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Development of a simple and sensitive fluorimetric method for isolation of coumaphos-hydrolysing bacteria
R L Harcourt1, I Horne, T D Sutherland
1Commonwealth Scientific and Industrial Research Organization (CSIRO) Entomology, GPO Box 1700, Canberra, ACT 2601, Australia.
Aims:
To develop a simple, rapid and sensitive fluorimetric assay to detect, isolate and characterize a soil bacterium capable of degrading the organophosphorus pesticide, coumaphos.
Methods And Results:
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.
Conclusions:
This method is 100-fold more sensitive than the currently used spectrophotometric method for coumaphos.
Significance And Impact Of The Study:
This is a unique and versatile tool to screen for bacteria possessing phosphotriesterase activity.