ASTM: D5392: E. Coli in Water

Summary
Analytes
Revision
Data and Sites

Official Method Name

Standard Test Method for Isolation and Enumeration of Escherichia Coli in Water by the Two-Step Membrane Filter Procedure

Current Revision

Reapproved 2000. Current edition approved April 15, 1993.

Media

WATER

Instrumentation

Membrane Filtration

Method Subcategory

Microbiological

Method Source

ASTM

Citation

Annual Book of ASTM Standards, Section 11, Water and Environmental Technology, Volume 11.02, Water (I)

Brief Method Summary

This two-step test method provides a direct count of bacterial colonies developing on the surface of the filter when placed on a selective nutrient medium. The water sample is passed through a membrane filter that retains the bacteria. After filtration, the membrane filter containing the bacterial cells is placed on a selective, differential medium, mTEC. The membrane on the medium is first incubated at 35^oC for 2 h so that injured or stressed bacteria can be resuscitated and then the medium is incubated at 44.5^oC for 22 h. Following incubation the filter is transferred to a filter pad saturated with urea substrate. After 15 min all yellow or yellow-brown colonies are counted with the aid of 10 to 15X magnifier and a fluorescent lamp.

Scope and Application

This test method describes a membrane filter (MF) procedure for the detection and enumeration of Escherichia coli, a bacterium found exclusively in the feces of humans and other warm-blooded animals. The presence of these microorganisms in water is an indication of fecal pollution and the possible presence of enteric pathogens. These bacteria are found in water and wastewater in a wide range of densities. The detection limit of this procedure is one colony forming unit (CFU) per volume filtered.
This test method has been used successfully with temperate fresh and marine ambient waters, and wastewaters.

Applicable Concentration Range

None.

Interferences

Water with high levels of colloidal or suspended materials can clog the membrane filter pores and prevent filtration. Also, suspended materials cause spreading colonies that could interfere with target colonies and thereby prevent accurate counting.
In some samples, chemicals may have toxic effects on the target organism.

Quality Control Requirements

Yellow or yellow-brown colonies from the urease test can be verified as E. coli. Verification of colonies may be required in evidence gathering, and is also recommended as a QC procedure with initial use of the test and with changes in sample sites, lots of commercial media or major ingredients in media compounded in the laboratory.

Sample Handling

Sampling procedures are described in detail in the USEPA microbiological methods manual and in Practices D 3370.
Ice or refrigerate water samples at a temperature of 1 to 4^oC during transit to the laboratory. Use insulated containers to ensure proper maintenance of storage temperature. Take care that sample bottles are not totally immersed in water during transit or storage.

Maximum Holding Time

Examine samples as soon as possible after collection. Do not hold samples longer than 6 h between collection and initiation of analyses.

Relative Cost

Less than $50

Sample Preparation Methods

This method has 1 analyte associated with it.

Analyte	Detection Level	Bias	Precision	Pct False Positive	Pct False Negative	Spiking Level
E. coli(68583-22-2) E. coli E.coli Escherichia coli	1.000 CFU/Vol	N/A	25.00	9	1	30.00 CFU/Vol

Precision Descriptor Notes:	A collective study was conducted among eleven volunteer laboratories, each with two analysts who independently tested reagent water, local fresh and marine recreational waters and sewage treatment plant effluent samples, in duplicate. Because of the instability of microbial populations in water samples, each laboratory analyzed its own sample series and no full measure of recovery or bias was possible. However, all laboratories analyzed a single surrogate sample prepared from a freeze-dried culture of E. coli. The mean count (X) and the overall standard deviation of a single count (ST) including the variability among laboratories for this standardized E. coli sample, were 31.6 colonies/membrane and ST = 7.61 colonies/membrane, respectively.
Detection Level Note:	None.

Precision Descriptor Notes:

A collective study was conducted among eleven volunteer laboratories, each with two analysts who independently tested reagent water, local fresh and marine recreational waters and sewage treatment plant effluent samples, in duplicate. Because of the instability of microbial populations in water samples, each laboratory analyzed its own sample series and no full measure of recovery or bias was possible. However, all laboratories analyzed a single surrogate sample prepared from a freeze-dried culture of E. coli. The mean count (X) and the overall standard deviation of a single count (ST) including the variability among laboratories for this standardized E. coli sample, were 31.6 colonies/membrane and ST = 7.61 colonies/membrane, respectively.

Detection Level Note:

None.

Revision	PDF/Link
Reapproved 2000. Current edition approved April 15, 1993.

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ASTM: D5392: E. Coli in Water

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