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Full text of "Atlas and essentials of bacteriology"

THE PROPERTY OF 

H9lii!8Miiiil'" -'■" icftliePaGil, 



MEPICAL .SCInI®OL 





THE PROPERTY OF 

Hateo'inK-'-'^'-llglfionk Pacific 



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in 2007 with funding from 

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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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