The assay was performed using Staphylococcus aureus (ATCC 29213), an MSSA strain. Bacterial cells were cultivated in solid TSA broth (tryptone soya) at 36 °C and stirred at 200 rpm. S. aureus (106 cells) was exposed to LCC at its MIC value (12.5 µg/mL) in DMSO for 15 min in a 1.5 mL microcentrifuge tube in a total volume of 100 μL of TSA medium. Next, 900 μL of the TSA medium was added to the tube to dilute the compound and stop the reaction [59 (link)]. Cells were stained with the Live/Dead BacLight Kit (Thermo-Scientific® L7012, Carlsbad, CA, USA), which is composed of two nucleic acid dyes: SYTO9, which stains all cells, and propidium iodide, which penetrates cells with damaged membranes [60 (link)]. The stained cells were immobilized on agarose-covered slides as described by Martins [61 (link)]. Untreated S. aureus cells with intact membranes were used as negative controls, and S. aureus cells exposed to nisin (Sigma-Aldrich® N5764, St. Louis, MO, USA) were used as positive controls for membrane damage [62 (link)]. Microscopic analyses were carried out using an Olympus BX-61 microscope, equipped with a monochromatic OrcaFlash-2.8 camera (Hamamatsu, Japan), and the software CellSens Dimension Version 11 (Olympus).
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