Jr. Haines et al., MEASUREMENT OF HYDROCARBON-DEGRADING MICROBIAL-POPULATIONS BY A 96-WELL PLATE MOST-PROBABLE-NUMBER PROCEDURE, Journal of industrial microbiology, 16(1), 1996, pp. 36-41
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.