BOTULISM IN CATTLE 



77 



was then gently shaken and the liquid content removed to a 

 sterile flask. Small particles of visible silage were removed by 

 filtering through four layers of sterile gauze. The filtrate was 

 then seeded in shake agar culture and heated fifteen minutes to 

 80°C. to destroy vegetative bacteria. The inoculated tubes 

 were quickly placed in a cold water bath and allowed to solidify. 

 On the surface of the agar to a depth of 2 to 6 cm., equal parts 

 of agar and glycerol containing 1 per cent phenol were added to 

 insure anaerobiosis. Ten days later the cultures, after incubat- 

 ing at 22°C., were examined and in one of the fifteen dilutions 

 planted there was gas formation in the base of the tube, though 

 distinct colonies were not visible. Anaerobes encountered in 

 animal feeds, in our observations, are favored by the addition of 

 glucose to the media, yet the numerous saprophytes encountered 

 may outgrow and even disguise the presence of B. hotulimis- 

 like organisms. It is true that B. botuliniis does not thrive on 

 agar, yet it seems to develop slowly in plain agar shake cultures 

 at 22°C. to 25°C. with limited gas production. Subculturing in 

 glucose pork agar and transferring colonies to glucose pork broth 

 (faintly alkaline) was employed to determine the toxic character 

 of anaerobes cultivated in agar after ten days incubation in 

 vacuum or hydrogen atmosphere. The normal toxicity of 

 newly isolated B. botulinus-like organisms in broth cultures may 

 not be characteristic or fully acquired until the second or third 

 transfer at intervals of seven to ten days. The cultural char- 

 acters and toxic quality of B. hotulinus from silage as observed 

 in guinea pigs, is illustrated in table 1; and in figures 2 and 3. 

 All animals succumbed with the symptoms characteristic of 

 B. hotulinus intoxication. 



