Standard Methods: 9223B:  Enzyme substrate assay for measuring total coliforms and E. coli (ONPG-MUG test or CPRG-MUG test)

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Official Method Name
Enzyme Substrate Coliform Test: 9223B Enzyme Substrate Test
Current Revision
Standard Methods Online
Most Probable Number
Method Subcategory
Method Source
  Standard Methods
  Standard Methods Online - Standard Methods for the Examination of Water and Wastewater
Brief Method Summary
A specified volume of sample (typically 100 mL) is mixed with commercially prepared enzyme substrates and incubated at 35 +/- 0.5 C. Beta-galactosidase, an enzyme produced by total coliforms, is detected by hydrolysis of the chromogenic substrates ONPG (ortho-nitrophenyl-beta-D-galactopyranoside) and CPRG (chlorophenol red-beta-D-galactopyranoside). Beta-glucuronidase, an enzyme produced by E. coli, is detected by hydrolysis of the fluorescent substrate MUG (4-methylumbelliferyl-beta-D-glucuronide). Hydrolyzed ONPG is seen as a yellow color after incubation for 24 to 28 h; hydrolyzed CPRG is seen as a red or magenta color after incubation for 28 to 48 h - either condition is positive for total coliforms. No color from ONPG, or a yellow color from CPRG, is negative for total coliforms. Hydrolyzed MUG is seen as blue fluorescence when viewed under long-wavelength (366-nm) ultraviolet light, indicating a positive test for E. coli. Requirements: methods are available commercially They include, but are not limited to, Colilert and Colisure (Standard Methods 9223 is specific to Colilert and Colisure as it is currently written in the 20th edition). Check with the manufacturer for instrumentation requirements. (IDEXX Laboratories, Westbrook, Maine). 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. EPA Fed Reg (Aug 2001) for E coli, ambient only: fresh, marine, or estuarine surface waters; applicability must be demonstrated for other matrices. Approved for presence/absence of total coliforms and E. coli in drinking water. 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: a) Total Coliform Rule: presence/absence of total coliforms and E coli b) Surface Water Treatment Rule: enumeration of total coliforms
Applicable Concentration Range
For samples in wells (excluding dilution factors), using IDEXX Colilert or Colisure kits: 1 to 2419 MPN/100 mL for 97-well Quanti-Tray; 1 to 200 MPN/100 mL for 51-well Quanti-Tray
E. coli interferences: some strains of Shigella spp. may produce a positive fluorescence response. Because Shigella spp. are overt human pathogens this is not considered a detriment for testing the sanitary quality of water. Total coliform interferences: More than one million CFU/100 mL of noncoliform bacteria (Aeromonas, Pseudomonas)
Quality Control Requirements
(Standard Methods 20th ed. Section 9020 B.8 and 9) 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. Duplicate analyses--Perform duplicate analyses on 10% of samples. Check with the manufacturer's instructions for any additional requirements.
Sample Handling
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. 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.
Maximum Holding Time
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]
Relative Cost
Less than $50
Sample Preparation Methods