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Assessment of bacterial viability status by flow cytometry and single cell sorting
G N Caron1, P Stephens, R A Badley
1Unilever Research, Colworth Laboratory, Sharnbrook, Bedfordshire, UK. hard.nebe-von-caron@unilever.com
Journal of Applied Microbiology
|August 26, 1998
Summary
A new triple fluorescent stain method accurately detects bacterial viability and physiological states. This approach overcomes limitations of previous methods, enabling better characterization of cell damage and death for improved bacterial detection.
Area of Science:
- Microbiology
- Cell Biology
- Biotechnology
Background:
- Bacterial viability assessment is crucial for various applications.
- Existing methods for bacterial detection and viability measurements have limitations, including dye extrusion and cell dormancy.
- Distinguishing between metabolically active, intact, and permeabilized cells is challenging.
Purpose of the Study:
- To develop a fast triple fluorochrome staining procedure for enhanced bacterial detection and viability measurements.
- To overcome limitations of existing viability assays, such as active dye extrusion and cell dormancy.
- To enable further cellular characterization of intact cells based on dye uptake and exclusion.
Main Methods:
- A novel triple fluorochrome staining protocol using ethidium bromide (EB) and bis-oxonol (BOX).
- Flow cytometry and single-cell sorting (EPICS Elite) for analyzing stained bacterial cells.
- Validation using aged Salmonella typhimurium cells and subsequent culture on agar plates.
Main Results:
- The triple staining method differentiates four physiological states: reproductively viable, metabolically active, de-energized with a polarized membrane, and depolarized.
- Active exclusion of EB indicates metabolic activity.
- Uptake of EB with exclusion of BOX indicates de-energized cells with a polarized membrane.
- Uptake of both EB and BOX indicates depolarized cells.
- Permeabilized cells were identified by propidium iodide (PI) uptake.
- Sorted cells showed recovery, confirming the method's ability to distinguish viable and damaged cells.
Conclusions:
- The developed triple fluorochrome staining procedure offers a rapid and accurate method for bacterial viability assessment.
- Cellular depolarization is a sensitive indicator of cell damage but not a definitive measure of cell death.
- This method enhances the characterization of bacterial physiological states, improving diagnostic capabilities.