Evaluation of mineral agar plates for the enumeration of hydrocarbon-degrading bacteria

Abstract

The enumeration of hydrocarbon-degrading bacteria is important in the evaluation and monitoring of the bioremediation of petroleum spills. Colony formation on mineral agar plates incubated in the presence of hydrocarbons is often used for such enumerations. We evaluated this method by inoculating mineral agar plates with dilutions of gasoline-contaminated ground water and of unpolluted surface water and incubating the plates either with or without toluene-xylene (TX) fumes. Approximately the same number of colonies formed on both sets of plates, and the colony-forming units calculated were much higher than the most probable number of TX-degrading bacteria or of TOL plasmid-containing bacteria estimated using liquid mineral media containing TX or meta-toluate. Colonies that grew on both the fume-incubated and the control plates were examined for growth in TX fumes or on meta-toluate on transfer to fresh mineral agar, and for the presence of the TOL plasmid using a dioxigenin-dUTP labelled whole TOL plasmid probe. About 98% of the colonies from plates inoculated with unpolluted surface water were negative for all three characters even when they came from TX-incubated plates. Many of the colonies from fume or control plates inoculated with gasoline-contaminated ground water possessed one or more of these phenotypic characters. We conclude that many environmental bacteria, which are not hydrocarbon degraders, can form colonies on mineral agar plates in the presence of hydrocarbons, and that the use of this type of medium may result in the over-estimation of the population of hydrocarbon-degrading bacteria.