5.6. Plasma Membrane Permeability

Plasma membrane integrity was assessed as described by Suchodolski et al. [41]. Briefly, C. albicans and C. dubliniensis cells were harvested by centrifugation (10 min, 956× g) in the stationary phase and washed twice with PBS, followed by 2 h of incubation in PBS (1 × 10⁶ CFU/mL) with different concentrations of CTX and CTX-containing mouthwash (1.5 and 47 µg/mL). Cells were then washed twice with PBS, resuspended in 200 µL PI (6.0 µM), and incubated for 5 min, at room temperature. After incubation, treated cells were washed twice again with PBS. The pellets were resuspended in 0.25 mL of PBS and 4 µL of samples were visualized with a LEICA^® DM-2000 optical microscope coupled with a LEICA^® DFC-280 camera and LAS^® version 3.3.0 software (Leica Microsystem, Wetzlar, Germany). Images were captured at 40× magnification, under standardized microscopy parameters. The Plugin “Cell Counter” from ImageJ software was used for counting of cells on 3 images from 3 different experiments. The number of cells were counted in brightfield in 10 random 40× fields, until a total of 100 cells. Each image was independently analyzed for the total area (20 mm²) of darkfield/brightfield and results expressed by the percentage of stained cells. The percentage of cell death was assessed by measuring the area ratio of the fluorescent regions to the dark regions (a mask of the bright field was subtracted from the fluorescent image to isolate the objects under analysis). Cell death control (positive control) consisted of cells treated with 1% SDS, while cell viability control (negative control) consisted of cells mock-treated with PBS alone.