Change in mTEC agar used for Escherichia coli determinations

In Reply Refer To:
Mail Stop 412                                          October 9, 2001

OFFICE OF WATER QUALITY TECHNICAL MEMORANDUM 2002.01

Subject:  Change in mTEC agar used for Escherichia coli determinations

                                  POLICY

The Ocala Water Quality& Research Laboratory (OWQRL) has been distributing
dehydrated mTEC agar since 1983.  The dehydrated mTEC is fully quality
assured using U.S. Environmental Protection Agency (USEPA) procedures and
previous quality-assurance tests by OWQRL have shown that the Accumedia
brand was the most reliable of the commercially available dehydrated mTEC
agar. However, investigators in a few Water Resources Discipline Districts
have noticed problems with the dehydrated mTEC agar used to enumerate
Escherichia coli (E. coli) that may lead to unreliable and ambiguous
results.  This memo describes background information so that project
personnel are aware of potential problems with dehydrated mTEC agar and
includes a solution if problems are encountered.

The Office of Water Quality (OWQ) recommends that project personnel
carefully evaluate their current use of mTEC agar for E. coli analyses for
the types of problems discussed below.  If any of these problems are
apparent, more reliable results may be obtained by substituting hydrated
mTEC agar supplied by the Ohio District Microbiology Laboratory (ODML) for
the dehydrated mTEC agar provided by Ocala.  Ocala will continue to supply
the dehydrated mTEC agar to those that have not experienced the problems
listed below.  Questions regarding this recommendation should be directed
to Stephen Sorenson (sorenson) in OWQ or Donna Francy (dsfrancy) or Rebecca
Bushon (rnbushon) in the Ohio District.

                                BACKGROUND

The mTEC agar method has been widely used for determination of E. coli in
water since publication of the method in 1986. The primary growth medium,
mTEC agar, is supplied commercially in a dehydrated form.  The Ohio
District started using this method in 1987, but began experiencing problems
with the medium in 1991.  The problems encountered included the following:

    Typical yellow to yellow-brown E. coli colonies turn purple near the
end of the 22 to 24-hour incubation period on mTEC agar at 44.5 C with no
other change in morphology.  These colonies remain purple after the urease
test resulting in a high false-negative rate.
    Colonies exhibit confluent growth (grow in a large mass), resulting in
plates that are difficult or impossible to read.
    High numbers of purple colonies that are not E. coli and constitute
background growth are present.

Because problems have been observed with the dehydrated mTEC agar, the ODML
began making mTEC agar from the published ingredient list in 1994 and using
it in the "fresh" or hydrated form. Limited side-by-side comparison of the
dehydrated and hydrated mTEC showed that the hydrated mTEC provided more
interpretable results on samples where the dehydrated mTEC was observed to
have had anomalous results. The hydrated mTEC agar has produced long term
excellent and consistent E. coli results.  The ODML recommends the use of
hydrated mTEC and has been supplying it on a limited basis to several
Districts over the last few years.  Other Districts have reported that E.
coli test results using hydrated mTEC agar result in no color reversal,
reduced background growth, and better colony definition than the dehydrated
mTEC, all of which make accurate enumeration possible.

                               IMPLICATIONS

The use of fresh hydrated mTEC agar for E. coli determinations will 
require
some operational changes.  The agar can be purchased from the ODML in
dilution bottles containing 100-mL of hydrated agar.  The agar must be kept
near 4C under normal refrigeration or on ice in coolers until use and has a
"shelf life" of 6 months from time of preparation.  The only preparation
step needed by the user is to melt the agar in a water-filled beaker over a
hot plate and pour into the plates.  The agar will be autoclaved before
shipment and will not need to be further sterilized in the District office.
The hydrated agar has the exact formulation that is listed in the published
USEPA procedure; the only difference is the physical state in which it is
stored.  Because these changes do not involve changes in the actual
published USEPA method, existing parameter codes will still be valid and no
changes in the database for new data are needed.

                                 REFERENCE

U.S. Environmental Protection Agency, 1986, Test method for Escherichia
   coli and enterococci in water by the membrane-filter procedure:
   Cincinnati, Ohio, EPA 600/4-85/076, 24 p.

                         Stephen K. Sorenson /s/
                         Acting Chief, Office of Water Quality

This memorandum does not supersede any other OWQ Technical Memorandum.

Distribution:  District Chiefs, District and Regional Water-Quality
          Specialists, OWQ Staff, NAWQA Leadership Team, NAWQA Synthesis
          Project Chiefs, NAWQA 91, 94, and 97 Study-Unit Project Chiefs

(See attached file: mTECmemo02.01.htm)
************************************************
Nana L. Snow
USGS/Office of Water Quality
412 National Center
Reston, VA 20192
703/648-6862
703/648-5722 (fax)
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