Combine one volume of DMAO and two volumes of EthD-III in a microcentrifuge tube, mix thoroughly and add 8 volumes of 0.85% NaCl solution to derive 100X dye solution.
For each 100 uL of bacterial suspension, add 1 uL of the dye mixture.
Mix thoroughly and incubate at room temperature in the dark for 15 minutes.
Mount 5 uL of the stained bacterial suspension on a slide with an 18 mm square coverslip.
Observe under a fluorescence microscope. The fluorescence from both live and dead bacteria may be viewed simultaneously with any standard FITC long-pass filter set. Alternatively, the live (green fluorescent) and dead (red fluorescent) cells may be imaged separately with FITC and Cy®3 or Texas Red® band-pass filter sets.