MycoLight™ Bacterial Viability Assay Kit

Thaw kit components at room temperature and centrifuge briefly before starting your experiment.

Note: The Kit has been tested at logarithmically growing cultures of the following bacterial species: Bacillus cereus, B. subtilis, Clostridium perfringens, Escherichia coli, Klebsiella pneumoniae, Micrococcus luteus, Mycobacterium phlei, Pseudomonas aeruginosa, P. syringae, Salmonella oranienburg, Serratia marcescens, Shigella sonnei, Staphylococcus aureus and Streptococcus pyogenes. Agrobacterium tumefaciens, Edwardsiella ictaluri, Eurioplasma eurilytica, Lactobacillus sp., Mycoplasma hominus, Propionibacterium sp., Proteus mirabilis and Zymomonas sp.

Note: The following is the recommended protocol for bacterial staining. The protocol only provides a guideline and should be modified according to the specific needs.

1. Add equal volume of MycoLight™ Green (Component A) and Propidium Iodide (Component B) in a microfuge tube and mix them well.

   Note: Prepare the working solution fresh each time. 

1. Grow bacteria in any appropriate medium. Best results for healthy bacteria are obtained from log-phase cultures. Dilute the bacterial culture to ~ 106 to 108 cells per mL in 0.85% NaCl or appropriate buffer. Prepare sufficient suspension to provide 500 µL per test for flow cytometry or 100 µL per test for 96-well plate.

   Note: Remove traces of growth medium before staining bacteria. A single wash step is usually sufficient to remove significant traces of interfering media components from the bacterial suspension. Phosphate wash buffers are not recommended because they appear to decrease staining efficiency. 

2. Add 4 µL of the dye working solution (250X) to each mL of the bacterial suspension. Mix well and incubate at room temperature for 15 minutes. Protect from light. 

3. The stained bacterial cells can be analyzed by a fluorescence microscope, fluorescent microplate reader or flow cytometry. 

4. The fluorescence from both live and dead bacteria may be viewed simultaneously with any standard fluorescein long pass filter set. Alternatively, the live (green fluorescent) and dead (red fluorescent) cells may be viewed separately with FITC and Texas Red filter sets.