376 J. RUSSELL ESTY 



In 1917 Bull and Pritchett discovered that Clostridium Welchii 

 produced a soluble toxin which was capable of causing the lesions 

 characteristic of infections by this organism and which was 

 neutralized by a specific immune serum. These facts have been 

 confirmed by this investigation which was carried on in 1916, 

 1917, and 1918, to study the biology, pathogenicity, and distri- 

 bution of the gas bacillus. 



II. ISOLATION 



The media selected for the isolation of Clostridium Welchii 

 were glucose broth, glucose-liver broth, glucose-liver-agar and 

 milk. The liver broth and agar were prepared by the method 

 given in Standard Methods for Water Analysis 1912. Ordinary 

 market milk adjusted to + 1 to phenolphthalein was used. Before 

 sterilization, the surfaces of the media were covered with a layer 

 of paraffin oil (albolin) . 



Pure cultures were obtained by inoculating tubes of sterile 

 milk, previously covered with oil, with the material supposed to 

 contain Clostridium Welchii and heating to 82° to 85° for fifteen 

 minutes. The tubes were then cooled to 37° and incubated at 

 that temperature. The presence of the bacillus in market milk 

 was determined by transferring small portions of the milk to 

 other sterile tubes; or the tubes of market milk were heated 

 directly to 82° to 85° for fifteen minutes, cooled to 37°, and after 

 covering the surface with sterile paraffin wax, incubated. 



A positive reaction showed "stormy fermentation," i.e., coagu- 

 lation of the casein, copious formation of gas, fragmentation of 

 the curd, derangement of the cream layer and a detectable odor 

 of butyric acid. However, milk cultures sometimes contained 

 gas bacilli and yet failed to show this typical reaction due to 

 various reasons. Cultures which showed a positive reaction 

 were inoculated into tubes of glucose-liver broth and incubated 

 from four to six hours, at which time the culture presented a 

 "steaming" appearance. At this time plates were made on 

 glucose-liver-agar, employing anaerobic technique, and incubated 

 at 37°. After sufficient incubation (twelve to twenty hours) 



