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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:


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

Image of bacteria stained with the LIVE/DEAD® BacLight™ Bacterial Viability Kit
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 (in nm) 480/500 490/635
Standard filter set FITC Texas Red®
Storage conditions ≤20°C, protect from light ≤20°C, protect from light