DEVELOPMENT OF SENSITIVE ASSAYS FOR BIOINDICATORS IN GROUNDWATER

Scope of Study. To develop sensitive assays for selected bioindicators which could be used to monitor the quality of ground water supplies.; Findings and Conclusions. Two classes of bioindicators were studied: (1) enzymes and (2) respiratory cofactors. The enzymes were lactate dehydrogenase, alkaline phosphatase, catalase, and adenylate kinase; the respiratory cofactors were the pyridine nucleotides, ATP, FMN, and iron porphyrins.; One-hour incubation assays were more sensitive than continuous assays for all enzymes (a 20-fold improvement for alkaline phosphatase (20 ng to 1 ng) to a 200-fold improvement for catalase (200 ng to 1 ng)). The enzymatic activities were measured in toluene-treated Escherichia coli using the one-hour incubation assays and the limit of detection was 10('6)-10('7) cells/ml for the four enzymes.; The respiratory cofactors were measured by several methods and the sensitivity of each method determined. Pyridine nucleotides were quantitated spectrophotometrically, fluorometrically, and in a coupled reaction with bacterial luciferase. Enzymatic cycling was used to amplify the fluorescence response and decreased the limit of detection to 0.05 pmol of NADP('+) and 5 pmol of NAD('+). Pyridine nucleotides were extracted from E. coli using boiling Tricine buffer and measured using enzymatic cycling. The limit of detection was 2.4 x 10('5) cells/assay.; Adenosine triphosphate was quantitated by a coupled enzyme assay which produced NADPH that was measured fluorometrically or by enzymatic cycling. Cycling improved the sensitivity from 200 pmol to 0.2 pmol. ATP was extracted from E. coli using boiling Tricine buffer and measured using enzymatic cycling. The limit of detection was 10('4) cells/assay. The results of the ATP studies were compared to studies using firefly luciferase to measure ATP.; FMN was measured by a luminescence assay using a crude bacterial luciferase preparation with FMN reduction carried out enzymatically using NADH as electron donor. The limit of detection was 1 ng FMN.; Iron porphyrins were measured by their catalytic action on the rate of chemiluminescent oxidation of luminol. Hemoglobin samples were quantitated over a range of 2 pg - 10 ng using aluminum disks to attenuate the intensity of the light emission. E. coli were quantitated by the peak height and integration methods of luminescence measurement with a limit of detection of 2 x 10('3)/ml and 1 x 10('3)/ml, respectively.; A sensitive assay of lactate dehydrogenase was developed by combining a one-hour incubation with enzymatic cycling of the reaction product, NAD('+). The limit of detection was improved 1000-fold (1 ng to 1 pg) and the number of E. coli cells which could be detected was decreased 10-fold (10('5) to 10('4)/assay).; It is concluded that several of the assays developed--ATP and lactate dehydrogenase using enzymatic cycling and iron porphyrins--could be used to detect changes in their respective bioindicators in environmental samples such as ground water which have as little as 10('4) cells/liter. Measurements of other bioindicators--pyridine nucleotides, catalase, adenylate kinase, and alkaline phosphatase could be used effectively for samples with higher cell concentrations.