores. 
 
 5. Beer wort (not neutralized) is allowed, after 
 sterilization, to settle for a few weeks, then poured 
 off clear into test tubes, and again sterilized. 
 
 6. Nutrient bouillon. 
 
 (a) From meat : 500 gm. lean beef are boiled upon 
 the flame for one-half hour with 1,000 gm. of water in 
 an enamelled pot, filtered, the filtrate reduced to 1,000 
 gm. and 10 gm. peptone with 5 gm. sodium chloride 
 added; this is placed in the steam chamber until 
 dissolved, and the whole is then neutralized with 
 
TECHNICAL APPENDIX. 181 
 
 normal soda lye (indicator, phenolphthalein).* We 
 then filter, pour into test tubes, and sterilize. 
 
 (h) From meat extract : 10 gm. meat extract are dis- 
 solved in 1,000 gm. water, 5 gm. sodium chloride and 
 10 gm. peptone are added, the solution is neutralized 
 and well sterilized several times. 
 
 7. Potato water for tubercle bacilli : 500 gm. peeled 
 potatoes are rubbed upon a grater, allowed to remain 
 over night in 500 gm. water in the refrigerator, de- 
 canted, filled up to 1000 gm., cooked for an hour in the 
 water-bath, filtered, four per cent glycerin is added, 
 and the mixture sterilized. 
 
 8. Gelatin nutrient media. 
 
 (a) Meat water-peptone gelatin (ordinary "gela- 
 tin" or "nutrient gelatin" of the laboratories). 
 
 To 1,000 gm. bouillon (vide nutrient bouillon) are 
 added 100 gm. gelatin, 10 gm. peptone, 5 gm. sodium 
 chloride, the mixture is heated in the steam chamber 
 until all the ingredients are liquefied, neutralized 
 with normal soda lye, sterilized, and filtered. After 
 the melted gelatin is placed in test tubes it is again 
 sterilized. 
 
 (b) Meat- water gelatin: the same as under (a), but 
 without peptone and sodium chloride. 
 
 (c) Beer-wort gelatin is made by adding ten per 
 cent gelatin to the wort ; it should not be neutralized. 
 
 (d) Plum-decoction gelatin : 500 gm. dried plums 
 are cooked in 500 gm. water, the fluid is poured off, 
 and the plums are again cooked with 500 gm. water. 
 
 * Illustration : Ten cubic centimetres bouillon require for sat- 
 uration 2.2C.C. one-tonfih normal soda lye; 1,000 c.c. bouillon 
 require for saturation 220 c.c. one-tenth normal soda lye, or 22 
 c. c. normal soda lye. 
 
182 ATLAS OF BACTERIOLOGY. 
 
 Both fluids are then mixed, filtered, and ten per cent 
 gelatin is added. Not to be neutralized. 
 
 (e) Herring gelatin. Two salt herring, unwashed, 
 are boiled in 1,000 gm. water and ten per cent gelatin 
 is added to the filtrate ; not to be neutralized. 
 
 (/) Potato-water gelatin, according to Holz, for 
 bacterium typhi: 500 gm. potatoes are thoroughly 
 washed, peeled, finely grated, and squeezed through 
 a linen cloth. The opaque juice may be allowed to 
 settle for twenty -four hours and then filtered, or, as 
 we always prefer, filtered at once through pure 
 animal charcoal. After heating one hour in the 
 steam chamber ten per cent gelatin is added to the 
 clear fluid, this is again heated in the steam chamber, 
 filtered, poured into test tubes and sterilized on three 
 successive days. 
 
 (g) Potassium iodide potato-water gelatin (Eisner) : 
 One per cent iodide is added to the gelatin. The 
 best way is to add a well-sterilized solution in the 
 requisite amounts to gelatin which has just been 
 made ready for use. 
 
 9. Nutrient agar. To 1,000 gm. bouillon add 
 10 gm. very finely divided agar, boil for one hour 
 on the fire in a glass retort until completely dis- 
 solved; the water which has evaporated is replaced 
 and then 10 gm. peptone and 5 gm. sodium chloride 
 are added. After heating again in the steam cham- 
 ber the fluid is neutralized, filtered by means of the 
 hot-water funnel, placed in test tubes, and again 
 sterilized. 
 
 10. In order to make grape-sugar or milk-sugar 
 agar, two per cent of the corresponding substance is 
 added with the peptone and sodium chloride. As 
 
TECHNICAL APPENDIX. 183 
 
 bouillon agar generally contains traces of grape 
 sugar, we have for some time made a milk-sugar 
 agar which is free from grape sugar, according to the 
 plan described under A. 
 
 11. Glycerin agar. To the nutrient agar is added 
 five per cent glycerin, the mixture poured into test 
 tubes and sterilized. 
 
 12. Sugar-chalk agar. Mix melted sugar agar 
 with finely powdered, dry, sterilized carbonate of 
 lime until the mixture becomes cloudy and opaque, 
 inoculate the bacteria into it, and pour out in plates. 
 
 13. Potatoes. After careful washing the potatoes 
 are peeled, cut into discs 1 cm. thick, and sterilized 
 several times in high Petri's dishes. We may also 
 perforate the peeled potato with a large cork borer 
 and divide \lie cylinder by an oblique cut into two 
 wedges. The pieces are then placed in a test tube 
 at the bottom of which is a little dry cotton (to ab- 
 sorb the water ci condensation) and sterilized several 
 times in the steam chamber. 
 
 14. Blood serum. The blood, taken from the 
 slaughtered animal under proper precautions, is al- 
 lowed to stand for twenty -four hours in well cleaned 
 glass cylinders in the refrigerator; on the following 
 day the serum is removed by means of large sterile 
 pipettes. It is placed in bottles, one per cent chloro- 
 form is added, and is then allowed to stand for a few 
 weeks, being shaken occasionally. For use, we place 
 the serum, which has been poured into tubes, in the 
 incubating chamber for a few days in order that the 
 chloroform may escape completely. It is employed 
 either in the fluid state or after it has been made rigid 
 at a temperature of 65°. 
 
