Membrane Permeability Assay

Cytoplasmic membrane damage was assayed with the Live/Dead BacLight bacterial viability kit (Invitrogen L7012) according to the manufacturer’s instructions (Horváth et al., 2015). Bacteria (S. enterica and L. monocytogenes) were grown to mid-logarithmic phase and the optical density was set to OD₆₀₀ = 0.1 in LB and washed gently with PB buffer. Bacterial cells resuspended in PB buffer were treated with 10 μM NCR247, 5 μM NCR335, and 5 μM PMB at room temperature for 60 min. Untreated cells served as negative control. Then the cells were stained with 7.5 μM SYTO-9 and 30 μM propidium iodide (PI). After 15 min incubation in dark the cells (5 μL) were spotted on microscope slide and covered with 2% (w/v) agar slices and observed with Olympus Fluoview FV 1000 confocal laser microscope with 60× magnification objective. 488 nm laser was used for excitation, and emission was detected at 500–530 nm for SYTO9. Excitation and emission wavelengths were 543 and 555–655 nm for PI, respectively. Sequential scanning was used to avoid crosstalk of the fluorescent dyes.