Standard Methods: 9222A,B,C: Coliforms, total, by Membrane Filtration (MF) Plating

Summary
Analytes
Revisions
Data and Sites

Official Method Name

Membrane Filter Technique for Members of the Coliform Group 9222A. Introduction. 9222B. Standard Total Coliform Membrane Filter Procedure. 9222C. Delayed Incubation Total Coliform Procedure.

Current Revision

1998

Media

WATER

Instrumentation

Membrane Filtration

Method Subcategory

Microbiological

Method Source

Standard Methods

Citation

Standard Methods Online - Standard Methods for the Examination of Water and Wastewater

Brief Method Summary

Filter sample through a membrane filter in the field, immediately after collection. Place filter in transport medium and ship to laboratory in sealed container. Transfer membrane to les-ENDO or m-ENDO agar containing basic Fuchsin as indicator. Incubate at 35 +/- 0.5 C for 20 to 22 h. Red colonies with a metallic (golden) sheen are coliform positive. Requirements: ingredients for transport media, mENDO, lesENDO; buffer for rinsing and dilutions. Culture dishes (50x10mm); 0.45 micron membrane filters or equivalent, certified to completely retain coliform bacteria. Refrigeration; autoclave; manifold and sterile filter funnel. Magnifying glass; forceps, alcohol; incubator at 35+-0.5C; a cool white fluorescent light source to read results; sterile pipet. Cost of analysis (USEPA Fed. Reg. Aug 2001): E coli $22 ($10 to $35) .

Scope and Application

Ambient, compliance monitoring: fresh and saline waters; drinking water. 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

Applicable Concentration Range

USEPA Total Coliform Rule: presence/absence. USEPA Surface Water Treatment Rule: 20 - 80 CFU/100 mL for enumeration; typical of drinking water samples without dilution. Enumerate from 1 to 80 CFU/100 mL in general. Maximum: 200 CFU/100 mL; dilute samples that exceed this level.

Interferences

Overgrowth by fungi, including yeasts in transit: add cycloheximide to m-ENDO preservative medium for transport. Moisture loss in transit or during holding before analysis. General 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. Section 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

Sample Handling

Preservatives: mEndo medium, sodium benzoate solution, cycloheximide, m-ST medium; 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 9020B8 and B9, 9060A. 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. Recommended sample volumes according to water type are: 100 mL for drinking water, lakes, reservoirs, wells, springs; 50 mL for drinking water sources. 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

Sample can be kept on holding/preservative medium for maximum of 72 h before culturing and enumeration in lab.

Relative Cost

Less than $50

Sample Preparation Methods

This method has 1 analyte associated with it.

Analyte	Detection Level	Bias	Precision	Pct False Positive	Pct False Negative	Spiking Level
Total coliforms(E761700) Total coliform Total coliforms	1.000 CFU/100 mL	N/A	45.00			20.00 CFU/100 mL

Precision Descriptor Notes:	Precision: 45 percent at 20 CFU per volume filtered. 22 percent at 80 CFU per volume filtered. Consistent results reported between 9222C (delayed) and 9222B (direct), applied to both fresh- and salt-water samples. Even for presence/absence determinations, method advises enumeration of coliforms in repeat sampling situations, as in assessing a biofilm problem in a distribution system, or in determining the magnitude of a contaminating event.
Detection Level Note:	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.

Precision Descriptor Notes:

Precision: 45 percent at 20 CFU per volume filtered. 22 percent at 80 CFU per volume filtered. Consistent results reported between 9222C (delayed) and 9222B (direct), applied to both fresh- and salt-water samples. Even for presence/absence determinations, method advises enumeration of coliforms in repeat sampling situations, as in assessing a biofilm problem in a distribution system, or in determining the magnitude of a contaminating event.

Detection Level Note:

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	PDF/Link
1998
Standard Methods 18th, 19th, 20th ed.
Standard Methods 19th Edition (1995)
Standard Methods 18th Edition (1992)

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Standard Methods: 9222A,B,C: Coliforms, total, by Membrane Filtration (MF) Plating

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