 % 
 
184 ATLAS or BACTERIOLOGY. 
 
 15. Loffler's serum mixture for diphtheria bacilli. 
 Three parts of beef or sheep serum are mixed with 
 one part calf's bouillon, which contains one per cent 
 grape sugar, one per cent peptone, and one-half per 
 cent sodium chloride. 
 
 16. Entirely different from the other media is that 
 first devised by Kiihne, modified by various writers, 
 and finally made somewhat more practicable by 
 Stutzer and Burri. We refer to the silicic acid nu- 
 trient medium. Gelatinous silicic acid, whieh is 
 merely mixed with a few salts, is an important nu- 
 trient medium for certain organisms (for example, the 
 nitrate-producers) on account of the lack of organic 
 nutrient substances. For the somewhat complicated 
 manipulation, vide Stutzer and Burri (C. B., Yol. I., 
 Part v., 722). 
 
 2. The Employment of the Duterent Nutrient 
 Media Depends upon the Following View- 
 Points : 
 
 I. Fluids (bouillon, sugar bouillon, milk, non- 
 albuminous nutrient solution). 
 
 1. To produce cultures en masse. 
 
 2. To obtain bacterial solutions containing an ac- 
 curately determinable number of bacteria (counting 
 by means of plates). 
 
 3. To observe the development of membrane and 
 sediment. 
 
 4. To study the metabolic products. 
 II. Solid Nutrient Media. 
 
 1. Gelatinous nutrient media. The most exten- 
 sive use is made of gelatinous, transparent nutrient 
 
TECHNICAL APPENDIX. 186 
 
 media (agar and gelatin) and for the following rea- 
 sons : 
 
 (a) They may be used as fluids and as solid media : 
 as fluids they permit the separation, as solid sub- 
 stances the fixation, of the isolated germs and their 
 separate growth into colonies. 
 
 (b) On account of their transparency they permit a 
 macroscopic as well as a microscopic observation of 
 the cultures; they permit a differential diagnosis of 
 the varieties and an early recognition of any im- 
 purities. 
 
 They are used particularly : 
 
 (a) For plate cultures, *.e., as a proof of positive 
 separation and for the enumeration of individuals and 
 varieties. 
 
 (b) To secure characteristic macroscopic cultures, 
 which will serve for differential diagnosis. 
 
 (c) For permanent cultures or collections of living 
 bacteria. 
 
 The special advantages of agar and gelatin are : 
 
 (a) Gelatin. Advantages : Easily produced, easily 
 formed into plates (at 25°) ; its property of liquefac- 
 tion by certain bacteria possesses great diagnostic 
 importance. Disadvantages : As it melts at 25°, it 
 cannot be used in hot weather and at incubating tem- 
 perature. 
 
 (b) Agar. Advantages : Practicable at incubating 
 temperature (i.e., for the rapid culture of bacteria 
 [spores] and particularly of thermophile bacteria). 
 Disadvantages: Difficulty of preparation; not so 
 easily formed into plates. The cultures are often not 
 very characteristic. 
 
 2. Blood serum and glycerin agar. Used for the 
 
186 ATLAS OF BACTERIOLOGY. 
 
 culture of pathogenic varieties, which thrive with 
 difficulty or not at all upon other nutrient media. 
 Plate cultures are only possible with glycerin agar 
 and mixtures of agar and serum. 
 3. Potatoes. 
 
 (1) To obtain macroscopically characteristic cul- 
 tures of great durability and for differential diag- 
 nosis. 
 
 (2) Occasionally for the development of spores. 
 
 3. A Few Words on the Manipulation of Ordinary 
 Cultures. 
 
 The platinum needle must be brought to a glow 
 throughout its entire length each time before using 
 and before putting it away. 
 
 {a) Fluid cultures are inoculated with a loopful of 
 pure culture. 
 
 (b) Gelatin and agar stick cultures are made with a 
 straight needle without a loop, only one puncture to 
 each tube but extending nearly to the bottom. 
 
 (c) Agar and gelatin streak cultures and potato cul- 
 tures are made by a gentle superficial stroke upon the 
 surface with the platinum loop. In the case of the 
 potato it is sometimes necessary to rub the culture in. 
 
 (d) Gelatin plate cultures. 
 
 1. To isolate definite germs in the pure culture. 
 We melt three gelatin tubes ; put into the first, after 
 it has been cooled to 30°, a loopful of a fluid culture 
 or a trace of a solid culture; shake the tube while 
 turning it upside down, and then convey from this 
 one or two loopfuls of liquefied gelatin into a second 
 tube. After shaking this, two to three loopfuls are 
 
TECHNICAL APPENDIX. 187 
 
 placed in a third tube, and tlie contents are then 
 poured into three dry sterilized plates, lifting the 
 cover briefly and gently inclining the plate to and 
 fro, in order that the gelatin may be distributed as 
 uniformly as possible. In making inoculations from 
 one tube to another it is advisable to hold them in an 
 inclined position in order to guard against the en- 
 trance of foreign germs. The plates are then placed 
 in the culture chamber at a constant temperature of 
 22° (or they may be kept at the temperature of the 
 room) and at the end of two to three days the indi- 
 vidual colonies which have developed are observed 
 macroscopically and also microscopically with low 
 (50) magnifying powers. As a general thing only 
 two of the three plates are serviceable for observa- 
 tion, one at least is sown too thick or too thin. 
 
