Flow cytometric detection and quantification of mycobacteria in metalworking fluids

Mycobacteria are relevant target microorganisms in metalworking fluids (MWFs), owing to their recent recovery from industrial systems and their potential implication in respiratory illness. Whereas a number of detection technologies are available to monitor microbial contamination in MWF, no methods have been developed for the detection of mycobacteria in these matrices. We report here a novel method for detecting Mycobacterium parafortuitum in a semi-synthetic MWF with nonspecific nucleic acid dyes using flow cytometry (FCM). Following disaggregation of bacteria through (probe) sonication, the emulsion was destabilized using isopropanol (IPA) to minimize the nonspecific binding of the fluorescent dye to MWF components. The quantitative results show good correlation with direct counts using epifluorescent microscopy (EFM) within the range of 2.31×104 to 2.31×107 cells ml−1. The time required to complete FCM and EFM enumeration was 10 and 300 s sample−1, respectively. These results lend support to the applicability of FCM, a multiparametric data acquisition technique, for microbial detection in these complex fluids.