Rapid identification of bacterial viability, culturable and non-culturable stages by using flow cytometry
The protocol details methods developed to optimize staining procedures and instrument settings for the rapid identification and unbiased enumeration of viable but non-culturable (VBNC) and viable-culturable (VC) cells as well as the membrane integrity of bacteria by flow cytometry (FCM). To detect viability, various Gram-negative bacteria were stained with numerous fluorescent membrane permeable (SYTO 9, SYTO 13, SYTO 17, SYTO 40) and impermeable (propidium iodide, PI) probes, and then quantified using the FCM. FCM data are then integrated with specific plate count results to calculate VC cells in different growth states. The cells were also exposed to heat (72° C) to monitor cellular membrane integrity. At late log phase (at 18 h incubation) of bacterial culture, the culturable, non-culturable and cells with damaged membranes varied from 40-98%, 1 to 64% and 0.7% to 4.5%, respectively. In this study, the cells with damaged cell membranes were considered dead (non-viable). This robust method preserves cell viability and takes a little more than an hour allowing the simultaneous quantification of phenotypes or cellular functions that is compatible with downstream cell-sorting and RNA-based assays.
Figure 1
This is a list of supplementary files associated with this preprint. Click to download.
Histograms of cells stained with propidium iodide (PI) before (0-min) and after heating for 5-min: E. coli O157:H7 (a, b), P. aeruginosa (c, d), P. syringae (e, f) and S. enterica serovar Typhimurium (g, h). P1 gate indicates the PI stained cell region and P5 gate shows the fluorescent liquid counting bead region.
(1) Supplementary Table S1. Operating conditions and settings defining the protocols for specific cell types and dyes. The threshold was applied on specific dyes used to stain particular cells (2) Supplementary Table S2. The calculation of VBNC and VC cells after staining with different dyes. The values after ‘‘+/-' symbol represent the standard error of means calculated from true independent replications (three to four separately grown cultures). The order of dyes was ranked based on the highest number of culturable and non-culturable cells
Posted 01 Oct, 2020
Rapid identification of bacterial viability, culturable and non-culturable stages by using flow cytometry
Posted 01 Oct, 2020
The protocol details methods developed to optimize staining procedures and instrument settings for the rapid identification and unbiased enumeration of viable but non-culturable (VBNC) and viable-culturable (VC) cells as well as the membrane integrity of bacteria by flow cytometry (FCM). To detect viability, various Gram-negative bacteria were stained with numerous fluorescent membrane permeable (SYTO 9, SYTO 13, SYTO 17, SYTO 40) and impermeable (propidium iodide, PI) probes, and then quantified using the FCM. FCM data are then integrated with specific plate count results to calculate VC cells in different growth states. The cells were also exposed to heat (72° C) to monitor cellular membrane integrity. At late log phase (at 18 h incubation) of bacterial culture, the culturable, non-culturable and cells with damaged membranes varied from 40-98%, 1 to 64% and 0.7% to 4.5%, respectively. In this study, the cells with damaged cell membranes were considered dead (non-viable). This robust method preserves cell viability and takes a little more than an hour allowing the simultaneous quantification of phenotypes or cellular functions that is compatible with downstream cell-sorting and RNA-based assays.
Figure 1
© Research Square 2021