302 WATER HYGIENE 
3. If typical colon-hke 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 twenty- 
four hours, the test cannot yet be considered definitely negative, since 
it not infrequently happens that members of the B. colt group fail to 
form typical colonies on Endo’s medium or lactose-litmus-agar plates, 
or that the colonies develop slowly. In such cases, it is always neces- 
sary to complete the test as directed under C 2 and 3. 
C. CompLeTep TEst 
1. From the Endo’s medium or lactose-litmus-agar plate, made as 
prescribed under B, fish at least two typical colonies, transferring each 
to an agar slant and a lactose broth fermentation tube. 
2. If no typical colonies appear upon the plate within twenty-four 
hours, the plate should be re-incubated another twenty-four 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 
incubated until gas formation is noted; the incubation not to exceed 
forty-eight hours. The agar slants shall be incubated at 37° C. for 
forty-eight 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 sat- 
isfactory completed test, demonstrating the presence of a member of 
the B. cols group. 
The absence of gas-formation in lactose broth or failure to demon- 
strate non-spore-forming bacilli in a gas-forming culture constitutes a 
negative test. 
Application of Presumptive, Partially Confirmed, and Completed 
Tests. 
A. The Presumptive Test. 
1. When definitely positive, that is showing more than 10 per cent 
(10%) of gas in twenty-four -hours, is sufficient. 
(a) As applied to all except the smallest gas-forming portion 
of each sample in all examinations. 
(6) As applied to the smallest gas-forming portion in the 
examination of sewage or of water showing relatively 