 2. If we wish to ascertain the number of colonies, 
 for example, in a specimen of water, we place in three 
 test tubes of melted gelatin, 1 c.c, 0.5 c.c, and 0.1 c.c. 
 of the water, shake and pour into three dishes. To 
 ascertain the number of germs, we use Wolffhiigel's 
 counting apparatus if very many germs have devel- 
 oped. If the germs are few the following plan is 
 simpler: The plate is laid upside down (upon the 
 cover) , the bottom is divided with ink into sextants, 
 and each visible colony is marked with a dot. Plates 
 upon which the number of germs in drinking-water 
 are to be ascertained must be counted several times 
 (on the second, third, and fifth days). When the 
 fluid is very rich in germs (for example, sour milk, 
 ditch water, etc.), 1 c.c. is first placed in 100 c.c. of 
 sterilized water and the mixture then manipulated as 
 described above. Solid bodies are first rubbed up in 
 
188 ATLAS OF BACTERIOLOGY. 
 
 water. "WTien air is to be examined a definite volume 
 is sucked through a tube of sterilized sand, the latter 
 carried into sterilized water, and plates are then 
 formed. 
 
 (e) Agar plate cultures are made in the same way. 
 The agar should not be poured into the dishes when 
 too cool, because otherwise it coagulates at once into 
 an irregular surface ; if used when too warm, the in- 
 oculated bacteria will die*. In recent times it has 
 been recommended that in making agar plates the 
 nutrient medium should first be allowed to become 
 rigid in the dish, and then the mass to be examined 
 is smeared superficially upon it with a sterilized 
 platinum loop, a strip of filtering paj^er or a xjlatinum 
 brush. In this way we obtain only characteristic 
 superficial colonies. 
 
 (/) Sugar-agar-agitation cultures : The contents of 
 the tube are melted in the water-bath, then cooled to 
 about 40°; a loopful of pure culture is then intro- 
 duced, the tube well shaken, and when it becomes 
 rigid the culture is placed in the incubating chamber. 
 
 4. Anaerobic Cultures. 
 
 We have employed almost exclusively Buchner's 
 method, i.e., the absorption of oxygen by pyrogallic 
 acid and potash lye.* 
 
 (a) For stick cultures : Upon the bottom of a glass 
 cylinder, which must be somewhat longer and wider 
 than a test tube, is placed a heaping teaspoonful of 
 pyrogallic acid and 20 c.c. of a three-per-cent potash 
 
 * Sensitive varieties are said to thrive still better in a hydrogen 
 atmosphere. 
 
TECHNICAL APPENDIX. 189 
 
 lye; place in it the infected stick culture and close 
 the cylinder at once with a soft-rubber stopper or a 
 ground-glass stopper which is sealed with paraffin. 
 According to Kitasato the anaerobics which are less 
 sensitive to oxygen may be cultivated in saccharine 
 agar in a high stick culture, even without pyrogallic 
 acid. A wire with a small loop is pushed into the 
 layer of sugar agar (8 to 10 c.c. high), and the wire 
 turned on its long axis before withdrawal. 
 
 (b) For i^late cultures we use, instead of the glass 
 cylinder, a wide exsiccator with a ground cover; fill 
 the lower part with sand and the pyrogallic-acid mix- 
 ture, and then manipulate as before. 
 
 III. EXPERIMENTS ON ANIMALS. 
 A. Infectioyi. 
 
 1. Subcutaneous inoculation. A shallow incision 
 is made with a pair of scissors on some part of the 
 skin, after it has been washed with a 0.1-per-cent 
 solution of corrosive sublimate; the inoculating 
 matter is carried beneath the skin by means of a 
 stout platinum wire with a loop. Mice are generally 
 inoculated above the root of the tail ; they are simply 
 held by the tip of the tail, and allowed to hang into a 
 glass which is covered up in great part by a piece 
 of wood. Guinea-pigs and rabbits are inoculated on 
 the side of the thorax. 
 
 2. Subcutaneous injection is generally effected by 
 means of Koch's rubber ball injection syringe or 
 Strohschein's syringe. A fold of skin is picked up 
 at some part of the body, and the needle inserted in 
 the longitudinal direction. If several cubic centi- 
 
190 ATLAS OF BACTERIOLOGY. 
 
 metres are to be injected, the following simple method 
 may be adopted: A short piece of rubber tube pro- 
 vided with an injection needle is fastened to a grad- 
 uated pipette, the entire apparatus sterilized, the 
 pipette filled, and the fluid blown' in by the aid of the 
 mouth or a rubber bulb. 
 
 3. Peritoneal injection is made by perforating with 
 a sterilized hollow canula, at one puncture, the ab- 
 dominal wall, then cautiously advancing the needle 
 and injecting the fluid. 
 
 B. Observation. 
 
 Mice may be kept in sterilized glass vessels closed 
 with cotton and wire netting ; larger animals must be 
 kept in sterilized cages or stalls. 
 
 G. Autopsy and Disposal of the Cadaver. 
 
 Autopsies must be made immediately after death, 
 or, at least, the animal placed on ice. The animal, 
 lying on the back, is tied or nailed through the legs 
 to a board, the abdomen and chest are throughly 
 moistened with corrosive sublimate, and then the ab- 
 dominal cavity is opened with a previously sterilized 
 knife. The abdominal walls are separated and from 
 the spleen, liver, and kidneys some blood (or tissue 
 juice) is removed with a sterilized platinum loop. 
 This is smeared at once upon prepared agar plates. 
 The organs are carefully cut out, avoiding contact 
 with the intestines, and are placed in absolute alcohol 
 for further examination. Then the thorax is opened 
 with a pair of scissors, blood taken from the heart and 
 lungs, and these organs are placed in alcohol. Be- 
 fore each operation the instruments must be carefully 
 
TECHNICAL APPENDIX. 191 
 
 brought to a glow. It is better to have on hand 
 numerous instruments which have been sterilized at 
 130°. The hands must be kept perfectly clean. 
 
 After the autopsy it is best to cremate the cadaver. 
 If this is not feasible, the body is wrapped in cloths 
 dipped in a solution of corrosive sublimate and buried 
 in a hole in the ground at least one-half metre deep, 
 which is filled in with quicklime. 
 
ALPHABETICAL INDEX OF ILLUSTRATIONS. 
 
