Required Equipment and Materials10 μl, 1000 μl pipettes, incubator, Microscope (blue excitation filter and red emission filter) or flow cytometer (488 nm blue laser)

Fig. 1 Cell staining mechanism

Staining procedure1. Allow CFDA solution to stand at room temperature for 30 minutes to thaw. Solution should be protected from light.2. Resuspend the organism with an appropriate buffer (phosphate buffer, saline, etc.) ^a) and adjust the number of cells to 106 cells/mL(flow cytometry) or 108-109 cells/mL(microscopy).3. Add CFDA solution into the microbial cell suspension and vortex gently to mix. Use 5 μl for flow cytometry and 15 μl for microscopy analysis. The maximum wavelengths of the dye are 493 nm for excitation and 515 nm for emission.4. Incubate the microbial cell at 37ºC for 5 minutes ^b).5. Fix the microbial cell by addition of formaldehyde (1-4% final concentration).6. Remove the buffer by filtration or centrifugation, and resuspend with buffer.7. Analyze the stained-cells by a flow cytometer or a microscope.

a) Gram-negative bacteria tend to exhibit lower fluorescence intensity than Gram-positive bacteria because of their cell structure (outer membrane impedes penetration of CFDA). For the staining of Gram-negative bacteria, use 0.1 M phosphate buffer, 0.9 M NaCl, 0.5 mM EDTA, pH 8.5.b) If CFDA staining is not sufficient with 5 minutes of incubation, increase the incubation time.

1. N. Yamaguchi, et al., Flow cytometric analysis of bacterial respiratory enzymatic activity in the natural aquatic environment. J Appl Microbiol. 1997;83:43-52.2. M. Kawai, et al., Rapid Enumeration of Physiologically Active Bacteria in Purified Water Used in the Pharmaceutical Manufacuturing Process. J Appl Microbiol. 1999;86:496-504.

Staining Data

Fig. 2 B. cereus stained with CFDA and DAPI (left). S. epidermidis stained with CFDA and PI (right).