EPA Method 600-R-00-013

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EPA Method 600-R-00-013:
Membrane filtration plating of coliform bacteria on MI agar. Official Name: Membrane filter method for the simultaneous detection of total coliforms and Escherichia coli in drinking water.

Allows analysis of both total coliforms and E. coli. Filter sample through a membrane filter (Standard Methods 20th ed. Section 9222B), place membrane on MI agar, containing the enzyme substrates MUGal (4-methylumbelliferyl-D-galactopyranoside) for total coliforms and IBDG (indoxyl-D-glucuronide) for E. coli. Incubate at 35oC for 22-24 h. E. coli are counted as blue colonies under ambient light. Total coliforms are counted as colonies that fluoresce under a long-wave ultraviolet light source: blue-white fluorescence is positive for total coliforms other than E. coli; blue-green fluorescence is positive for E. coli; non-fluorescent blue colonies are also counted (breakdown of IBDG assumed to mask fluorescence). Requires an extensive dilution series for ambient waters. Requirements: Ingredients for MI agar; buffer for rinsing and dilutions. Culture dishes (50x10mm); 0.45 micron membrane filters; sterile pipets; manifold and sterile filter funnel; forceps, alcohol. Refrigeration; autoclave; incubator at 35+/-0.5oC with 90% humidity. Stereoscopic microscope (10-15X); a long-wave ultraviolet light source (366 nm) to count fluorescent colonies. Cost of analysis (USEPA Fed. Reg. Aug 2001): E coli $22 ($10 to $35) total coliforms $22 ($15 to $48)

Ambient, compliance monitoring: non-compliance: all water - freshwater (ground and surface waters, runoff), recreational water, marine waters, municipal wastewater, swimming pool, drinking water, bottled water, soil, sludge. EPA Fed Reg (Aug 2001) for E coli, ambient only: fresh, marine, or estuarine surface waters; applicability must be demonstrated for other matrices. USEPA. 1999 (December 1). National primary and secondary drinking water regulations: analytical methods for chemical and microbiological contaminants and revisions to laboratory certification requirements; final rule. Fed. Reg. 64(230)67449-67467. Safe Drinking Water Act: a) Total Coliform Rule: presence/absence of total coliforms and E coli b) Surface Water Treatment Rule: enumeration of total coliforms 40 CFR 141, 143. 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. Membrane filter method for the simultaneous detection of total coliforms and Escherichia coli in drinking water. EPA-600-R-00-013. USEPA: Cincinnati, OH. 21 pp.

Colloidal or suspended particulate material that can clog the membrane filter or cause spreading of colonies and make enumeration difficult. Colonies to be excluded from counting: 1) non-E coli: tiny, flat or peaked pinpoint colonies (< 0.5 mm dia) that are blue in ambient light, in samples with < 200 CFU per filter; 2) non-coliforms: non-blue colonies in ambient light that fluoresce bright green. 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--a positive (E. coli) for total coliforms and E. coli and negative for total coliforms (Staphylococcus) or E. coli (Enterobacter) 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--validate analysis of a portion of these differentiated colonies according to Brenner and others (1993).

Maximum Holding Time:
Analyze samples as soon as possible after collection. Drinking water samples can be analyzed within 30 h of collection. Natural water samples should not be held longer than 6 h, and the analyses should be complete within 8 h of sample collection.



20 - 80 CFU/100 mL is considered ideal for enumeration, and typical of drinking water samples without dilution. Maximum: 200 CFU/100 mL; dilution is required for samples that exceed this level

Single-lab precision (Brenner et al 1993): E coli range: 3.3% at > 30 CFU/100 mL to 27% at < 10 CFU/100 mL total coliforms range: 2.5% at > 30 CFU/100 mL to 5.1% at 11 to 30 CFU/100 mL Multilab (19) precision (Brenner et al 1996): E coli range: 8.6% at > 30 CFU/100 mL to 40% at < 10 CFU/100 mL; total coliforms range: 6.9% at 11 to 30 CFU/100 mL to 28% at < 10 CFU/100 mL. Accuracy (Brenner et al.1993): E. coli: 4.3% false + and 4.3% false - Specificity: E coli 95.7%; total coliforms 93.1% Recovery, E. coli 97.9% of Heterotrophic Plate Count (HPC) 115% R2A spread plate count Recovery, total coliforms: Klebsiella pneumoniae 87.5%HPC and 89.3% R2A Enterobacteraerogenes 85.7% HPC and 85.8% R2A. Can recover E. coli that may not be recovered by other tests: from waters with high particulates and high non-coliform background; also, E. coli strains that are chlorine stressed, nutrient-deprived, temperature-sensitive and/or anaerogenic (EPA Fed Reg. Aug. 2001) Brenner, K.

Determined by the largest volume that can be plated, typically 100 mL, 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.

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