BacLight Bacterial Viability Kit

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:

Protocol

Culture conditions and preparation of bacterial suspensions

bacterial culture1. Grow 25 mL bacterial culture to late log-phase in nutrient broth
Centrifuge 2. Centrifuge at 10,000 × g for 10 minutes
decorative3. Remove the supernatant
decorative4. Resuspend the pellet in 2 mL of wash buffer
decorative5. Dilute 1 mL of the cell suspension by adding it to 20 mL of wash buffer
decorative6. Incubate at room temperature for 1 hour, mixing every 15 minutes
decorative7. Centrifuge at 10,000 × g for 10 minutes
decorative8. Resuspend pellet in 20 mL of wash buffer
decorative9. Centrifuge at 10,000 × g for 10 minutes
decorative10. 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

decorative1. Combine equal volumes of SYTO 9 and propidium iodide in a microfuge tube
decorative2. Add 3 µL of the dye mixture to each milliliter of the bacterial suspension
decorative3. Incubate at room temperature in the dark for 15 minutes
decorative4. Pipette 5 µL of the stained bacterial suspension onto a glass slide and cover with a coverslip
decorative5. Image cells with the appropriate filters listed below
Spectral information and storage
 SYTO 9Propidium iodide
Excitation/Emission480/500 nm490/635 nm
Standard filter setFITCTexas Red
Storage conditions≤20°C, protect from light≤20°C, protect from light