Staining of Escherichia coli for flow cytometry: Influx and efflux of ethidium bromide

In an attempt to develop procedures for nucleic acid staining of bacteria for clinical routine assays, the uptake of ethidium bromide (EB) in wild-type Escherichia coli was studied using flow cytometry. Phosphate-buffered saline (PBS) containing EDTA or Tris significantly increased the net uptake of EB compared to PBS only. However, in the majority of the cells, the net uptake reached a constant level that was only a few percent of that of fully permeabilized cells, apparently due to the activity of a metabolically driven efflux pump. When cells were exposed to cold shock (0 degrees C for 30 min) in the presence of Tris or EDTA, the net uptake of dye was similar to that of fully permeabilized cells, whereas it was about half that value in PBS. When cold shock was given in growth medium, the cells split up into four subpopulations, with a net dye uptake ranging from that of fully permeabilized cells to less than 1% of that value. As expected, metabolic inhibitors (Na-azide, 2-deoxy-D-glucose, and CCCP) reduced efflux activity. However, fluorescence of metabolically inhibited cells never exceeded more than about half the value of that of dead cells, possibly reflecting conformational changes in DNA structure as a result of cell death.