NEMI Method Summary

Method Summary Information

Method Number: D5392 Media: WATER
Revision: Reapproved 2000.  Current edition approved April 15, 1993.
Method Source: ASTM International
Subcategory: MICROBIOLOGICAL Analytes in this method
Official Name: Standard Test Method for Isolation and Enumeration of Escherichia Coli in Water by the Two-Step Membrane Filter Procedure
Descriptive Name E. Coli in Water
Source Info: ASTM International
100 Barr Harbor Drive West
Conshohocken, PA 19428
Telephone: (610) 832-9500
Fax: (610) 832-9555
Email: service@astm.org
Citation: ASTM Volume 11.02
 
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 35oC for 2 h so that injured or stressed bacteria can be resuscitated and then the medium is incubated at 44.5oC 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
Conc 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.
QC 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 4oC 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.
Max 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
URL to obtain ordering information for this method:
http://www.astm.org/cgi-bin/SoftCart.exe/DATABASE.CART/PAGES/D5392.htm?E+mystore
5462
Page 1