EPA-MICRO: 1103.1 (modified): Membrane filtration plating of E. coli on modified mTEC agar
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Official Method Name
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Test method for Escherichia coli and enterococci in water by the membrane-filter procedure |
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Current Revision
| 2000 |
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Media
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WATER |
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Instrumentation
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Membrane Filtration |
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Method Subcategory
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Microbiological |
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Method Source
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Citation
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USEPA, 2000 (March). Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and Escherichia coli EPA-821/R97/004. Section 10.3 Modified E coli method. USEPA: Washington, DC. 53 pp. |
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Brief Method Summary
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Filter sample through a membrane filter (Standard Methods 20th ed. Section 9222B), place membrane on modified mTEC agar containing the chromogenic enzyme substrate Magenta Gluc (5-bromo-6-chloro-3-indoyl-beta-D-glucuronide). Incubate at 35oC for 2 h to resuscitate stressed bacteria, then incubate at 44.5oC for 22 h (requires a two-hour resuscitory incubation prior to the regular incubation). Requires only one step. Colonies that are magenta are positive for E. coli, indicating enzymatic hydrolysis of the chromogenic substrate. Requirements: ingredients for modified mTEC agar; 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 +/- 0.5oC and 44.5 +/- 0.2oC Cost of analysis (USEPA Fed. Reg. Aug 2001): E coli $22 ($10 to $35) |
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Scope and Application
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Ambient, compliance monitoring: surface water, bathing 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. |
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Applicable Concentration Range
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20 - 80 CFU/100 mL is considered ideal for enumeration. Maximum: 200 CFU/100 mL; dilution is required for samples that exceed this level. |
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Interferences
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High turbidity and high bacterial densities. 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. |
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Quality Control Requirements
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(Standard Methods 20th ed. 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 |
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Sample Handling
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Sample preservation: chilled, 1 to 4o C; 0.0008% (w/w) Na2S2O3 added to chlorinated waters EPA Fed Reg (Aug 2001). Techniques for collection: Standard Methods for the Examination of Water and Wastewater, 20th Edition. L. Clesceri, A. Greenberg, and A. Eaton (editors). APHA: Washington, DC. 1998. Sections 9020 B8,B9, 9060B, 9222B. 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. Sample processing time 1 hour. Select sample volumes based on previous knowledge of the pollution level, to meet requirements for enumeration. Filter at least three volumes per sample. Sample volumes between 1 and 100 mL are normally analyzed at half-log intervals: 100, 30, 10 and 3 mL. To minimize the effects of high turbidity or bacterial densities, samples can be subdivided into fractions for analysis, and the results combined. |
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Maximum Holding Time
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Sample should be analyzed within 24 h for routine monitoring (Standard Methods 20th ed. Section 9060B); however, a 6 h holding time for all samples is highly recommended (Myers and Sylvester, 1997) [Drinking water can be 30 h] |
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Relative Cost
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Less than $50 |
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Sample Preparation Methods
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