Two-color bacterial viability assay
This kit is used to assess the viability of bacterial populations as a function of the membrane integrity of the cell. Cells with a compromised membrane that are considered to be dead or dying will stain red, whereas cells with an intact membrane will stain green.
This protocol can be used for:
- Identifying live and dead bacteria using a fluorescence microscope
This protocol should not be used for:
- Flow cytometry
You will need the following for this protocol:
- Bacteria growing in culture
- LIVE/DEAD BacLight Bacterial Viability Kit, for microscopy (Cat. No. L7012)
- Nutrient broth (e.g., LB Broth (Cat. No. 12780-052))
- 0.85% NaCl or other appropriate wash buffer
- Fluorescence microscope
Protocol
Culture conditions and preparation of bacterial suspensions
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Protocol tips
- Warm vials to room temperature and centrifuge briefly before opening
- Wash to remove all growth medium from bacteria before staining
- Phosphate wash buffers may decrease staining efficiency and are not recommended
Micrococcus luteus and Bacillus cereus stained with the LIVE/DEAD BacLight Bacterial Viability Kit (Cat. No. L7012). When incubated with the SYTO 9 stain and the propidium iodide nucleic acid stain provided in this kit, live bacteria with intact cell membranes fluoresce green and dead bacteria with compromised membranes fluoresce red.
Staining bacteria
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Spectral information and storage
SYTO 9 | Propidium iodide | |
---|---|---|
Excitation/Emission | 480/500 nm | 490/635 nm |
Standard filter set | FITC | Texas Red |
Storage conditions | ≤20°C, protect from light | ≤20°C, protect from light |