Rapid preparation procedure for staining of exponentially growing P. vulgaris cells with ethidium bromide: a flow cytometry-based study of probe uptake under various conditions

Abstract

For flow cytometry-based detection as well as susceptibility testing and counting of bacteria, staining of the cells is essential to distinguish them from other particulate matter in the sample and to extrude information on their physiological status. Since ethanol fixation is time-consuming, may mask phenomena associated with viability, and lead to uncontrolled loss and aggregation of cells, the aim of this study was development of rapid procedures which allow complete staining of cellular nucleic acids independent of fixation. In an attempt to develop a rapid preparatory procedure for staining of wild type P. vulgaris, the uptake of the nucleic acid binding dye ethidium bromide in viable cells was studied by flow cytometry under conditions intended to block probe efflux and increase cell wall permeability. Uptake of ethidium bromide was measured repeatedly after treating cells with metabolic inhibitors and cold shock (0°C) to increase permeability. Within a few minutes after staining, the fluorescence increased to above 80% of that found in fully stained ethanol-fixed controls. The dye uptake was homogeneous, and the fluorescence remained stable for hours. The fluorescence of these cells which was several hundred fold above that of cells stained at room temperature, presumably reflected the combined effect of enhanced outer membrane permeability and reduced efflux of dye. The total processing time, i.e. from harvesting to measurement, was only a few minutes. In contrast, cells which were stained in the absence of metabolic inhibitors, showed responses which were determined by the harvesting conditions, i.e. the cooling time in growth medium while in room temperature, i.e. from harvesting at 37°C to dilution in ice cold buffer. Cells which were transferred to ice cold buffer within 1 min after harvesting, stained heterogeneously. They accumulated in discrete populations with average fluorescence intensities differing several hundred fold, suggesting different levels of probe efflux activity. The fluorescence intensity of each population remained fairly constant with time, while cells appeared to be shifted to populations with higher dye uptake through the time span of the experiment. In contrast, cells which were allowed to cool for 5 min before transfer from growth medium to ice cold buffer showed homogeneous, low fluorescence, similar to that obtained for cells stained at room temperature. The fluorescence of such cells remained constant with time. Parallel plating experiments demonstrated an inverse relationship between fluorescence and viability.