ASSESSMENT OF BACTERIAL VIABILITY STATUS BY FLOW-CYTOMETRY AND SINGLE-CELL SORTING

Rapid bacterial detection and viability measurements have been greatly enhanced by recent advances in the use of fluorescent stains in cytom etry. It has previously been shown that four physiological states can be distinguished: reproductively viable, metabolically active, intact and permeabilized. Previous sorting experiments have shown that not al l intact cells readily grow, but some intact cells can grow even when they fail to show metabolic activity, as determined by esterase turnov er. To circumvent the limitations imposed by active dye extrusion or c ell dormancy on viability measurements used to date (e.g. enzyme activ ity or cell polarization), a fast triple fluorochrome staining procedu re has been developed that takes account of these problems. This allow s further cellular characterization of intact cells by: active exclusi on of ethidium bromide (EB) (metabolically active cells), uptake of EB but exclusion of bis-oxonol (BOX) (de-energized but with a polarized cell membrane) and uptake of both dyes (depolarized). Permeabilized ce lls were identified by propidium iodide (PI) uptake. The method was va lidated using an electronically programmable single cell sorter (EPICS Elite(R)) and aged Salmonella typhimurium cells. Reproductive viabili ty was determined by sorting single cells to their staining pattern di rectly onto agar plates. Most polarized cells could be recovered as we ll as a significant fraction of the depolarized cells, demonstrating t hat depolarization is a sensitive measure of cell damage but a poor in dicator of cell death.