Both P. aeruginosa and S. aureus cells obtained from overnight cultures were at the end of the exponential growth phase and the beginning of the stationary phase, and thus used for preparation of live and dead cells. The cultures (50 mL) were centrifuged (7000 g, 10 min, 22°C) and the pellet was resuspended in 1 mL 0.9% NaCl solution. 0.5 mL of the cell suspension was added to 20 mL of 70% isopropanol to obtain dead cells and 0.5 mL to 20 mL of 0.9% NaCl to obtain live cells. Cells were incubated for 1 hour at room temperature before being spun down and washed with 0.9% NaCl once. OD595 was adjusted to 1.2 for P. aeruginosa with a total cell number of 2.0 × 109 cells per mL and 2.5 for S. aureus with a total cell number of 2.0 × 108 cells per mL. Further dilution was done to reach indicated ODs. Mixtures of different proportions of live/dead cells were prepared prior to staining.
Viable cell numbers were determined by spotting 5 μl of a 1:5 dilution series on Tryptic Soy Agar.
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