Determination of Bacteria in Water 245 



fermentation tubes, and sterilized in an autoclave at 15 lb. (i2o°C.) 

 for fifteen minutes after the pressure reaches 15 lb. 



The tubes thus prepared are inoculated with appropriate quantities 

 of the water — say 10 cc, i cc. and Ko cc- — and then incubated at 

 37°C. for forty-eight hours. If at the end of twenty-four hours more 

 than 10 per cent, of gas occurs in the closed arm of the fermentation 

 tube the presumptive test is positive. If no gas is formed or if 

 there be less than 10 per cent, the incubation shall be continued to 

 forty-eight hours. The presence of gas in any amount in such a 

 tube, constitutes a doubtful test; the absence of gas after forty-eight 

 hours incubation constitutes a negative test. 



B. The Partially Confirmed Test. — This consists in making one or 

 more Petri dish cultures, using either Endo's medium (q.v.) or lactose- 

 litmus-agar (q.v.) and water from the fermentation tube showing gas 

 with the smallest amount of water tested. 



The plates are incubated at 37°C. for eighteen to twenty-four 

 hours. If typical red colon-like colonies have developed upon the 

 plate within this period, the confirmed test may be considered to 

 be positive. If no typical colonies develop within twenty-four 

 hours the test cannot yet be considered negative as sometimes the 

 members of the B. coli group fail to form typical colonies in Endo's 

 medium or Utmus-lactose-agar within that time. In such cases it is 

 necessary to carry the test to a third step. 



C. The Completed Test. — From the Petri dish cultures upon the 

 Endo's medium or the litmus-lactose-agar, two typical colonies are 

 transferred, one to an agar slant, the other to a lactose broth fermen- 

 tation tube. If no typical colonies have developed at the end of the 

 twenty-four hour period, the dishes are reincubated for another 

 twenty-four hours, after which two, at least, of the colonies consid- 

 ered to be most like B. cob, are transplanted to agar slants and 

 lactose broth fermentation tubes. 



The transplants are incubated at 37°C. as usual, the formation of 

 gas being noted up to a period not exceeding forty-eight hours in the 

 fermentation tubes and microscopical examinations made of the agar 

 cultures. The formation of gas in the fermentation tube s and the 

 demonstration of non-spore-bearing bacilli on the agar may be con- 

 sidered a satisfactory completed test demonstrating the presence of 

 a number of the B. coU group. Absence of gas formation in lactose 

 broth or failure to demonstrate non-spore-forming bacilli in a gas- 

 forming culture constitutes a negative test. 



The potability of water is not determined by the bacteriological 

 analysis alone, but also by chemical analysis, optical condition, and 

 odor. So far as the bacteriology itself is concerned the standard 

 adopted in the Public Health Reports, Nov. 16, 1914, Vol. xlv. No. 29, 

 P' 29S9) requires that the total number of bacteria shall not exceed 

 100 per cubic centimeter when grown upon standard agar-agar plates 

 and counted after twenty-four hours incubation at 37°C. Further 



