Ohio District's Microbiology laboratory

Clostridium perfringens,  using the mCP agar method

Updated May 2005

 The mCP agar method is a two-step membrane-filtration method for the detection of Clostridium perfringens (C. perfringens) in environmental waters. This method cannot be done in the field; it must be done in a laboratory.

THEORY: The first step is anaerobic incubation of mCP agar plates at 42^°C for 24 hours. The mCP agar contains selective and differential agents. D-cycloserine and polymyxin B sulfate inhibit background growth. Bromocresol purple identifies colonies that can ferment sucrose. Phenolphthalein diphosphate identifies colonies that produce acid phosphatase.

For the second step, after incubation on mCP, filter membranes containing yellow straw-colored colonies are transferred to pads saturated with ammonium hydroxide.  After 15 seconds, colonies that turn dark pink to magenta are counted as C. perfringens. The phosphate is cleaved from the phenolphthalein diphosphate substrate by the action of acid phosphatase, and typical colonies of C. perfringens turn dark pink to magenta after exposure to ammonium hydroxide. Discerning which colonies are positive for C. perfringens takes experience with the method.

USE:  The mCP method can be used for monitoring all types of waters. C. perfringens is present in large numbers in human and animal wastes, and its spores are resistant to wastewater-treatment practices, extremes in temperature, and environmental stress. The method has been recommended for use for examination of (1) chlorinated waters and untreated water containing industrial wastes lethal to non-sporeforming bacteria, (2) sewage sludge, and (3) situations in which the detection of remote as well as recent pollution is desirable.

See mCP method directions (Appendix I).

MEDIA:  The mCP medium is not commercially available. The ODML prepares the medium in 100-mL quantities. The agar medium may be stored at 4^°C for up to 6 months. Before agar plates are poured, five reagents must be added to the tempered medium. Once poured, the plates can be stored at 4^°C for no more than one month. See preparation instructions (Appendix I).

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: 

Armon, R., and Payment, P., 1988, A modified m-CP medium for enumerating Clostridium perfringens from water samples: Canadian Journal of Microbiology, v.34, p.78-79.

Bisson, J.W., and Cabelli, V.J., 1979, Membrane filter enumeration method for Clostridium perfringens: Applied and Environmental Microbiology, v. 37, no.1, p. 55-66.

Bisson, J.W., and Cabelli, V.J., 1980, Clostridium perfringens as a water pollution indicator: Journal of the Water Pollution Control Federation, v. 52, no. 2, p. 241-248.

U.S. Environmental Protection Agency, 1996, EPA Information Collection Rule microbial laboratory manual: Washington, D.C., EPA/600/R-95/178, section XI.

NWIS PARAMETER CODES:

90915 C. perfringens on mCP agar, colonies per 100 mL