102 



3. The formation within 24 hours of gas occupying more than 10 

 per cent. (10%) of the closed arm of fermentation tube constitutes a 

 positive presumptive test. 



4. If no gas is formed in 24 hours, or if the gas formed is less than 

 10 percent (10%), the incubation shall be continued to 48 hours. The 

 presence of gas in any amount in such a tube at 48 hours constitutes a 

 doubtful test, which in all cases requires confirmation. 



5. The absence of gas formation after 48 hours' incubation consti- 

 tutes a negative test. (An arbitrary limit of 48 hours' observation doubt- 

 less excludes from consideration occasional members of the B. coli group 

 which form gas very slowly, but for the purposes of a standard test the 

 exclusion of these occasional slow gas forming organisms is considered 

 immaterial ) . 



B. Partially Confirmed Test. 1. Make one or more Endo's 

 medium or lactose-litmus-agar plates from the tube which, after 48 hours' 

 incubation, shows gas formation from the smallest amount of water tested. 

 (For example, if the water has been tested in amounts of 10 c. c., 1 c. c., 

 and 0.1 c. c. gas is formed in 10 c. c., and 1 c. c., not in 0.1 c. c. the test need 

 be confirmed only in the 1 c. c. amount). 



2. Incubate the plates at 37 C., 18 to 24 hours. 



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

 within this period, the confirmed test may be considered positive. 



4. If, however, no typical colonies have developed within 24 hours, 

 the test cannot yet be considered definitely negative, since it not infre- 

 quently happens that members of the B. coli group fail to form typical 

 colonies on Endo's medium or lactose-litmus-agar plates, or that the colon- 

 ies develop slowly. In such case, it is always necessary to complete the 

 test as directed under "C" 2 and 3. 



C. Completed Test. 1. From the Endo's medium or lactose-litmus- 

 agar plate made as prescribed under "B", fish at least two typical colon- 

 ies, transferring each to an agar slant and a lactose broth fermentation 

 tube. 



2. If no typical colonies appear upon the plate within 24 hours, the 

 plate should be reincubated another 24 hours, after which at least two 

 of the colonies considered to be most likely B. coli, whether typical or 

 not, shall be transferred to agar slants and lactose broth fermentation 

 tubes. 



3. The lactose broth fermentation tubes thus inoculated shall be in- 

 cubated until gas formation is noted; the incubation not to exceed 48 

 hours. The agar slants shall be incubated at 37 C. for 48 hours, when 

 a microscopic examination shall be made of at least one culture, selecting 

 one which corresponds to one of the lactose broth fermentation tubes which 

 has shown gas-formation. 



The formation of gas in lactose broth and the demonstration of non- 

 spore-forming bacilli in the agar culture shall be considered a satisfac- 

 tory completed test, demonstrating the presence of a member of the B. coli 

 group. 



