Flow Cytometric Screening Reveals Nucleic Acid Dyes Aberrantly Stain Peptidoglycan Sacculi

Flow cytometry is
an essential tool to discern phenotypic
and functional
differences in single cell mixtures. Most efforts to develop and deploy
multicolor flow panels have focused on mammalian cells, whereas a
more limited palette of tools exists for microbiota and pathogen-focused
analyses. Here, we describe a systematic screen of commercially available
nucleic acid dyes to aid in the development of multicolor panels for
both Gram-negative and Gram-positive bacteria. We found that dyes
show responses that varied by orders of magnitude across two model
bacteria. When examining whether dyes can help to discriminate intact
cells from noncellular debris, we discovered that certain compounds
also bind to purified peptidoglycan with intensities comparable to
those observed with bacterial binding. Together, these data will aid
in the selection of specific reagents to use in the development of
larger scale, multicolor panels for microbial flow cytometry.