Standard Methods: 9222D: Membrane filtration test for fecal coliforms
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Official Method Name
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Membrane Filter Technique for Members of the Coliform Group: 9222D. Fecal Coliform Membrane Filter Procedure. 9222G. MF Partition Procedures |
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Current Revision
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Media
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WATER |
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Instrumentation
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Membrane Filtration |
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Method Subcategory
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Microbiological |
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Method Source
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Citation
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Brief Method Summary
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Filter sample through a membrane filter. Place membrane on mFC agar containing aniline blue as indicator. Incubate at 44.5C for 22-24 h. Colonies that are various shades of blue are positive for fecal coliforms. The blue color indicates the capability to ferment lactose to acid. Requirements: ingredients for mFC agar; buffer for rinsing and dilutions; culture dishes (50x10mm); 0.65 micron membrane filters micron membrane filters, certified to fully retain fecal coliforms. Refrigeration; autoclave; manifold and sterile filter funnel; sterile pipets; forceps, alcohol. Incubator at 44.5+-0.2C. A low power (10-15 magnification) binocular wide-field dissecting microscope or equivalent. Cost of analysis (USEPA Fed. Reg. Aug 2001): fecal coliforms $21 ($15 to $35) |
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Scope and Application
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ambient, compliance monitoring. 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. 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: Total Coliform Rule: presence/absence of total coliforms, fecal coliforms or E coli |
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Applicable Concentration Range
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20 - 60 CFU/100 mL is considered ideal for enumeration of fecal coliforms (restricted by large colony size on FC medium). A reduced sample volume is required for samples above this range (Table 9222:III). |
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Interferences
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Very selective for fecal coliforms. Few nonfecal coliform colonies will be observed because of the selective action of the elevated temperature and addition of rosolic acid salt reagent. 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. |
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Quality Control Requirements
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(Standard Methods 20th ed. Section 9020 B.8 and 9; Myers and Sylvester, 1997) 1. Control culture--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 Standard Methods, 20th ed., 9020B. 9 or using a commercial multi-test system |
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Sample Handling
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Sample preservation: chilled, 1 to 4 C; 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 and B. Britton, L.J.; Greeson, P.E. 1987. Methods for collection analysis of aquatic biological and microbiological samples. USGS Techniques of Water Resources Investigations. Book 5. Chapter A4. 363 pp. p.37 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 to be diluted to 100 mL and filtered, according to water type, are: 100 mL lakes, reservoirs, wells, springs; 50 mL drinking water sources, natural bathing waters; 1 - 10 mL, sewage treatment; 0.1 - 1 mL, farm ponds and streams, stormwater runoff; 0.01 - 0.1 mL, raw municipal sewage and feedlot runoff; 0.001 - 0.01 mL sewage sludge. 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. |
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Maximum Holding Time
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Sample should be analyzed within 6 h for compliance or 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] |
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Relative Cost
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Less than $50 |
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Sample Preparation Methods
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