 Actinomyces, pi. 63 
 Anthrax bacillus, pi. 38-40 
 Arthrospores, pp. 67, 76 
 Bacillus acidi lactici, pi. 13 
 
 anthracis, pi. 38-40 
 
 butyricus, pi. 42, V.-VI. 
 
 Chauvoei, pi. 46 
 
 coli, pi. 14, 15 
 
 cyanogencs, pi. 23, 24 
 
 diplitheriae, pi. 20 
 
 erysipelatos suum,pl.34,I. 
 
 fluorescens liquefaciens, 
 pi. 28 
 
 fluorescens non - liquefaci- 
 ens, pi. 22 
 
 haemorrhagicus, pi. 21, 
 VIL, VIII. 
 
 influenzae, pi. 63, V. 
 
 janthinus, pi. 27 
 
 kiliensis, pi. 26 
 
 latericius, pi. 21, I. -VI. 
 
 leprae, pi. 63, I. -III. 
 
 mallei, pi. 19 
 
 megatherium, pi. 35 
 
 mesentericus fuscus, pi. 
 42, 43, VIII., IX. 
 
 mesentericus vulgatus, pi. 
 44 
 
 murisepticus, pi. 34, II.- 
 X. 
 
 13 
 
 Bacillus mycoldes, pi. 41-42, 
 I.-IV. 
 
 oedematis maligni, pi. 47 
 
 pneumonia;, pi. 12 
 
 prodigiosus, pi. 25 
 
 putidus, pi. 22 
 
 pyocyaneus, pi. 29 
 
 septicacmiae haemorrhagi- 
 cae, pi. 18 
 
 subtilis. pi. 36, 37 
 
 syncyaneus, pi. 24 
 
 tetani, pi. 45 
 
 typhi, pi. 16, 17 
 
 violaceus, pi. 27 
 
 vulgatus, pi. 43 
 
 Zopfii, pi. 30, 31 
 Bacteria, forms of, p. 66 
 
 in soft chancre, pi. 63, IV. 
 Bacterium acidi lactici, pi. 13 
 
 coli commune, pi. 14, 15 
 
 erysipelatos suum, pi. 34, 1. 
 
 haemorrhagicum, pi. 21, 
 VIL, VIII. 
 
 influenzae, pi. 63, V. 
 
 janthinum, pi. 27 
 
 kiliense, pi. 26 
 
 latericium, pi. 21, I. -VI. 
 
 mallei, pi. 19 
 
 murisepticum, pi. 34, II.- 
 IX. 
 
194 
 
 ATLAS OF BACTERIOLOGY. 
 
 Bacterium pediculatum, p. 
 73 
 pestis, pi. 63, VI., VII. 
 pneumoniae, pi. 13 
 prodigiosum, pi. 35 
 putidum, pi. 33 
 pyocyaneum, pi. 39 
 septicaemiae hsemorrbagi- 
 
 cae, pi. 18 
 syncyaneum, pi. 34 
 typhi, pi. 16, 17 
 violaceum, pi. 37 
 vulgare, pi. 33 
 vulgare /5 mirabilis, pi. 33 
 Zopfii, pi. 30, 31 
 Butyric acid bacillus, pi. 43, 
 
 V.-VII. 
 Capsule bacillus, Friedlander's, 
 pi. 13 
 coccus, Fraenkel's, pi. 5 
 formation of, p. 73 
 Chain coccus, pi. 6 
 Chicken cholera, pi. 18 
 Cholera bacillus, pi. 49-53 
 reaction, pi 54, IV. 
 vibrio, pi. 49-53 
 Chromogenous sarcinae, pi. 9- 
 
 11 
 Cladothrix dichotoma Auto- 
 rum non Cohn, pi. 61 
 Comma bacillus of cholera, pi. 
 49-53 
 bacillus of Finkler, pi. 53, 
 
 VI., 56 
 bacillus of Metsclmikoff, 
 pi. 53, V. 
 Corynebacterium diphtherise, 
 
 pi. 30 
 Diphtheria bacillus, pi. 30 
 
 Diplococcus gonorrhoeae, pi. 3, 
 
 VL, Vl.a, Yl.b 
 Diplococcus lanceolatus, pi. 5 
 pneumoniae, pi. 5 
 roseus, pi. 4 
 Endogenous spores, p. 79 
 Erysipelas streptococcus, pi. 6 
 Farcin de boeuf, pi. 60 
 Fermentation tubes, p. 155 
 Finkler 's comma bacillus, pi. 
 
 56, 53, VI. 
 Flagella types, p. 73 
 Fluorescens liquefaciens, pi. 38 
 
 non-liquefaciens, pi. 33 
 Fluorescent bacteria, pi. 33, 
 
 38, 39 
 Fowl cholera, pi. 18 
 Fraenkel's pneumonia coccus, 
 
 pi. 5 
 Friedlander's pneumonia ba- 
 cillus, pi. 13 
 Germination of spores, p. 73 
 Glanders bacillus, pi. 19 
 Gonococcus, pi. 3, VI., VI. «, 
 
 VL6 
 Gonorrhoea, pi. 3, VI., VI. a, 
 
 VI. 6 
 Green pus, pi. 39 
 Hanging drop, p. 167 
 Hauser's bacterium, pi. 33, 33 
 Hay bacillus, pi. 36, 37 
 Hog erysipelas, pi. 34, I. 
 Indol reaction in cholera, pi. 
 
 54. 4 
 Influenza bacillus, pi. 63, V. 
 Involution forms of anthrax, 
 pi. 40, V. 
 forms of cholera, pi. 53, IV. 
 Kiel water bacillus, pi. 36 
 
ALPHABETICAL INDEX OF ILLUSTRATIONS. 
 
