E. COLI ENUMERATION BY MEMBRANE FILTRATION
For detecting E. coli in recreational waters, EPA recommends
the membrane filtration method, Escherichia coli in Water by the
Membrane Filter Procedure Method 1103.1 1985 developed by Dufour,
Strickland, and Cabelli (Dufour and others 1981). This method is commonly used
in Vermont by volunteer monitoring groups and the Vermont Department of
Environmental Conservation laboratory.
Water samples for E. coli enumeration must be stored at 1-4oC
and analyzed as soon as possible, but absolutely within 6 hours of
collection. Temperature and holding requirements ensure
that the population of E. coli in the sample is representative of
the water at the time of collection.
Water samples, which may vary by volume or may be diluted, are vacuum
filtered through sterile 47 mm diameter grid-marked membrane filters
with 0.45 um pores. The membrane filters containing bacteria
filtered from known volumes of water are placed on a selective medium,
mTec, in sterile petri plates and incubated at 35oC for 2
hours to resuscitate injured cells. Next, plates are incubated at
44.5oC for 22 hours to promote growth of E. coli and
inhibit growth of non-thermotolerant bacteria. Fecal coliforms,
including E. coli, produce acid resulting in yellow colonies.
Following incubation, filter membranes are transferred to sterile
pads saturated with urea broth at a pH of 5.0. Organisms which
break down urea produce alkaline by-products which change the color of
colonies to purple. E. coli does not have the enzyme which
breaks down urea; E. coli colonies remain yellow or
yellow-brown. After 15 minutes, yellow and yellow-brown colonies
are counted under a fluorescent lamp at 5X magnification to give the
number of organisms in the original filtered volume of
water. Each colony is assumed to have developed from a single
organism. Ideally, filters with 20-80 colonies are counted and the
dilution factor, if any, calculated to obtain E. coli/100
ml.
This method has a
selectivity of 9% false positives (organisms counted as E. coli when not)
and less than 1% false negatives (E. coli not counted). Other
tests for fecal coliforms test for a whole group of organisms of which some species are ubiquitous in the
environment. In contrast, the EPA’s membrane filtration method for E. coli is selective for a particular species
which is solely fecal in origin.
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