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Two-color assay for bacterial gram sign determination

The LIVE BacLight Bacterial Gram Stain Kit provides a novel one-step fluorescence assay for the determination of gram sign in living bacteria. When gram-negative and gram-positive bacteria are simultaneously stained with the membrane-permeant SYTO 9 dye and hexidium iodide; gram-negative bacteria fluoresce green and gram-positive bacteria fluoresce red.

This protocol can be used for:

  • Identifying gram-negative versus gram-positive bacteria using microscopy

This protocol should not be used for:

  • Flow cytometry

You will need the following for this protocol:


1. Add 50 μL of late log-phase bacterial culture to 1 mL of filter-sterilized water
2. Centrifuge for 5 minutes at 10,000 × g
3. Wash the cells by removing the supernatant and resuspending the pellet in 1 mL of filter-sterilized water
4. Prepare the cell staining solution by adding equal volumes of SYTO 9 (Component A) and hexidium iodide (Component B) to a microcentrifuge tube and mixing thoroughly
5. Add 3 μL of the dye mixture per mL of bacterial suspension and mix thoroughly
6. Incubate at room temperature in the dark for 15 minutes
7. Put 5 µL of the stained bacterial suspension onto a glass slide and cover with a coverslip
8. Image cells with the appropriate filters listed below
Spectral information and storage
 SYTO 9Hexidium iodide
Excitation/Emission480/500 nm518/600 nm
Standard filter setGFPRFP
Storage conditions–20°C, protect from light–20°C, protect from light


Protocol tips

  • Bacteria concentration should be 108–109 bacteria/mL
  • Filter water through a 0.2 μm pore-size filter to sterilize and to remove particulate matter that might interfere with the assay
  • Dye stock solutions should be stored frozen
  • BacLight mounting oil has a refractive index of 1.517 ± 0.003 at 25°C. DO NOT USE FOR IMMERSION OIL
  • BacLight mounting oil may be stored at room temperature