 196 
 
 Lactic acid bacillus, pi. 13 
 Lepra bacillus, pi. 63. L-IIL 
 Leptothrix epidermidis, pi. 59 
 Loffler's bacillus, pi. 20 
 Malignant oedema, pi. 47 
 Malleus, pi. 19 
 Membrane, thickening of, in 
 
 bacteria, p. 73 
 Mesentericus fuscus, pi. 44 
 
 vulgatus, pi. 43 
 Metschnikoff's vibrio, pi. 53, 
 
 V. 
 Micrococcus agilis, pi. 3, I.-V. 
 badius, pi. 11, VII. 
 candicans, pi. 2, IV.-VIII. 
 gonorrhoeae, pi. 3, VI. 
 luteus. pi. 8. I.-V. 
 pyogenes a aureus, pi. 1 
 pyogenes y albus, pi. 2, 
 
 I.-IL 
 pyogenes (3 citreus, pi. 2, 
 
 III. 
 roseus, pi. 4 
 tetragenus, pi. 7 
 Morbus Werlhofii, pi. 21, VIL, 
 
 VIII. 
 Mouse septicaemia, pi. 34 
 
 typhoid, pi. 17, XL 
 Mycobacterium leprae, pi. 63, 
 L-IIL 
 tuberculosis, pi. 48 
 Oospora bo vis, pi. 62 
 chromogenes, pi. 61 
 farcinlca, pi. 60 
 Pediococcus tetragenus, pi. 7 
 Plague bacillus, pi. 63, VL, 
 
 VIL 
 Plasmolysis, according to 
 Fischer, 70 
 
 Pneumonia bacillus, pi. 12 
 
 coccus, pi. 5 
 Potato bacillus, pi. 42, VIII. , 
 
 IX., 43, 44 
 Prodigiosus, pi. 25 
 Proteus mirabilis, pi. 32 
 
 vulgaris, pi. 33 
 Pseudodichotomy in bacilli, 
 69 
 in streptococci, 69 
 Pus, green, blue, pi. 29 
 Pyocyaneus, pi. 29 
 Rabbit septicaemia, pi. 18 
 Rauschbrand, pi, 46 
 Recurrens spirilli, pi. 58, 
 
 VIIL, IX. 
 Root bacillus, pi. 41, 42, L-IV. 
 Sarcina aurantiaca, pi. 10. 
 canescens, pi. 11, VIIL 
 cervina, pi. 11, I. 
 erythromyxa, pi. 11, III. 
 flava, pi. 9 
 lutea, pi. 11, IV. 
 pulmonum, pi. 8 
 rosea, pi. 11, VL 
 Septicaemia haemorrhagica, pi. 
 
 18 
 Spirilli from the gums, pi. 58, 
 VII. 
 from the nasal mucous 
 membrane, pi. 58, III., 
 IV. 
 Spirillum concentricum, pi. 
 
 57, VI. , VIIL 
 Spirillum Obermeieri, pi. 58, 
 VIIL, IX. 
 rubrum, pi. 47, I.-V. a 
 serpens, pi. 58, I. 
 undula, pi. 58, V. 
 
196 
 
 ATLAS OF BACTERIOLOGY. 
 
 Spirocbtete Obermeieri, pi, 58, 
 VIII., IX. 
 of the gums, pi. 58, VII. 
 Spores, development of, 77 
 germiDation of, 78 
 types of, 77 
 Staphylococcus pyogenes al- 
 bus, pi. 2, L,IL 
 pyogenes aureus, pi. 1. 
 pyogenes citreus, pi. 2, 
 III. 
 Streptococcus brevis, pi. 6, X. 
 conglomerates, pi. 6, XI. 
 longus, pi. 6, IX. 
 meningitidis cerebrospi- 
 nal is, pi. 3, VIL, VIII. 
 of erysipelas, pi. 6 
 Streptococcus pyogenes, pi. 6 
 Streptothrix, pi. 60 
 
 Structure of the bacterium 
 cell, 70 
 
 Tetanus bacillus, pi. 45. 
 
 Tetragenus, pi. 7 
 
 Tuberculosis, pi. 48 
 
 Typhoid bacillus, pi. 16, 17 
 
 Vibrio albensis, pi. 54 
 
 aquatilis, pi. 55, II., VIL, 
 
 VIII., IX. 
 berolinensis,pl. 55, V., VI. 
 cholerse, pi. 49-53 
 danubicus, pi. 55, I. -III. 
 Finkler, pi. 53, VI., 56 
 fluorescent, from the Elbe, 
 
 pi. 54 
 Metschnikoff, pi. 53, V. 
 proteus, pi. 53, VI., 56 
 spermatozoides, pi. 58, VI. 
 
 Violet bacillus, pi. 37 
 
IWDEX 
 
 Abbe's illuminating appara- 
 tus, 166 
 Abrin, 135, 163 
 Absolute immunity, 157 
 Acclimatization of anthrax, 99 
 Aceton, 150 
 Acid, acetic, 150 
 
 agar, 89 
 
 butyric, 150 
 
 formic, 150 
 
 media, use of, 90 
 
 propionic, 150 
 Active immunization, 157 
 Adenin, 81 
 
 Al^robic races of anaerobic va- 
 rieties, 97 
 Aerobics, facultative, 96 
 
 strict, 95 
 Aerotaxic figures, 112 
 ^thyl alcohol, 149 
 Agar* cultures, 189 
 Albuminoids in bacteria, 80 
 
 labile, 135 
 Alcohol, 150 
 
 production of acids from, 
 156 
 Aldehyde, 150 
 Agitation cultures, 101 
 Alexin, 163 
 
 Alkali, production of, by bac- 
 teria, 130 
 
 Alkaline agar, 89 
 Alkaloids, putrefaction, 132 
 Alternating fission in different 
 
 planes, 75 
 Alum carmine, 169 
 Amidoacids, 133 
 Amines. 130, 133 
 Ammonia, demonstration of, 
 141 
 
 production of, 130, 141 
 Ammonium bases, 133 
 
 carbonate in water as a 
 nutrient, 85 
 Amygdalin, 123 
 Anaerobic cultures, 188 
 Anaerobics, conversion of, into 
 aerobics, 97 
 
 facultative, 96 
 
 strict, 96 
 Aniline fuchsin, 168 
 
 gentian, 169 
 
 oil, 169 
 
 water, 169 
 Animals, experiments on, 189 
 Antagonistic action in the ani- 
 mal body, 157 
 
 bacteria, 104 
 Anthrax spores, viability of, 
 
 108 
 Antisepsis, 90 
 Antisubstances, 164 
 
198 
 
 INDEX. 
 
