The microbiological indicators tested only involve fecal contamination indicator bacteria such as coliforms, heat-resistant coliforms, and Escherichia coli. The traditional detection method is the multi-tube fermentation method, which has a long detection cycle and short storage time of the culture medium. A positive result requires a verification test, and the operation steps are relatively cumbersome. As a rapid detection method, the enzyme substrate method can better make up for the shortcomings of traditional methods with its fast and simple characteristics.
| Official Method Name | Enzyme Substrate Coliform Enzyme Substrate Test |
| Current Revision | Standard Methods Online |
| Media | WATER |
| Instrumentation | Most Probable Number |
| Method Subcategory | Microbiological |
| Method Source | Standard Methods |
| Citation | 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. |
| 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. |
| Applicable Concentration Range | For samples in wells (excluding dilution factors), using Colitech kits: 1 to 2419 MPN/100 mL for 97 well detection tray; 1 to 200 MPN/100 mL for 51 well detection tray |
| Interferences | 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 |