Defined Substrate Technology Method for Rapid and Specific Simultaneous Enumerationof Total Coliforms and Escherichia coli from Water: Collaborative Study

Abstract

The defined substrate technology (DST) method is a reagent system designed to enumerate specific target microbes(s) from a mixture of bacteria. The system simultaneously enumerates total coliforms and Escherichia coli directly from a water sample. The reagent contains o-nitrophenyl-beta-D-galactopyranoside (ONPG), which is hydrolyzed by total coliforms to produce a yellow chromogen, and 4-methylumbeilliferyl-beta-D-glucuronide (MUG), which is hydrolyzed and fluoresces when E. coli organisms grow. Noncoliform bacteria are suppressed and cannot metabolize the indicator nutrients. Nine laboratories participated in a field evaluation of the method, which covered a wide range of surface and subsurface water sources and water-processing modalities, including the examination of natural samples. The DST system was compared to multiple-tube fermentation (MTF) (quantitative) and presence-absence (P-A) (qualitative) Standard Methods formats. Comparison of water samples from natural sources by using the most probable number (MPN) procedure showed that the DST test was equivalent to the currently used MTF test. Results from the DST and the qualitative P-A procedure showed that these tests agreed with each other in 94% of the water samples analyzed. Specificity of the DST method was established by subculturing a species consistent with a total coliform or E. coli from each positive tube. Eight laboratories participated in a collaborative study of the method. Each laboratory received 3 concentrations of E. coli (organisms/100 mL); 10 (low); 60 (medium); and 120 (high). The DST test was inoculated from a split sample of each bacterial density in parallel with Standard Methods brilliant green lactose broth.(ABSTRACT TRUNCATED AT 250 WORDS)