 Antitoxic effects, 164 
 
 Antitoxin, 164 
 
 Aromatic metabolic products 
 
 of bacteria, 142 
 Arthrospores, 67. 76 
 Ascitic fluid, 159 
 Asepsis, 90 
 Ash, amount of, in bacteria, 
 
 80 
 Assimilation of nitrogen, 147 
 Attenuation of spores, 159, 
 of virulence, 90, 159 
 
 Bacillus ^thaceticus, 157 
 amylobar^ter, 153 
 anthracis, 79, 97, 99, 102, 
 
 122, 141, 145, 159 
 aquatilis, 85 
 
 butyrious Hilppe, 81, 152 
 Cliauvoei, 96 
 De Baryanus, 77 
 denitrificans I., 147 
 denitrificans II,, 147 
 diplitheriae, 157 
 crythrosporus, 85 
 fluorescens liquefaciens, 
 
 122, 132, 140 
 kiliense. 122 
 leptosporus, 79 
 limosus, 77 
 macrosporus, 77 
 megatherium, 111, 122 
 mesentericus, 99, 113 
 raycoides, 101, 145 
 oedematis maligni, 96, 
 
 161 
 oxalaticus, 71 
 perlibratus. 113 
 radicicola, 147 
 
 Bacillus sessilis, 80 
 
 Solmsii, 77 
 
 subtilis, 80, 101, 111, 113, 
 120, 141 
 
 tetani, 87, 96, 106 
 
 thermophilus, 99 
 
 tuberculosis, 
 
 urese, 131 
 
 viscosus sacchari, 81 
 
 vulgatus, 98 
 Bacteria, antagonism between, 
 104 
 
 chemiccl composition of, 
 80 
 
 chemical effects, 115 
 
 definition, 65 
 
 growth in groups, 67 
 
 mechanical and electrical 
 effects of, 100 
 
 mechanical effects, 111 
 
 optical effects, 111 
 
 resistance of, to deficiency 
 of food and water, 93 
 
 solitary growth of, 67 
 
 thermic effects, 115 
 
 vital conditions of, 84 
 Bacterial proteins, 135 
 Bacterio-fluorescin, 128 
 Bacterio-trypsin, 117 , 
 Bacteroids, 148 
 Bacterium aceti, 156 
 
 acidi lactici, 86, 97 
 
 Bischleri, 152 
 
 cholerse gallinarum, 94 
 
 coli, 110, 141, 145, 147 
 
 cuniculicida, 87 
 
 erysipelatos suum, 87 
 
 indigonaceum, 128 
 
 janthinum, 128 
 
IKDEX. 
 
 199 
 
 Bacterium kiliense, 127, 130 
 mallei, 122 
 murisepticum, 87 
 pediculatum, 73 
 PflUgeri, 98, 105 
 phosphorescens, 114 
 pneumonice, 82, 122 
 prodigiosum, 82, 102, 121, 
 
 145 
 putidum, 102, 104 
 pyocyaneum, 121 
 pyogenes fatidum, 122 
 syncyaneum, 129 
 synxanthium, 122 
 typhi, 145, 147 
 violaceum, 122, 127 
 vulgare, 119, 160 
 vulgare /9 Zenkeri, 144 
 
 Beer wort, 180 
 
 Beggiatoa, 81 
 
 Beozaldehyde, 123 
 
 Bilineurin, 133 
 
 Bismarck brown, 169 
 
 Blood serum, 183 
 
 Blue milk, 128 
 
 Bouillon culture, 141 
 
 Brieger's method of isolating 
 ptomains, 134 
 
 Brownian molecular move- 
 ments, 112 
 
 Bunge's granules, 71 
 mordant, 170 
 
 Butter, rancidity of, 143 
 
 Butyl alcohol, 152 
 
 Butyric acid, 152 
 
 Cadaverin, 133 
 Capsule bacteria, 72 
 preparation of, 172 
 
 Carbohydrates, production of 
 
 acids from, 148 
 Carbolized fuchsin, 168 
 Carbonic acid, action on bac- 
 teria, 97 
 Carolin, 127 
 Cedar, oil of, 167 
 Cell structure of bacteria, 68 
 Cellulose, 81 
 
 decomposition of, by bac- 
 teria, 153 
 Central body of bacteria, 71 
 
 fluid of bacteria, 70 
 Chemical composition of bac- 
 teria, 80 
 
 effects of bacteria, 115 
 
 ferments, 116 
 Chemotaxis, 112 
 Cholera as a nitrite poisoning, 
 158 
 
 diblastic theory of, 106 
 Cholesterin, 80 
 Cholinbilineurin, 133 
 Chromogenic functions of bac- 
 teria, 129 
 Chromogenous bacteria, 110 
 Cinnamic acid, 163 
 Clostridium butyricum, 152 
 Club-shaped bacteria, 67 
 Comma bacteria, 67 
 Congenital immunity, 162 
 Counting bacteria, 105 
 Creolin, 92 
 Cultures, 179 
 
 manipulation of, 186 
 
 anaerobic, 188 
 
 Decomposition of cellulose by 
 bacteria, 153 
 
200 
 
 INDEX. 
 
