MEASUREMENT OF HYDROCARBON-DEGRADING MICROBIAL-POPULATIONS BY A 96-WELL PLATE MOST-PROBABLE-NUMBER PROCEDURE

A 96-well microtiter plate most-probable-number (MPN) procedure was de veloped to enumerate hydrocarbon-degrading microorganisms, The perform ance of this method, which uses number 2 fuel oil (F2) as the selectiv e growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an establis hed 24-well microtiter plate MPN procedure (the Sheen Screen), which u ses weathered North Slope crude oil as the selective substrate and det ects positive wells by emulsification or dispersion of the oil, Both p rocedures gave similar estimates of the hydrocarbon-degrader populatio n densities in several oil-degrading enrichment cultures and sand samp les from a variety of coastal sites, Although several oils were effect ive substrates for the 96-well procedure, the combination of F2 with I NT was best, because the color change associated with INT reduction wa s more easily detected in the small wells than was disruption of the c rude oil slick, The method's accuracy was evaluated by comparing hydro carbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures, For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result, T hus, this and other hydrocarbon-degrader MPN procedures might underest imate the hydrocarbon-degrading population, even for culturable organi sms.