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.