TESTS FOR B COLI 301 
either (A) the Presumptive, (B) the Partially Confirmed, or (C) the 
Completed test, as hereafter defined, each test being applicable under 
the circumstances specified. 
A. Presumptive Test 
1. Inoculate a series of fermentation tubes with appropriate gradu- 
ated quantities of the water to be tested. 
2. Incubate these tubes at 37° C. for forty-eight hours. Examine 
tube at twenty-four and forty-eight hours, and record gas-formation. 
The records should be such as to distinguish between: 
(a) Absence of gas-formation. 
(6) Formation of gas occupying less than 10 per cent (10%) of the 
closed arm. 
More detailed records of the amount of gas formed, though desirable 
for purposes of study, are not necessary for carrying out the standard 
tests prescribed. 
3. The formation within twenty-four hours of gas occupying more 
than 10 per cent (10%) of the closed arm of fermentation tube con- 
stitutes a positive presumptive test. 
4, If no gas is formed in twenty-four hours, or if the gas formed is 
less than 10 per cent (10%), the incubation shall be continued to forty- 
eight hours. The presence of a gas in any amount in such a tube at 
forty-eight hours constitutes a doubtful test, which, in all cases, requires 
confirmation. 
5. The absence of gas-formation after forty-eight hours’ incubation 
constitutes a negative test. (An arbitrary limit of forty-eight hours’ 
observation doubtless excludes from consideration occasional members 
of the B. colt group which forms gas very slowly, but for the purposes of a 
standard test the exclusion of these occasional slow gas-forming organ- 
isms is considered immaterial.) 
B. Parrratty CONFIRMED TEST 
1. Make one or more Endo’s medium or lactose-litmus-agar plates 
from the tube which, after forty-eight 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., 1 ¢.¢., and 0.1 ¢.c., and 
gas is formed in 10 ¢.c., and 1 ¢.c., and not in 0.1 ¢.c., the test need be 
confirmed only in the 1 c.c. amount.) 
2. Incubate the plates at 37° C., eighteen to twenty-four hours. 
