In Reply Refer To:
Mail Stop 412 October 9, 2001
Subject: Change in mTEC agar used for Escherichia coli determinations
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.
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.
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 4°C 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.
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
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