EXPERIMENTAL WORK. 



The strain of Bacillus coli which was selected was 

 taken from a stock culture. To confirm the identity of 

 the organism it was subjected to the following tests; 



a. The organism was a small rod- shaped organism, 

 with morphology, as regards the usual criteria, typical 

 of Bacillus coli. 



b. It was negative to Gram's method of staining. 



c. It produced abundant acid and gas when grown in 

 nutrient broth containing one per cent lactose. It pro- 

 duced acid and gas also in dextrose and sucrose broth. 



d. It did not liquefy gelatin. 



e. It was readily agg3.utinated by high dilution 

 (up to 1:2500) of B. coli antiserum. 



In order that a sufficiently large quantity of 

 material be obtained, the cultures were grown in liter 

 flasks. About thirty flasks were used at one time. To 

 each flask was added ten grams of peptone, four grams of 

 sodium chloride and one liter of tap water. This titra- 

 table acidity of such medium varied for each lot, but 

 usually fell between 1.5 to 2.0$ acid, using phenolphtha- 

 lein as indicator. This comparatively strong acid re- 

 action was not adjusted, because, as the Bacillus coli 

 produces abundant ammonia in sugar- free protein media, 

 the ammonia gradually neutralizes the acids present. 

 This particular species usually thrives in a medium whose 

 initial reaction is not greater than 2.5$ acid. On the 

 other hand, its growth ceases when the reaction reaches 

 an alkalinity of 2.5$. It is obvious, therefore, that 

 with a comparatively strong initial acid reaction, the 

 longer will be the time interval before the maximum alka- 

 line reaction is reached; and presumably, therefore, the 

 greater will be the number of bacteria produced; and it 

 is a large number of bacteria that is necessary for the 

 work. The flasks were not plugged with cotton, but were 

 capped with three thicknesses of wrapping paper. They 

 were then sterilized in the autoclave for two hours at 

 fifteen pounds pressure. Then they were allowed to stand 

 at room temperature for a few days, at the end of which 

 time were discarded any flasks which revealed the 

 presence of bacterial growth. The paper caps were next 

 washed with bichloride of mercury solution 1:1000. The 

 flasks were then inoculated with a broth culture of the 



