Optimization Of Bacterial Cell Viability Assays With The Fluorophores SYTO 9 And Propidium Iodide And Its Mechanism Based On Flow Cytometry

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.Here,we selected Escherichia coli and Brevibacillus brevis as representative strains of Gram-negative bacteria and Gram-positive bacteria,respectively.By investigating the staining kinetics,impacts of potential stains toxicity on cell physiology and morphology,bacterial dyeing characteristics of SYTO 9 and PI,and the potential toxicity of dyes on cells were studied to optimize staining conditions.In order to provide basic data support for the accurate assessment of bacterial viability using SYTO 9 and PI as dyes by flow cytometry.The main results were as follows:The solo staining effects were different due to the differences in cell wall structure of the two strains.SYTO 9 can penetrate the cell wall and membrane of B.brevis immediately to bind nucleic acid,while E.coli cells could only be fully stained with SYTO 9 after 0.5 m M EDTA permeabilization treatment and were slow to bind with nucleic acid,reaching balance after 20 min.In turn,the entry of PI into B.brevis was time-consuming,while the nucleic acid binding of PI to E.coli was rapid.Both SYTO 9 and PI signals were affected by fluorescence resonance energy transfer and background fluorescence.The dynamic binding and releasing processes between two dyes and nucleic acids were conducive to obtaining accurate results.Based on maximized signal to background distinction,the optimal B.brevis and E.coli staining concentrations are 2.5?M SYTO 9 and 9.0?M PI at 10~6 cells/m L cell abundance,and incubated at room temperature for 10 min and 20 min,respectively.Derivative spectrometry was used to quantify the binary solution to analyze the staining kinetics.The results showed that the dual staining characteristics of the dyes on two strains were consistent.SYTO 9 directly entered cells slowly in dual staining,while PI trended to adhere to the cell wall before entering the cell due to electrostatic force.Studies on the potential toxicity of excessive dye staining on cells indicated that excessive SYTO 9 altered the synthesis of phospholipids,showing an increase in C16:0 and C18:0 and the ratio of unsaturated fatty acids,which facilitated breakthrough of PI dye by changing membrane permeability,resulting in erroneous results.Excessive PI staining caused irreversible damage to the basic bacterial structural characteristics.