Microtiter Platebased Mpn Techniques

As a result of the limitations of traditional solid-agar-based enumeration methods, liquid culture methods have been developed by employing the most-probable-number (MPN)

FIGURE 6.2 Bacterial colonies growing on an agar plate with a thin film of phenanthrene applied by the spray-plate technique. The cleared halo surrounding colonies is evidence of phenanthrene biotransformation.

Bacterial Contamination Plate Images

FIGURE 6.2 Bacterial colonies growing on an agar plate with a thin film of phenanthrene applied by the spray-plate technique. The cleared halo surrounding colonies is evidence of phenanthrene biotransformation.

procedure (32). MPN is a statistical method based upon dilution of a sample to the point of extinction; i.e., multiple replicates of various dilutions of a sample are analyzed and the results are compared with statistical tables to determine the MPN of microorganisms in the original sample. The development of 96-well microtiter plates gave the opportunity to miniaturize such procedures. The sheen screen method, introduced by Brown and Braddock (23), represented the start of the miniaturized MPN method for oil degraders. This method was specific for crude oil as a substrate (Fig. 6.3), and a similar method was published for no. 2 fuel oil as the substrate (49). Wrenn and Venosa (131) developed a

96-well microtiter plate MPN procedure to separately enumerate aliphatic and aromatic hydrocarbon degraders in separate plates. The alkane-degrader MPN method uses M-hexade-cane as the carbon source, growth is scored by turbidity, and the reduction of iodonitrote-trazolium violet to iodonitrotetrazolium-formazan (red precipitate) is used as an indicator of electron transport activity. A recent variant on the technique (21) is similar to that of Wrenn and Venosa but purports to be simpler because of the inclusion of diphenyl tetrazolium violet in the growth medium.

An inherent limitation in all the 96-well microtiter plate MPN techniques is also one of lack of bioavailability. Given the long, narrow

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