1. Inoculate a series of fermentation tubes with appropriate graduated
quantities of the water to be tested. In every fermentation tube there
must always be at least three times as much medium as the amount of
water to be tested. When necessary to examine larger amounts than 10 cc.
as many tubes as necessary shall be inoculated with 10 cc. each.
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.)