The use of blue-excitable nucleic-acid dyes for the detection of bacteria in well water using a simple field fluorometer and a flow cytometer

The blue-excitable nucleic acid dyes Coriphosphine O (CPO), YOYO-1, and YOPRO-1 were evaluated to rapidly detect the presence of bacteria in well water samples using a simple field fluorometer (Turner Designs, Sunnyvale, CA, Model 10-AU-005) and a tabletop flow cytometer (Coulter Epics XL). The dyes were first titrated on the Turner Designs Model 10-AU field fluorometer with log-fold dilutions of Esherichia coli, since this organism is the indicator organism for water contamination. A detection limit of 10(4) Colony Forming Units per ml (CFU/ml) was established for YOPRO-1 and 10(5) CFU/ml for YOYO-1. The detection limit with CPO was determined to be 10(7) CFU/ml due to the high background fluorescence of the dye. The dyes were also evaluated with ragweed pollen to gauge the effect of a biological interferent. Ten well-water samples were subsequently analyzed using the technique. The results showed that only YOYO-1 correctly detected all the samples that were positive according to the reference laboratory. YOPRO-1 correctly detected only one of four positive samples. Analysis with the CPO dye was inconclusive due to high background fluorescence. The samples were then subjected to analysis on the flow cytometer. Results obtained with YOYO-1 compared well to those obtained on the fluorometer and by the reference techniques. YOPRO-1 performed better on the flow cytometer than with the simple fluorometer, correctly detecting three of four positive samples. Although the CPO results showed a very slight increase of green fluorescence with positive samples, they were largely indistinguishable from negative samples. This study suggests YOYO-1 could be useful with either a simple fluorometer or with a tabletop flow cytometer in screening water samples for the presence of bacterial contamination.