Flow cytometry for rapid assessment of viability after exposure to a quaternary ammonium compound

Abstract

The use of flow cytometry to rapidly assess the viability of Pseudomonas spp. and Staphylococcus spp. after exposure to a quaternary ammonium compound (QAC) was investigated using rhodamine 123 (Rh 123), Stain A (LIVE Stain) accumulating in viable but not in dead cells (Live/Dead Baclight bacterial viability kit, Molecular Probes Inc., Eugene, OR, USA), and Sytox green (Molecular Probes) accumulating in dead but not viable cells. Staining conditions were optimized for each stain. The fraction of viable cells after exposure to benzalkonium chloride was determined by using the three staining techniques and colony counts on agar medium. For all Staphylococcus spp. tested there was a high correlation the methods based on flow cytometry and colony counts irrespective of which stain was used. Although viable, all Pseudomonas spp. tested accumulated Rh 123 poorly and about 30% failed to accumulate LIVE stain as well. However, the correlation between colony counts and Sytox green labelling of Pseudomonas spp. was high. Our results indicate that flow cytometry together with live or dead cell labelling can be used to study the bactericidal effect of QACs. The methods based on LIVE stain and Sytox green were simpler and less time consuming than Rh 123 labelling. Only Sytox green could be used with all strains of Staphylococcus and Pseudomonas tested.