Colilert -18 ( Standard Method 9223B ) is an enzyme substrate test utilizes hydrolyzable substrates for the simultaneous detection of total coliform bacteria and Escherichia coli enzymes. When the enzyme technique is used, the total coliform group is defined as all bacteria possessing the enzyme β-D-galactosidase, which cleaves the chromogenic substrate, resulting in release of the chromogen. Escherichia coli are defined as bacteria giving a positive total coliform response and possessing the enzyme β-glucuronidase, which cleaves a fluorogenic substrate, resulting in the release of the fluorogen.

Colilert-18 is similar to the original Colilert method: the chromogenic substrate ortho-nitrophenyl-β-D-galactopyranoside (ONPG) is used to detect an enzyme produced by coliform bacteria. Samples containing coliform bacteria will hydrolyze the ONPG substrate to release a yellow-colored compound. E. coli are detected by using the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide (MUG). Samples containing E. coli bacteria will hydrolyze the MUG substrate to produce a fluorescent compound when viewed under long-wavelength ultraviolet light. This combination of substrates allows detection of both total coliforms and E. coli after an incubation period of 18 to 22 hours at 35°C. (The original Colilert method requires a minimum incubation time of 24 hours.) According to the manufacturer, Colilert-18 can detect 1 CFU/100mL with as many as 2 million heterotrophic bacteria/100mL present.

EPA 353.2 Method : The Nitrate (NO3) is quantitatively reduced by passage of the sample through a copperized cadmium column. The nitrite, reduced nitrate plus original nitrite ( NO3 + NO2 , or NOX) is then determined by diazotizing with sulfanilamide followed by coupling with N(1-napthyl) ethylenediamine dihydrochloride. The resulting water soluble dye has a magenta color which is read at 520 nm. Nitrite alone also can be determined by removing the cadmium column.

Nitrate (NO3) and Nitrite ( NO2) as N are measured by Flow Injection Analysis (FIA) using LACHAT Quikchem 8500 Series 2 . It is based on the injection of a liquid sample into a moving, non-segmented continuous carrier stream of a suitable liquid. The injected sample forms a zone, which is then transported toward a detector that continuously records the absorbance electrode potential or other physical parameter as it continuously changes due to the passage of the sample material through the flow cell.