1. Allow PI solution a) to stand at room temperature for 30 minutes to thaw. Solution should be protected from light.
2. Resuspend the organisms with PBS(-) or saline and adjust the number of cells to 106 cells/mL(flow cytometry) or 108-109 cells/mL(microscopy).
3. Add 10 μl of PI solution into 1 mL of microbial cell suspension and vortex gently to mix. Formaldehyde-fixation may be carried out, if necessary.
4. Incubate the microbial cells at room temperature for 5 minutes.
5. Analyze the stained-cells with a flow cytometer or a microscope. The maximum wavelengths of the dye are 530 nm for excitation and 620 nm for emission.
a) Since PI may be carcinogenic, be careful when handling and disposing.
1. N. Yamaguchi, et al., Flow cytometric analysis of bacterial respiratory and enzymatic activity in the natural aquatic environment. J Appl Microbiol. 1997;83:43-52.
S. epidermidis stained with CFDA and PI (left).
E. coli stained with CFDA and PI (middle).
S. epidermidis stained with DAPI and PI (right).