SELECTIVE ENUMERATION OF AROMATIC AND ALIPHATIC HYDROCARBON-DEGRADINGBACTERIA BY A MOST-PROBABLE-NUMBER PROCEDURE

A most-probable-number (MPN) procedure was developed to separately enu merate aliphatic and aromatic hydrocarbon degrading bacteria, because most of the currently available methods are unable to distinguish betw een these two groups. Separate 96-well microtiter plates are used to e stimate the sizes of these two populations. The alkane-degrader MPN me thod uses hexadecane as the selective growth substrate and positive we lls are detected by reduction of iodonitrotetrazolium violet, which is added after incubation for 2 weeks at 20 degrees C. Polycyclic aromat ic hydrocarbon degraders are grown on a mixture of phenanthrene, anthr acene, fluorene, and dibenzothiophene in a second plate. Positive well s turn yellow to greenish-brown from accumulation of the partial oxida tion products of the aromatic substrates and they can be scored after a 3-week incubation period. These MPN procedures are accurate and sele ctive. For pure cultures, heterotrophic plate counts on a nonselective medium and the appropriate MPN procedure provide similar estimates of the population density. Bacteria that cannot grow on the selective su bstrates do not produce false positive responses even when the inoculu m density is very high. Thus, this method, which is simple enough for use in the field, provides reliable estimates for the density and comp osition of hydrocarbon-degrading microbial populations.