Introduction to Microplate MPN Enumeration of Hydrocarbon Degraders

Abstract

The number of hydrocarbon-degrading microorganisms in environmental samples or liquid cultures can be determined by the most probable number (MPN) methods. These procedures are based on serial dilution of the sample and subsequent determination of the presence or absence of degrader cells for several subsamples of each dilution. The 96-well format of the microplate is very suitable for such replicate detection of growth of hydrocarbon degraders. Growth-positive wells can be distinguished from growth-negative wells by several means, for instance, by increased optical density, by emulsification of a crude oil film, by the metabolic conversion of colorless tetrazolium compounds into the corresponding colored formazans, or by mineralization of 14C-labeled substrates. Detailed instructions are given for (1) the preparation of dilution series; (2) MPN enumeration of total oil degraders, alkane degraders, monoaromatic hydrocarbon degraders, and diaromatic hydrocarbon degraders; (3) MPN enumeration of polycyclic aromatic hydrocarbon (PAH) degraders; (4) a radiotracer MPN method based on the conversion of 14C-labeled substrate into 14CO2 which is quantified by autoradiography and digital image analysis; and (5) how to interpret results and calculate