Ohio District's Microbiology laboratory

Enterococci, using the mEI method

Updated May 2005

The mEI agar method (USEPA Method 1600) is a one-step membrane-filtration method that allows the detection of enterococci in 24 hours with an incubation at 41°C.  It is recommended for use in place of the old enterococci method, the mE agar method (USEPA Method 1106.1), which is a two-step method that takes 48 hours to complete with an incubation at 35°C.  This method can be done in the field or laboratory.

THEORY:  The mEI medium is similar to the mE medium except that it contains a reduced amount of triphenyltetrazaolium chloride (TTC) and contains a substrate, indoxyl b-D-glucoside, that turns blue when cleaved by an enzyme present in enterococci (b-glucosidase).  All colonies with any blue halo are recorded as enterococci, regardless of colony color.  Magnification with a dissecting microscope is used for counting to give maximum visibility of colonies.

USE:  The mEI test for enterococci can be applied to potable, fresh, estuarine, marine, and shellfish-growing waters. The mE method is also suitable for these types of waters; however, it involves the use of two types of media and takes longer to complete. 

MEDIA:  The mEI agar medium is available from Government Scientific Source, Inc. (800/248-8030, Cat 214885). Indoxyl β-D-glucoside, nalidixic acid, and 1% presterilized TTC must also be purchased if the medium is made from dehydrated ingredients. Indoxyl β-D-glucoside can be purchased from Sigma-Aldrich Corp. (800/325-3010, Cat I3750-2G (2g) or Cat I3750-1G (1g)). Nalidixic acid can be purchased from Hach (800/227-4224, Cat 24071-24). The 1% presterilized TTC can be purchased from Hach (Cat 24060-42).

See preparation instructions for details on media composition and preparation (Appendix H).

Prepoured plates can be purchased from Microbiology International (800/EZ-MICRO).

Use phosphate buffered dilution water and 0.45 mm membrane filters.  Buffer can be purchased from Hardy Diagnostics (800/346-2766, Cat D699 (99mL) or Cat U193 (500mL)).  See buffer preparation (Appendix M).

REFERENCES: 

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.

U.S. Environmental Protection Agency, 1997.  Method 1600—Membrane filter test method for enterococci in water: Washington, D.C., EPA/821/R-97/004 May 1997.

U.S. Environmental Protection Agency, 2000.  Improved enumeration methods for the recreational water quality indicators: Enterococci and Escherichia coli: Washington, D.C., EPA/821/R-97/004 March 2000.

The last two references can be obtained at  http://www.epa.gov/nerlcwww/index.html
Under enterococci.

NWIS PARAMETER CODES:

90909 Enterococci on mEI agar (preferred method), colonies per 100 mL
31649 Enterococci on mE agar (acceptable), colonies per 100 mL