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

Summary:
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.

Scope:
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.

Citation:
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.

Interferences:
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]

Media:
WATER

Subcategory:
Microbiological

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

Precision:
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.

Detection:
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.

Revision Number:
2000