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Development of a simple and sensitive fluorimetric method for isolation of coumaphos‐hydrolysing bacteria
Author(s) -
Harcourt R.L.,
Horne I.,
Sutherland T.D.,
Hammock B.D.,
Russell R.J.,
Oakeshott J.G.
Publication year - 2002
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1046/j.1472-765x.2002.01078.x
Subject(s) - coumaphos , bacteria , chromatography , chemistry , isolation (microbiology) , biology , microbiology and biotechnology , pesticide , ecology , genetics
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.