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 (Cat. No. L-7005)
- Filter-sterilized water
- Fluorescence microscope with GFP and RFP filters
Protocol
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Spectral information and storage
SYTO 9 | Hexidium iodide | |
---|---|---|
Excitation/Emission | 480/500 nm | 518/600 nm |
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