Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure

Abstract

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbon-degrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The method's accuracy was evaluated by comparing hydrocarbon-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. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.

Bioaccumulation and translocation factors of petroleum hydrocarbons in Aeluropus littoralis

“…A possible explanation for this might be the presence of metal cations (e.g., nickel and vanadium) in waste oil. The findings of the current study are consistent with those of other researchers who found elevated electrical conductivity in soils contaminated with petroleum hydrocarbons (22). Another finding was that crude oil degradation in soil increased the organic matter percentage and organic carbon content of the contaminated soil compared to the control soil.…”

Multidrug resistance in hydrocarbon-tolerant Gram-positive and Gram-negative bacteria

“…The number of viable bacteria in Poeni oily sludge (Teleorman County, Romania) was estimated by a modifi ed most probable number (MPN) procedure (Haines et al, 1996). Aliquots of 20 μl were added to ten separate dilution series in 96-microwell plates (Iwaki).…”

“…Bacteria with specifi c metabolic capabilities, such as hydrocarbon degradation, can be enumerated based on their ability to grow on selective media, using a 96-microwell plate MPN procedure (Haines et al, 1996). The number of total viable hydrocarbon-degrading bacteria per g of Poeni oily sludge was between 10 5 and 10 7 (data not shown).…”

Adaptative Response ofShewanella PutrefaciensandPseudomonas Aeruginosato Toxic Organic Solvents

“…Isolation and characterization of Shewanella putrefaciens IBB Ct4 and Pseudomonas aeruginosa IBB Ct5 strains Bacteria with specific metabolic capabilities, such as oil degradation, can be enumerated based on their ability to grow on selective media, using 96-well microtiter plate MPn procedure (14). the number of total viable hydrocarbondegrading bacteria in seawater sample was between 10 6 and 10…”

“…organic solvents with the logarithm of the partition coefficient in n-octanol and water mixture (log P oW ) betweenusing 96-microwell plates (iwaki, london, United Kingdom) (13,14). isolation of iBB ct4 and iBB ct5 bacterial strains from seawater was carried out on M 9 -Mg medium (41), using the enriched cultures method, with 1% (v/v) organic solvents as a single carbon source.…”