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Method Article
Mohiuddin Md. Taimur Khan
Department of Civil and Environmental Engineering, Washington State University
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
viable but non-culturable (VBNC), viable-culturable (VC), flow cytometry (FCM), fluorescent probes, cellular functions, rapid identification of bacterial viability, cell damage
Figure 1
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigureS1.jpg
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.
- SupplementaryTablesNatPro.pdf
(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
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Subject Areas
Biotechnology
Cancer
Biological techniques
Ecology
Molecular biology
Environmental sciences
Diseases
Medicinal chemistry
Engineering
Drug discovery
Chemical biology
Biological physics
Cheminformatics
Associated Publications
Specific and Rapid Enumeration of Viable but Nonculturable and Viable-Culturable Gram-Negative Bacteria by Using Flow Cytometry
by Mohiuddin M. Taimur Khan, Barry H. Pyle, Anne K. Camper
Applied and Environmental Microbiology (11 June, 2010)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Mohiuddin Md. Taimur Khan
Department of Civil and Environmental Engineering, Washington State University
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 01 Oct, 2020
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
- SupplementaryFigureS1.jpg
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.
- SupplementaryTablesNatPro.pdf
(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
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Associated Publications
Specific and Rapid Enumeration of Viable but Nonculturable and Viable-Culturable Gram-Negative Bacteria by Using Flow Cytometry
by Mohiuddin M. Taimur Khan, Barry H. Pyle, Anne K. Camper
Applied and Environmental Microbiology (11 June, 2010)