Evaluating Fungal Cell Permeability

The assessment of PM permeability was performed as described previously [53 (link)], with modifications. Briefly, 3 mL of Candida spp. cell suspensions (OD₆₀₀ = 0.1) in PBS were mixed with ILs (concentrations equivalent to MIC₉₀ against each fungi). Samples were incubated for 2 h at 37 °C, washed with PBS and stained with PI to the final dye concentration of 6 × 10⁻⁶ M for 5 min. Then, cell suspensions were washed with PBS and observed under the Zeiss Axio Imager A2 microscope equipped with the Zeiss Axiocam 503 mono microscope camera and Zeiss HBO100 mercury lamp. The percentage of permeabilized cells was evaluated by counting PI positive cells out of one hundred in three independent repetitions for each experiment.