Detection of microbial growth on polycyclic aromatic hydrocarbons in microtiter plates by using the respiration indicator WST-1

We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 [4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate], in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.