Bacterial Detection and Live/Dead Discrimination by Flow Cytometry

Flow cytometry, a technique first applied to eukaryotic cells, has been adapted to the analysis of viability, metabolic state, and antigenic markers of bacteria.1-4 In particular, flow cytometry can be readily applied to the enumeration of viable bacteria in a sample.

Live cells have intact membranes and are impermeable to dyes such as propidium iodide (PI), which only leaks into cells with compromised membranes. Thiazole orange* (TO) is a permeant dye and enters all cells, live and dead, to varying degrees. With gram-negative organisms, depletion of the lipopolysaccharide layer with EDTA greatly facilitates TO uptake. Thus a combination of these two dyes provides a rapid and reliable method for discriminating live and dead bacteria. If enumeration of the bacteria is important, BD Liquid Counting Beads (BD Biosciences, San Jose, CA), a flow cytometry bead standard, can be used to accurately quantify the number of live, dead, and total bacteria in a sample.