Development of a simple and sensitive fluorimetric method for isolation of coumaphos-hydrolysing bacteria.

To develop a simple, rapid and sensitive fluorimetric assay to detect, isolate and characterize a soil bacterium capable of degrading the organophosphorus pesticide, coumaphos. A high throughput microtitre plate-based method was used to quantify coumaphos hydrolysis by the bacterium. The fluorescent hydrolysis product of coumaphos, chlorferon, was detected at levels as low as 10 nmol l(-1). Incorporation of coumaphos into agar plates allowed the rapid detection of coumaphos-hydrolysing bacteria when exposed to an excitation wavelength of approximately 340 nm. The coumaphos-hydrolysing enzyme could be visualized when bacterial cell extracts were separated on SDS-PAGE, incubated with coumaphos and exposed to an excitation source as above. This method is 100-fold more sensitive than the currently used spectrophotometric method for coumaphos. This is a unique and versatile tool to screen for bacteria possessing phosphotriesterase activity.