Development of a simple and sensitive fluorimetric method for isolation of coumaphos-hydrolysing bacteria.
OBJECTIVE
To develop a simple, rapid and sensitive fluorimetric assay to detect, isolate and characterize a soil bacterium capable of degrading the organophosphorus pesticide, coumaphos.


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.


CONCLUSIONS
This is a unique and versatile tool to screen for bacteria possessing phosphotriesterase activity.
Harcourt
R L
RL
Commonwealth Scientific and Industrial Research Organization (CSIRO) Entomology, GPO Box 1700, Canberra, ACT 2601, Australia.
Horne
I
I
Sutherland
T D
TD
Hammock
B D
BD
Russell
R J
RJ
Oakeshott
J G
JG
D001419:Bacteria / Q000201:enzymology* / Q000302:isolation & purification*; D003372:Coumaphos / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D007306:Insecticides / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012988:Soil Microbiology; D014468:Umbelliferones / Q000737:chemistry
D007306:Insecticides; D014468:Umbelliferones; C305311:chlorferron; D003372:Coumaphos