Optimization of staining with SYTO 9/propidium iodide: interplay, kinetics and impact on Brevibacillus brevis

Fluorophores SYTO 9 and propidium iodide (PI) are extensively applied in medicine, food industry and environmental monitoring to assess the viability of bacteria. However, the actual performance of these dyes remains largely unknown. In addition, their effects on the physiology of cells have not been elucidated. Here we characterized the effects of these two dyes on Brevibacillus brevis under optimized staining. We found that SYTO 9 entered cells continuously while PI tended to adhere to the cell wall before entering the cell. In addition, results showed that a high amount of the dyes altered the physicochemical properties of membranes, improving their breakthrough. These results provide new perspectives and ideas for improving the characterization of bacterial viability using flow cytometry. METHOD SUMMARY Investigation of the interplays among dyes, the targeted organism and the staining conditions was achieved by flow cytometry. Quantification of dyes was performed by measuring the absorbance using derivative spectrometry. Variations in phospholipid synthesis and cell morphology after excessive dye exposure were determined by gas chromatography-tandem mass spectrometry and scanning electronic microscopy.