2. Incubate these tubes at 37° C. for 48 hours. Examine each tube at 24 and 48 hours, and record gas-formation. The records should be such as to distinguish between: (a) Absence of gas-formation. (b) Formation of gas occupying less than ten per cent. (10%) of the closed arm. (c) Formation of gas occupying more than ten 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 24 hours of gas occupying more than ten 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 ten per cent. (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 constitutes _a negative test_. (An arbitrary limit of 48 hours’ observation doubtless 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 cc., 1 cc., and 0.1 cc., and gas is formed in 10 cc., and 1 cc., not in 0.1 cc., the test need be confirmed only in the 1 cc. 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 infrequently 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 colonies 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 colonies, 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.