MycoLight™ Flow Cytometric Live Bacteria Assay Kit

Protocol summary

1. Prepare 100X dye stock solution.
2. Prepare bacteria samples.
3. Incubate bacteria samples with MycoLight™ 520 at 37°C for 5-10 minutes or room temperature for 60 minutes in dark.
4. Analyze sample by flow cytometry with FITC channel.

Important
Thaw one of each kit component at room temperature before starting the experiment.

1. MycoLight™ 520 stock solution (100X):
Add 100 µL of DMSO (Component C) into one vial of MycoLight™ 520 (Component A) to make 100X stock solution.

1. Prepare bacteria sample with concentration in range of 10⁶ to 10⁸ cells/ml. Grow bacteria into late log phase in appropriate medium. Remove medium by centrifugation at 10,000 x g for 10 minutes and re-suspend the pellet in Assay Buffer (Component B).  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. Treat cells with test compounds as desired. Remove treatments by centrifugation at 10,000 x g for 10 minutes and re-suspend the pellet in appropriate amount of Assay Buffer (Component B) so the concentration of bacteria in the treated sample is the same as the live.  Note:  Determine the concentration of the bacterial culture before starting the treatment. Note: Dead bacteria can serve as negative control, it is recommended to kill bacteria with 70% ethanol for 30 min followed by 60 min of boiling. 
                                                                                                                                                                                                                                      
3. Example of Live/Dead bacterial mixtrue preparation: Mix seven different propotions of the bacterial suspensions as in Table 1 for a total volume of 200 µL for each sample.                                                                                                                                                                                                                                                   
4. Add 2 µL of the 100X MycoLight™ 520 stock solution to 200 µL of the bacterial sample in Assay Buffer.                                                                                                                                                                                                              
5. Mix well and incubate in dark for 5-10 min at 37°C or 60 min at RT for optimum staining results.                                                                                                                                                                                                   
6. Add 300 µL of Assay Buffer (Component C) to increase volume before analyzing the cells with a flow cytometer.                                                                                                                                                                       
7. Monitor fluorescence of bacteria with a flow cytometer through FITC channel (488/530 nm).  Note: To exclude debris, it is recommended to set the threshold of the flow cytometer as the following: FSC >10,000, SSC>5,000. Note: The efficiency of MycoLight™ 520 is highly strain dependent and the staining conditions should be optimized accordingly.

Table 1. Example Volumes of Live and Dead samples to mix to achieve various Live/Dead ratios.