EPA Method 1103.1

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EPA Method 1103.1:
Membrane filtration plating of E. coli on modified mTEC agar. Official Name: Test method for Escherichia coli and enterococci in water by the membrane-filter procedure

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.

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.

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.

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.

QC Requirements:
(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

Maximum Holding Time:
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]



20 - 80 CFU/100 mL is considered ideal for enumeration. Maximum: 200 CFU/100 mL; dilution is required for samples that exceed this level.

Accuracy data cited in Table 3 of 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. 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.

Determined by using the largest volume plated. For example, if 100 mL was the largest volume plated, the detection would be 1 colony per 100 mL.

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