Introduction to microplate MPN-enumeration of hydrocarbon degraders

Most-probable-number (MPN) methods are used for determining the number of specific types of microorganisms in environmental samples or liquid cultures. Here we present three specific methods for applying the MPN method to quantify hydrocarbon-degrading populations in samples from contaminated environments. Two of those use specific coloration of active hydrocarbon-degrading communities enriched in the MPN procedure, namely the INT-method and a protocol that uses the tetrazolium compound WST-1 for detection of growth-positive wells. Finally, we describe an MPN enumeration based on mineralization of ¹⁴C- labeled substrates, which is probably the most reliable quantification method for hydrocarbon degraders. In this method, a ¹⁴C-hydrocarbon is mineralized to ¹⁴CO₂ in growth-positive wells, and the produced ¹⁴CO₂ is captured in alkaline traps and quantified. In the procedure described below, we have modified the method for a 96-well microplate setup, where the ¹⁴CO₂ is quantified simultaneously from all wells by autoradiography followed by digital image analysis.