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:
- Late log–phase bacteria grown in culture
- LIVE BacLight™ Bacterial Gram Stain Kit (L-7005)
- Filter-sterilized water
- Fluorescence microscope with GFP and RFP filters
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® 9 | Hexidium Iodide | |
|---|---|---|
| Excitation/Emission (in nm) | 480/500 | 518/600 |
| Standard filter set | GFP | RFP |
| 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
For Research Use Only. Not for use in diagnostic procedures.
