522 THE ANAEROBIC BACTERIA 
resulted from the ingestion of the toxin of this baciUus by animal 
experimentation. 
Morphology. B. botulinus is a rather large bacillus, measuring from 
0.9 to 1.2 microns in diameter by 4 to 6 microns in length, with rounded 
ends; it occurs singly or in pairs, less commonly in short chains-of 3 to 6 
elements. Old cultures of this organism and those incubated above 
36° C. show involution forms which appear as long, intertwined fila- 
ments. The organism is sluggishly motile and has from 4 to 8 peri- 
trichic flagella. It forms oval spores, slightly greater in diameter 
than the rod and situated near one end of it. The organism stains 
readily with anilin dyes and is Gram-positive. 
Isolation and Culture.— B. botulinus growls characteristically in 
slightly alkaline glucose gelatin incubated at 25° C. under strictly 
anaerobic conditions. The colonies, which grow with moderate 
rapidity, are pale yellow in color, nearly transparent and are composed 
of coarse granules. As they reach their maximum development the 
colonies become brown and opaque and send out lateral radiations 
which are irregular in outline and distribution; these may be seen with 
the lower powers of the microscope. 
Growth in Artificial Media.— The organism grows well in the ordinary 
nutrient media which are slightly alkaline in reaction, better when 
glucose is added, but only under anaerobic conditions. A strong odor 
of butyric acid is characteristic of growths of the organism in artificial 
media. It is necessary to transfer large amounts of material to insure 
growth of B. botulinus. Gelatin is liquefied by most cultures. The 
growth on agar is very similar to that in gelatin. A slight turbidity is 
developed in plain broth after twenty-four hours' incubation, a heavy 
turbidity in glucose broth. The organism does not grow very well in 
milk; it produces a slightly acid reaction but neither coagulation nor 
peptonization. 
The organism is an obligate anaerobe, whose optimum temperature 
of growth is 22° to 30° C. It grows but slowly at 35° C. Incubation 
at the latter temperature leads to the development of involution forms 
and an inhibition of spore formation and toxin production. The spores 
are not particularly resistant to heat or disinfectants, and cultures die 
out in three to four weeks unless transferred to fresh media within that 
time. The spores are killed by an exposure at 80° C. for sixty minutes. 
Five per cent carbolic acid kills them within a week. If the spores 
are protected from oxygen and sunlight, they may retain their viability 
for several months, either in a moist condition or dried. ^ 
Products of Growth.— The majority of cultures, belonging to types 
A and B particularly, liquefy gelatin with considerable rapidity. 
Type C. seems to be quite devoid of proteolytic activity.- It forms 
acid and gas in glucose broth; bioses and polysaccharides are not fer- 
1 See Localization of B. botulinus Spores and Relation to Type of Preserved Vege- 
tables. Geiger: PubUc Health Reports, 1923, 38, No. 29. 
2 Wagner, Dozier and Meyer: Jour. Infec. Dis., 1924, 34, 63. 
