Detection of organophosphorus pesticide detoxifying bacterial colonies, using UV-photography of parathion-impregnated filters

Abstract

A rapid, filter-lift assay was developed for the identification of bacteria capable of degrading organophosphorus pesticides. Filter pads impregnated with parathion were applied to the surface of plates containing potential parathion-degrading colonies. Positive colonies capable of converting parathion to 4-nitrophenol attain a visible yellow coloration after 30 min of exposure; however, the identification of selected individuals was difficult when large numbers of colonies were screened on a single plate. An enhancement of this screening method was achieved with the use of UV-photography, which allowed for the detection of a single mutant or cured colony among 500 parathion-degrading colonies per plate. In addition, it was possible to detect a single parathion-degrading colony among 10,000 non-degrading colonies per plate. The efficiency of the technique was validated with a parathion-degrading strain ofPseudomonas diminuta from which phenotypically negative isolates were selected, subjected to plasmid isolation, evaluated for detoxifying activity, and tested by a battery of microbiological criteria to confirm the parental strain phenotype. All non-productive colonies were identical to the parental strain except for the lack of detoxifying capability and the loss of a large plasmid found in the parental strain.