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
 1. Grow 25 mL bacterial culture to late log-phase in nutrient broth |
 2. Centrifuge at 10,000 × g for 10 minutes |
 3. Remove the supernatant |
 4. Resuspend the pellet in 2 mL of wash buffer |
 5. Dilute 1 mL of the cell suspension by adding it to 20 mL of wash buffer |
 6. Incubate at room temperature for 1 hour, mixing every 15 minutes |
| 7. Centrifuge at 10,000 × g for 10 minutes |
 8. Resuspend pellet in 20 mL of wash buffer |
| 9. Centrifuge at 10,000 × g for 10 minutes |
| 10. Resuspend pellet in 10 mL of wash buffer |
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
 1. Combine equal volumes of SYTO 9 and propidium iodide in a microfuge tube |
| 2. Add 3 µL of the dye mixture to each milliliter of the bacterial suspension |
 3. Incubate at room temperature in the dark for 15 minutes |
 4. Pipette 5 µL of the stained bacterial suspension onto a glass slide and cover with a coverslip |
 5. Image cells with the appropriate filters listed below |
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 |