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

R L Harcourt, I Horne, T D Sutherland, B D Hammock, R J Russell, J G Oakeshott

Index: Lett. Appl. Microbiol. 34(4) , 263-8, (2002)

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Abstract

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.