EPA-MICRO: 600-R-00-013: Membrane filtration plating of coliform bacteria on MI agar

Summary
Analytes
Revision
Data and Sites

Official Method Name

Membrane filter method for the simultaneous detection of total coliforms and Escherichia coli in drinking water.

Current Revision

2000

Media

WATER

Instrumentation

Membrane Filtration

Method Subcategory

Microbiological

Method Source

EPA-MICRO

Citation

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.

Brief Method Summary

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 35^oC 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+/-0.5^oC 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)

Scope and Application

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.

Applicable Concentration Range

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

Interferences

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.

Quality Control 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).

Sample Handling

Sample preservation: chilled, 1 to 4^oC; 1 mL of 10% Na₂S₂O₃ per liter drinking water or chlorinated effluent. Techniques for collection: 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. Standard Methods for the Examination of Water and Wastewater, 20th Edition. L. Clesceri, A. Greenberg, and A. Eaton (editors). Sections 9020B8 and B9, 9060A, 9222B. APHA: Washington, DC. 1998.
Sample processing time 1 hour. Requires an extensive dilution series for ambient waters. 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

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.

Relative Cost

Less than $50

Sample Preparation Methods

This method has 2 analytes 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	98% Rec (ML)	40.00	4	4	10.00 CFU/100 mL
Total coliforms(E761700) Total coliform Total coliforms	1.000 CFU/100 mL	86% Rec (ML)	28.00			10.00 CFU/100 mL

Precision Descriptor Notes:	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.
Detection Level Note:	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.

Precision Descriptor Notes:

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.

Detection Level Note:

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.

Revision	PDF/Link
2000

The Water Quality Portal contains sample results from sites that use this method.

Download WQP data

Select data:

Sites only
Sample results only

File format:

Comma-separated
Tab-separated
MS Excel 2007+

Sites where this method was used

Loading Sites ...

EPA-MICRO: 600-R-00-013: Membrane filtration plating of coliform bacteria on MI agar

WHO WE ARE

LEGAL