 Decomposition of fats, 143 
 
 Definition of bacteria, 65 
 
 Degeneration forms of bac- 
 teria, 80 
 
 Demonstration of indol, 142 
 of nitrites, 141 
 of phenol, 143 
 
 Desiccation experiments, 94 
 
 Deuteroalbumose, 135 
 
 Diastatic ferments, 121 
 
 Dichotomy, 68 
 
 Diethylamin, 133 
 
 Dimethylamin, 133 
 
 Dimethylethylendiamin, 133 
 
 Diphtheria antitoxin, 164 
 
 Disinfectants, combination of, 
 90, 93 
 
 Distilled water, action on bac- 
 teria, 93 
 
 Dry bacteria, viability of, 
 94 
 
 Drying nutrient media, 93 
 
 Dulcite, 156 
 
 Ehrlicii's solution, 169 
 Electric arc action on bac- 
 teria, 102 
 Enantobiosis, 104 
 Endospores, 76 
 
 staining of, 174 
 Enzymes, 116 
 
 proteolytic, 117 
 Ernst's granules, 71 
 Ethyl, 150 
 Ethylamin, 133 
 Ethylendiamin, 133 
 Ethylidlactic acid, 151 
 Eubacillus multisporus, 66 
 Experiments on animals, 189 
 
 Extractive matters in bacteria, 
 80 
 
 Facultative aerobics, 96 
 
 anaerobics, 96 
 Fats, decomposition of, 143 
 Fermentation, definition of, 
 124 
 
 flask, 155 
 
 lactic acid, 151 
 
 oxidative, 125 
 Ferments, 116 
 
 diastatic, 121 
 
 inverting, 122 
 
 rennet, 123 
 Ferric oxide, 81 
 Fibrin, liquefaction of, 117 
 Filamentous bacteria, 67 
 Fission of bacteria, 75 
 Flagella, 73 
 
 mordants, 169 
 
 staining of. 172 
 Flagellates, 65 
 Flesh-water peptone gelatin, 
 
 87 
 Fluorescent pigments, 126 
 Formic acid, 156 
 Frog -spawn disease, 73 
 Fuchsin, 168 
 
 Gas, formation of, from carbo- 
 hydrates, 153 
 Gelatin, liquefaction of, 117, 
 119 
 neutral, 88 
 nutrient media, 181 
 various kinds of, 181, 182 
 Germination of spores, 78 
 Globulin in bacteria, 80 
 
INDEX. 
 
 201 
 
 Gly cerate of lime, 157 
 Glycerin, 156 
 
 agar, 87, 183 
 Gram's stain, 171 
 Granules, Bunge's, 71 
 
 Ernst's, 71 
 
 metachromatic, 71 
 
 sporogenous, 71 
 Granulobacter polymyxa Bey- 
 
 erinck, 152 
 Growth of bacteria, 67 
 Guanidin, 133 
 Guanin, 81 
 
 HALF-scREW-shaped bacteria, 
 67 
 
 Hanging drop, 167 
 
 Hay decoction, 180 
 
 Heat, production by bacteria, 
 115 
 
 Hemicellulose, 81 
 
 Herring gelatin, 182 
 
 Honeycomb structure of bac- 
 teria, 69 
 
 Hydrocarbons in bacteria, 81 
 
 Hydrogen peroxide, produc- 
 tion on illuminated cultures, 
 103 
 
 Immune proteidins, 164 
 Immunity, 157 
 Increase of virulence, 160 
 Indicator, 88 
 Indol, 133 
 
 demonstration of, 142 
 Inverting ferments, 122 
 Involution forms of bacteria, 80 
 Iodine -potassium iodide solu- 
 tion, 169 
 
 Iodoform, 150 
 Iris diaphragm, 166 
 Isatin sulphate, 140 
 Isolation of ptomains, 134 
 
 Knob bacteria, 147 
 Koch's tuberculin, 135 
 Koly sepsis, 90 
 
 Labile albuminoids, 135 
 Lactate of lime, 157 
 Lactic-acid fermentation, 151 
 Lecithin, 80 
 Leptothrix, 81 
 Leucin, 133 
 
 Leuconostoc mesenterioides, 81 
 Leuko substances, 140 
 Lieber's iodoform reaction, 
 
 150 
 Lime, glycerate of, 157 
 
 lactate of, 157 
 Lipochromata, 127 
 Liquefaction of gelatin, 119 
 Litmus, reduction of, 140 
 
 whey, 180 
 LOffler's methyl blue, 169 
 
 mordant, 169 
 Longitudinal fission, 75 
 Long rod-shaped bacteria, 67 
 
 screw-shaped bacteria, 67 
 
 Malignant oedema, viability 
 
 of spores, 108 
 Mallein, 135 
 Mannite, 156 
 Marsh gas, 145, 153 
 Membrane of bacteria, 68 
 Mercaptan, 139 
 Mesophilic bacteria, 99 
 
202 
 
 INDEX. 
 
 Metachromatic granules, 71 
 Metaphenylendiamin, 141 
 Methylamin, 133 
 Methyl blue, 168 
 
 guanidin poisoning, 158 
 Micrococcus acidi paralactici, 
 153 
 
 agilis, 112 
 
 cereus flavus, 126 
 
 gonorrhoeae, 85 
 
 mastitidis, 122 
 
 pyogenes, 104, 119, 157 
 
 tenuis, 123 
 
 tetragenus, 122, 161 
 
 urese Leube, 131 
 Microscopical technique, 166 
 Milk, 180 
 
 ferment, 116 
 Mitigation of virulence, 90 
 Mordant, Bunge's, 170 
 
 Loffler's, 169 
 Motile bacteria, sporulation 
 
 of, 77 
 Motion of bacteria, character 
 
 of. 111 
 Muscarin, 133 
 
 Naphthylamin, 141 
 Negative chemotaxis, 112 
 Neuridin, 133 
 Neutral agar, 89 
 
 bacteria, 148 
 
 gelatin, 88 
 Nicolle's stain, 177 
 Nitrates, reduction of, 140 
 Nitric acid, conversion into 
 
 free acid, 147 
 Nitrification, 145 
 Nitrite poisoning, 158 
 
 Nitrites, demonstration of, 141 
 Nitrogen, assimilation of, 147 
 Nitrosobacter, 146 
 Nitrosomonas, 146 
 Non-albuminous nutrient me- 
 dia, 179 
 Normal soda, 88 
 Nuclein, 81 
 Nucleus of bacteria, 69 
 Nutrient agar, 182 
 
 bouillon, 180 
 Nutrient media, 84, 179 
 
 acid, 89 
 
 albuminous, 117, 180 
 
 alkaline, 87 
 
 employment of, 184 
 
 gelatin, 181 
 
 neutral, 87 
 
 non-albuminous, 121, 179 
 
 saccharine, 122 
 
 Oil immersion lens, 166 
 
 of cedar, 167 
 Optical effects of bacteria. 111 
 Oval bacteria, 67 
 Oxidative fermentation, 125 
 Oxyfatty acids, 144 
 
