MycoLight™ Fluorescence Live/Dead Bacterial Imaging Kit

Protocol summary

1. Prepare 100X MycoLight™ 520 stock solutions
2. Prepare bacteria samples
3. Add MycoLight™ 520 and Propidium iodide
4. Incubate bacteria samples with MycoLight™ 520 and Propidium iodide at 37°C for 5-10 minutes or room temperature for 60 minutes in dark
5. Analyze sample by fluorescence microscope with FITC and TRITC filter sets

Important notes
Thaw all the kit components at room temperature before starting the experiment

MycoLight™ 520 stock solution (100X):
Add 100 µL of DMSO (Component D) into the vial of MycoLight™ 520 (Component A). Note: Store stock solution at -20°C, avoid light and repeat freeze-thaw cycles.

Preparation of bacterial sample

1. Prepare bacteria sample with concentration of 10⁶ to 10⁸ cells/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 Assay Buffer (Component C).
   
   
3. Dead bacteria can be prepared by treating with 70% ethanol for 30 minutes followed by 60 minutes of boiling.

Staining protocol

The following is a suggested protocol and should be optimized with different bacterial strain or other specific needs. An optional washing step can be added before imaging if higher background is observed.

1. Add 1 µL of the 100X MycoLight™ 520 stock solutions and 1 µL of 100X Propidium iodide (Component B) to 100 µL of the bacterial sample in Assay Buffer.
   
   
2. Mix well and incubate in dark for 5-10 minutes at 37°C or 60 minutes at room temperature for optimum staining results.
   
   
3. Monitor fluorescence of bacteria with a fluorescent microscope through FITC (Ex/Em = 488/530 nm) and TRITC (Ex/Em = 540/620 nm) channel.