BOTULISM OR ALLANTIASIS 485 



they reach their maximum development the colonies become brown 

 and opaque, and only those granules at the periphery of the colony 

 remain motile. 



Growth in Artificial Media The organism grows well in the ordinary 

 nutrient media, better when dextrose is added, but only under anae- 

 robic conditions. A strong odor of butyric acid is characteristic of 

 growths of the organism in artificial media. It is essential to transfer 

 large amounts of material to insure growth of the organism. Gelatin 

 is liquefied. The growth on agar is very similar to that in gelatin, 

 except that no liquefaction takes place and no motile granules appear 

 in the colonies. A slight turbidity is developed in plain broth after 

 twenty-four' hours' incubation, a heavy turbidity in dextrose broth. 

 The organism grows well in milk, producing a slightly acid reaction 

 but neither coagulation nor peptonization. 



The organism is an obligate anaerobe, whose optimum temperature 

 of growth is 22 to 25 C. It grows but slowly at 25 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 in twenty- 

 four hours and pickling in 10 per cent, salt solution kills them within 

 a week. If the spores are protected from oxygen and sunlight they 

 retain their vitality for several months, either in a moist condition or 

 dried. 



Products of Growth The organism produces an active soluble 

 gelatinase in plain broth cultures and in gelatin, particularly the 

 latter. It forms acid and gas in dextrose broth; bioses and polysac- 

 charides are not fermented. The acid formed is partly butyric, and the 

 gas consists principally of carbon dioxide and hydrogen. 



The most important product of B. botulinus, however, is a potent 

 extracellular toxin which is readily prepared by growing the organisms 

 anaerobically in sugar-free broth at 25 C. for two weeks. The 

 broth is filtered through sterile porcelain filters, preferably in an atmos- 

 phere of hydrogen, and the toxin is found in the filtrate, from which 

 it can be precipitated by the addition of a 3 per cent, aqueous solution 

 of zinc chloride in the proportions of two parts of zinc chloride to 

 one of broth. 1 The toxin deteriorates rather rapidly if it is exposed 



1 Brieger and Kempner, Deutsch. med. Wchnschr., 1897, xxxiii, 521. 