 Papayotin, 163 
 Parvolin, 133 
 Pasteuria, 75 
 Pathogenic bacteria, 110 
 Pathogenesis, 157 
 Pentamethylendiamin, 133 
 Peptone water, 180 
 Peptones, 118 
 Phagocytosis, 163 
 Phenolphthalein, 88 
 Phlogogenic albuminoids, 135 
 
INDEX. 
 
 203 
 
 Phosphorescent bacteria, 113 
 Photobacteriura, 114 
 Phycochromacea, 65 
 Pigment, formation of, 126 
 Plasma of bacteria, 69 
 Plasmolysis, 70 
 Polar flagella, 73 
 Positive chemotaxis, 112 
 
 thermotropism, 113 
 Predisposition, 157 
 Processes of reduction, 140 
 Production of acids from alco- 
 hols, 156 
 
 of acids from carbohy- 
 drates, 148 
 Proteidins, immune, 164 
 Proteolytic ferments, 117 
 Pseudodichotomy, 68 
 Pseudopodia, 73 
 Psychrophilic bacteria, 99 
 Ptomains, 133 
 Putrefaction, 144 
 
 alkaloids, 133 
 Putrescin, 133 
 Pyogenic albuminoids, 135 
 Pyridin, 133 
 
 Rabbit septicaemia, 158 
 Rancidity of butter, 143 
 Ranges of temperature for bac- 
 teria, 98 
 Reaction of nutrient media, 
 
 87 
 Red pigments, 136 
 Reduction of nitrates, 140 
 
 processes, 140 
 Relative immunity, 161 
 Rennet ferments, 133 
 Resistance, 157 
 
 Resistance of bacteria to de- 
 ficiency of food and wat- 
 er, 93 
 of spores, 108 
 
 Ricin 135 
 
 Rinderpest, 161 
 
 Saline solutions as nutrients, 
 
 86 
 Saprogenous bacteria, 110 
 Saprophytes, 85 
 Sarcina pulmonum, 106 
 Schizomycetes, 65 
 Section preparations, 176 
 Separation of acids produced 
 
 by bacteria, 150 
 Sepsm, 133 
 Short rod -shaped bacteria, 67 
 
 screw -shaped bacteria, 67 
 Silicic acid nutrient medium, 
 
 184 
 Simple nutrient media, 85 
 Skatol, 133 
 
 Smear preparations, 170 
 Solitary growth of bacteria, 67 
 Spermin, 163 
 Spherical bacteria, 67 
 Spindle-shaped bacteria, 67 
 Spirillum desulphuricans, 139 
 
 endoparagocicum, 107 
 Spores, attenuation of, 159 
 
 biological characters of, 
 106 
 
 germination of, 78 
 
 power of resistance of, 107 
 
 tests for, 109 
 Sporogenous granules, 71 
 Sporulation, 77 
 
 influences favoring, 107 
 
204 
 
 INDEX. 
 
 Sporule, preliminary stage, 71 
 Staining solutions, 168 
 Stellate fission, 75 
 Sterilization, 90 
 Strict ae^robics, 95 
 
 anaerobics, 96 
 Succinic acid, 156 
 Sugar, chalk agar, 183 
 
 fermentation of, 125 
 Sulphanilic acid, 141 
 Sulphates, 139 
 Sulphmethsemoglobin, 158 
 Sulphur granules, 81 
 Sulphuretted hydrogen, 98, 
 
 138 
 Sunlight, action on bacteria, 
 
 101 
 Susceptibility, 161 
 Symbiosis, 104 
 Syncyanin, 128 
 Synergetic bacteria, 104 
 
 Tests of disinfectants, 91 
 Tetanus antitoxin, 164 
 
 poison, 136 
 
 spores, viability of, 108 
 
 virulence of, 137 
 Tetrads, 68 
 
 Thermophilic bacteria, 99 
 Thermotropism, 113 
 Thiosulphite, 139 
 Titration, 88 
 Torula, 68 
 
 Toxalbumins, 134, 136 
 Toxins, 132, 134 j 
 
 Transverse fission of bacteria, | 
 75 I 
 
 Trimethylamin, 133 
 
 Triolein, 80 
 
 Tripalmitin, 80 
 
 Tristearin, 80 
 
 Tubercle bacilli, staining of, 
 
 175 
 Tuberculin, Koch's, 135 
 Tyrosin, 133 
 Tyrothrix tenuis, 171 
 
 Universal nutrient, 89 
 Urea fermentation, 131 
 Uschinsky solution, 86 
 
 Vegetative proliferation, 66, 
 
 75 
 Viability of dry bacteria, 94 
 
 of spores, 108 
 Vinylcholin, 133 
 Violet pigments, 127 
 Virulence oi bacteria, attenua- 
 tion of, 159 
 increase of, 160 
 Vital conditions of bacteria, 
 84 
 
 Water bacteria, 85 
 
 Xanthin, 81 
 Xylol, 168 
 
 Yellow pigments, 126 
 
 Ziehl's solution, 168 
 ZoSglcea. 73 
 Zymogenous spores, 110 
 
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