Standard Methods: 9222B,G: Membrane filtration plating of E. coli with MUG-fluorescent detection
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Official Method Name
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Membrane Filter Technique for Members of the Coliform Group 9222B. Standard Total Coliform Membrane Filter Procedure. 9222G. MF Partition Procedures 9221F. Escherichia coli Procedure (Proposed) |
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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 les-ENDO or m-ENDO agar containing basic Fuchsin as indicator. Incubate at 35C for 24 h. Red colonies with a metallic (golden) sheen are coliform positive. Transfer the membrane filter to EC-MUG or NA-MUG (nutrient agar containing MUG, the enzyme substrate 4-methylumbelliferyl-beta-D-glucuronide). Incubate for 4 h. Observe individual colonies using a long wave ultraviolet light source. Fluorescence on the periphery of the colony (a bright blue halo) indicates hydrolysis of MUG and is positive for E. coli (Standard Methods 20th ed. 9221F. Escherichia coli Procedure (Proposed) and 9222G. MF Partition Procedures 1. Escherichia coli Partition Methods). Requirements: ingredients for Nutrient agar with MUG (NA-MUG) or EC broth with MUG (EC-MUG); buffer for rinsing and dilutions. Culture dishes (50x10mm); 0.65 or 0.45 micron membrane filters, certified to fully retain E. coli. Refrigeration; autoclave; manifold and sterile filter funnel. Magnifying glass; forceps, alcohol; incubator at 35+-0.5C; a long-wave ultraviolet light source (366 nm) to read results; sterile pipet. Cost of analysis (USEPA Fed. Reg. Aug 2001): E coli $22 ($10 to $35) |
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Scope and Application
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Ambient, compliance monitoring: 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. |
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Applicable Concentration Range
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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 |
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Interferences
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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 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 |
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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. 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 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. |
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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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