Flow cytometry of bacterial cells: comparison between different flow cytometers and different DNA stains

Abstract

The DNA content and light scatter from individual Escherichia coli cells were measured in two flow cytometers with different configurations. The DNA content could be measured with similar resolution either in an Argus flow cytometer equipped with a mercury lamp, or in a FACStar flow cytometer with two argon lasers as light sources. In contrast, light scatter measurements appeared to be a good measure of cell mass only in the Argus instrument. Three DNA stains were compared:, DAPI, Hoechst 33258, and mithramycin A together with ethidium bromide. All three stains yielded DNA histograms of similar quality in both flow cytometers. Optimal results required that the stain and cell concentrations were kept similar, that a fixed rate of sample introduction was used, and that a period of equilibration was allowed during running of each sample. The results demonstrate that conventional, laser-based flow cytometers may be used for high-resolution measurements of bacterial DNA content, thereby making flow cytometry available to an increased number of research groups working with prokaryotes.