Enzyme substrate tests use hydrolyzable chromogenic and fluorogenic substrates to simultaneously detect enzymes produced by total coliforms and Escherichia coli (E. coli). In this method, total coliform bacteria produce the enzyme β-d-galactosidase, which cleaves the chromogenic substrate in the medium to release chromogen. Most E. coli strains produce the enzyme β-glucuronidase, which cleaves a fluorogenic substrate in the medium to release fluorogen. The release of chromogen indicates that coliform bacteria are present, and the release of fluorogen indicates that E. coli are present.
Multiple-tube, multi-well, or presence–absence (single 100-mL sample) formats are available for use with these enzyme substrate tests.
1. Principle
a. Total coliform bacteria: Colilert®, Colilert-18®, and Colisure® media use the chromogenic substrates ortho-nitrophenyl-β-d-galactopyranoside (ONPG) and chlorophenol red-β-d-galactopyranoside (CPRG), respectively, to detect the enzyme β-d-galactosidase, which is produced by total coliform bacteria. The β-d-galactosidase enzyme hydrolyzes the chromogenic substrate that produces a color change, thereby indicating the presence of total coliforms without additional procedures.
Although non-coliform bacteria (e.g., Aeromonas, Flavobacterium, and Pseudomonas species) may produce small amounts of the enzyme β-d-galactosidase, the growth of these organisms is suppressed so they generally will not produce a false-positive result unless >106 CFU/100 mL are present.
b. Escherichia coli: The fluorogenic substrate 4-methyl-umbelliferyl-β-d-glucuronide (MUG) is used to detect the enzyme β-d-glucuronidase, which is produced by most strains of E. coli. The β-d-glucuronidase enzyme hydrolyzes the fluorogenic substrate that produces bluish fluorescence when viewed under long-wavelength (365–366 nm) ultraviolet (UV) light. Together, the color change (due to β-d-galactosidase) and the fluorescence (due to β-d-glucuronidase) indicate that a sample contains E. coli.
Large numbers of some bacteria or strains of bacteria (e.g., some strains of Shigella and Salmonella spp.) may cause a sample to fluoresce but will not change its color because they lack β-d-galactosidase. Such samples would be considered negative for E. coli.
2. Applications
These enzyme substrate coliform tests are recommended for the analysis of drinking water, source water, groundwater, and wastewater samples. If a laboratory has not used this method before, it is desirable to conduct parallel testing (including seasonal variations) with the existing method to assess site-specific effectiveness and to compare results. The results of many method-performance studies are available in the literature and the rates of false-positive and -negative results differ among various media. Users should carefully select the medium and procedure that best fits their needs. See Section 9020B.11 for guidance on validating new methods.
Water samples containing humic or other material may be colored. If there is a natural background color, note what it is. If the water is yellow enough to be misinterpreted as a weak positive after incubation, use a medium that does not turn yellow (e.g., Colisure). Some waters’ high calcium-salt content can cause precipitation, but this should not affect the reaction. In samples with excessive chlorine, a blue flash may be seen while adding Colilert or Colilert-18 media. If this occurs, consider sample invalid and discontinue testing.
Do not use the enzyme substrate test to verify presumptive coliform cultures or membrane-filter colonies, because the substrate may be overloaded by the heavy inoculum of weak β-d-galactosidase-producing noncoliforms, causing false-positive results.