Standard Methods: 9213D: E. coli by Membrane filtration plating on mTEC agar

Summary
Analytes
Revisions
Data and Sites

Official Method Name

Recreational Waters. 9213D. Natural Bathing Beaches

Current Revision

Standard Methods Online

Media

WATER

Instrumentation

Membrane Filtration

Method Subcategory

Microbiological

Method Source

Standard Methods

Citation

Standard Methods Online - Standard Methods for the Examination of Water and Wastewater

Brief Method Summary

Filter sample through a membrane filter (Standard Methods 20th ed. Section 9222B). Place membrane on mTEC agar, containing bromocresol purple and bromophenol red. Incubate at 35C for 2 h to resuscitate stressed bacteria, then incubate at 44.5C for 22 h (requires a two-hour resuscitory incubation prior to the regular incubation). Transfer filter to a filter pad saturated with urea substrate and containing phenol red. After 15 min. count colonies using a fluorescent lamp and magnifying glass. Colonies that remain yellow or yellow-brown (occasionally yellow-green) are positive for E. coli. Requirements: Ingredients for mTEC agar and for urea substrate; buffer for rinsing and dilutions; culture dishes (50x10mm); 0.45 micron membrane filters. Refrigeration; autoclave; manifold and sterile filter funnel; sterile pipets; Fluorescent lamp; magnifying glass; forceps, alcohol; incubators at 35 +/- 0.5C and 44.5 +/- 0.2C. Cost of analysis (USEPA Fed. Reg. Aug 2001): E coli $22 ($10 to $35)

Scope and Application

Ambient, compliance monitoring. natural bathing waters (fresh, estuarine, marine) and surface waters. EPA Fed Reg (Aug 2001) for E coli, ambient only: fresh, marine, or estuarine surface waters; applicability must be demonstrated for other matrices USEPA. 2001 (August 30). Guidelines establishing test procedures for the analysis of pollutants; Analytical methods for biological pollutants in ambient water; proposed rule. Fed. Reg. 66(169)45811-45829. Clean Water Act section 401. 40 CFR 136.1(c). (state certification, licenses) for compliance monitoring in programs 303(c), 304(a), and 501(a). 136.3 Identification of test procedures.

Applicable Concentration Range

20 - 80 CFU/filter is considered ideal for enumeration; 200 CFU/filter is maximum. Sample size is typically 100 mL, but can be from 10 to 1000 mL, to meet requirements for enumeration.

Interferences

Commercial dehydrated media may degrade quickly. Presence of excessive background growth (blue colonies) may indicate media degradation. Sources of interference in MF methods (USEPA Fed Reg Aug. 2001): high turbidity, toxic compounds, or large numbers of non-coliform (background) bacteria, and organisms damaged by chlorine or toxic compounds.

Quality Control Requirements

(Standard Methods 20th ed. Section 9020 B.8 and 9; Myers and Sylvester, 1997) 1. Control cultures--positive (E. coli) and negative (Enterobacter) control cultures may be used to test the medium. 2. Repeat counts--monthly replicate counts for the same analyst should agree within 5% and between analysts within 10%. 3. Duplicate analyses--Perform duplicate analyses on 10% of samples. 4. Sterility check--a 50 to 100 mL aliquot of buffered dilution water is plated before each sample to assess contamination of equipment or media. 5. Verification--Verify a portion of these differentiated colonies according to USEPA (1985) or using a commercial multi-test system

Sample Handling

Sample preservation: chilled, 1 to 4 C [note method 9213D is intended for untreated, non-chlorinated water samples (not swimming pool water); therefore sodium thiosulfate is not added]. Techniques for collection: Collect water 0.3 m below surface and in areas of greatest bather load. Recommended sample volumes according to water type are: 100 mL lakes, reservoirs, wells, springs; natural bathing waters, 1 - 10 mL. Standard Methods for the Examination of Water and Wastewater, 20th Edition. L. Clesceri, A. Greenberg, and A. Eaton (editors). Sections 9020 B8,B9; 9060A,B; 9222B. APHA: Washington, DC. 1998. Dufour, A.P.; Strickland, E.R.; Cabelli, V.J. 1981. Membrane filter method for enumerating Escherichia coli. Appl. Environ. Microbiol. 41(5):1152-1158. Myers, D.N.; Sylvester, F.D. 1997. National field manual for the collection of water-quality data - biological indicators. USGS Techniques of Water Resources Investigations. Book 9, Chapter A7. 38 pp. USEPA, 1985. Test method for Escherichia coli and enterococci in water by the membrane-filter procedure. EPA-600/4-85/076. USEPA: Cincinnati, OH. 24 pp
Sample processing time 1 hour. To minimize interferences causing underestimation of organism density in MF methods (USEPA Fed Reg Aug. 2001): replicates of smaller sample dilutions/volumes may be filtered and the results combined.

Maximum Holding Time

Sample should be analyzed within 6 h for compliance or 24 h for routine monitoring (Standard Methods 20th ed Section 9060B); however, for all samples, holding less than 6 h is highly recommended (Myers and Sylvester, 1997)

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/100 mL	N/A	20.00	6	5

Precision Descriptor Notes:	Median within-sample RSD = 17%; median between-sample RSD = 20% (Francy and Darner, 1998). Values cited from: Francy, D.S.; Darner, R.A. 1998. Factors affecting Escherichia coli concentrations at Lake Erie public bathing beaches. USGS Water Resource Investigative Report 98-4241. 41pp. Francy, D. S.; Darner, R.A. 2000. Comparison of methods for determining Escherichia coli concentrations in recreational waters. Water Research. 34(10):2770-2778. General comments (USEPA Fed Reg Aug. 2001): Membrane filter (MF) methods are generally more precise in enumerating target organisms than MPN tests. Based on susceptibility to interferences, MF tests may underestimate the number of viable bacteria.
Detection Level Note:	Determined by using the largest volume plated , and the lowest number of organisms that can be counted. For example, if 100 mL was the largest volume plated, the detection would be 1 colony per 100 mL.

Precision Descriptor Notes:

Median within-sample RSD = 17%; median between-sample RSD = 20% (Francy and Darner, 1998). Values cited from: Francy, D.S.; Darner, R.A. 1998. Factors affecting Escherichia coli concentrations at Lake Erie public bathing beaches. USGS Water Resource Investigative Report 98-4241. 41pp. Francy, D. S.; Darner, R.A. 2000. Comparison of methods for determining Escherichia coli concentrations in recreational waters. Water Research. 34(10):2770-2778. General comments (USEPA Fed Reg Aug. 2001): Membrane filter (MF) methods are generally more precise in enumerating target organisms than MPN tests. Based on susceptibility to interferences, MF tests may underestimate the number of viable bacteria.

Detection Level Note:

Determined by using the largest volume plated , and the lowest number of organisms that can be counted. For example, if 100 mL was the largest volume plated, the detection would be 1 colony per 100 mL.

Revision	PDF/Link
Standard Methods Online
Standard Methods 18th, 19th, 20th ed.
Standard Methods 19th Edition (1995)
Standard Methods 18th Edition (1992)

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Standard Methods: 9213D: E. coli by Membrane filtration plating on mTEC agar

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