Bioluminescence Assay for Measuring the Number of Bacteria Adhering to the Hydrocarbon Phase in the BATH Test

A thorough validation of the bacterial adherence to hydrocarbons (BATH) test was performed by means of a bioluminescence assay. Ten different gram-negative strains were subjected to the BATH test. For the calculation of the adhesion index, several factors had to be taken into account: ATP leakage, the action of ATP-hydrolyzing enzymes, the change in the extraction efficiency of Nucleotide-Releasing Reagent for Microbial Cells (NRB; Lumac bv) after vortexing and the difference in light production after the addition of NRB. When the adhesion index values obtained by bioluminescence measurement were used as reference, the total plate count technique appeared to be unreliable in estimating the number of bacteria adhering to the hydrocarbon phase. A highly significant correlation was established, however, between those reference values and the adhesion index values obtained by the optical density reading for octane and especially for hexadecane. With xylene, no correlation was found between the optical density reading values and the total plate count or bioluminescence values.