method where the turbidity of a water sample precludes an accurate analysis by the
membrane-filter technique. The multiple-tube method requires a complete laboratory
setup, it requires a minimum of 48 hours (up to 4 days), and the results show only the
presence of and an approximation of the "most probable number" (MPN) of coliform
a. Procedure. Appropriate quantities of the water to be tested (usually five
10-ml samples) are placed in sterile tubes containing a lactose broth medium. The
tubes are incubated at 35oC for 24 hours and then examined for the presence of gas
formation (para 3-2d(1)(a)). If no gas is present, they are incubated for an additional
24 hours and examined again.
(1) Presumptive test. If no gas is present at the end of 48 hours, the test is
negative. If gas in any amount is present, it is a positive presumptive test.
(2) Confirmed test. Material from tubes showing fermentation (gas
formation) is transferred to tubes containing brilliant green lactose bile broth and
incubated for 48 hours at 35C. Absence of gas constitutes a negative test. Presence of
gas constitutes a confirmed positive test.
(3) Completed test. This procedure is not always done. Its purpose is to
demonstrate with certainty that the organisms giving positive results are really members
of the coliform group. The procedure is to streak one or more Endo or eosin methylene
blue (EMB) plates from each tube showing gas and incubate for 24 hours. A typical (or
the most nearly typical) colony from each plate is then transferred to both a secondary
lactose broth fermentation tube and an agar slant petri dish. After 48 hours of
incubation at 35oC (24 hours if gas is present), gram--stain slides are prepared from the
agar cultures of the colonies that formed gas in the secondary fermentation tubes.
Microscopic demonstration of gram--negative, non--spore-forming, rod--shaped,
bacteria, accompanied by gas formation, constitutes a completed test and demonstrates
the presence of coliform organisms.
b. Most Probable Number. The most probable number (MPN) reported by the
laboratory on a multiple-tube coliform determination is a statistical probability of the
number of organisms per 100-ml of the water tested. It can be seen that the results are
only approximate. The accuracy can be increased by using more tubes of varying
amounts of sample, such as 5 of 50 ml, 5 of 10 ml, and 5 of 1 ml. Even with this
increased size and number of portions, the accuracy can vary substantially.
The EPA states that effective 31 December 1990, this technique is no longer
to be used to determine coliform densities. It is to be replaced by the
Presence/Absence coliform test.
c. Presence-Absence Coliform Test. Colilert is one of several products that
may be used to rapidly determine the presence or absence of coliform in water. It is
another method for MTF and membrane filtration (MF) testing. Other method testing