MycoLight™ Rapid Fluorescence Bacterial Gram Stain Kit

1. Prepare bactrerial samples
2. Prepare and add MycoLight™ dye working solution to bacteria samples
3. Incubate with MycoLight™ dye working solution at room temperature in dark for 15 minutes
4. Analyze sample by fluorescence microscope or fluorescence spectroscopy with FITC and TRITC filter sets

Thaw all the kit components at room temperature before use.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Add 100 µL of ddH2O into one vial of MycoLight™ Red (Component B) and mix them well.

Note: Store stock solution at -20 °C, avoid light and store in smaller aliquots to avoid repeated freeze-thaw cycles.

Mix equal volume of MycoLight™ Green (Component A) and MycoLight™ Red stock solution in a tube and mix them well.

1. Prepare bacteria sample with concentration around 10⁷cells/ml. Grow bacteria into late log phase in appropriate medium.

   Note: Measure the optical density of the bacterial culture at wavelength = 600 nm (OD600) to determine the cell number. For E. coli culture, OD600 = 1.0 equals 8 x 10⁸ cells/ml.

2. Remove medium by centrifugation at 10,000 x g for 10 minutes and re-suspend the pellet in ddH₂O, adjust bacteria concentration to ~ 10⁷ cells/ml.

1. Add 2 µL MycoLight™ dye working solution to 100 µL of the bacterial suspension.

2. Mix well and incubate in dark for 15 min at room temperature.

3. Remove the working solution by centrifugation at 10000 g for 10 minutes.

4. Resuspend the bacteria pallate in ddH2O.

5. Monitor fluorescence of bacteria with a fluorescent microscope through FITC (Ex/Em = 488/530 nm) channel for gram-negative bacteria and Cy5 (Ex/Em = 650/669 nm) channel for gram-positive bacteria.

   Note: The protocol only provides a guideline, should be optimized with different bacterial strains or other specific needs.

   Note: Relative ratio of gram positive and gram negative bacteria in a population can also be estimated with fluorescence spectroscopy with this kit. A sample analysis is included with the figures.