Improved Sensitivity of the Bioluminescent Determination of Numbers of Bacteria in Milk Samples

Abstract

A procedure was developed for the sensitive measurement of bacterial ATP in raw milk samples using the firefly luciferase assay. A sensitivity of 105 bacteria/ml was achieved in the laboratory. Treatment of milk samples with a detergent (Lubrol PX) and a proteolytic enzyme (trypsin) allowed filtration of 50 ml of milk through a 0.4-μm Nucleopore filter; a 50-ml sample of milk was required to provide sufficient ATP for measurement. The bacterial ATP was released from the bacteria on the filter using the commercial product Nucleotide Releasing Reagent for Bacteria (NRB® from Lumac). The procedure allows determination of 105 bacteria/ml of raw milk in 20 min.