Determination of Membrane Permeabilization in C. tropicalis

To investigate the alteration of membrane permeabilization under protolichesterinic acid treatment, cell membrane impermeable fluorescent dye propidium iodide (PI) was used. C. tropicalis was treated with the concentrations of 0 (control), 0.5× MIC, MIC, and 2× MIC concentration of protolichesterinic acid in PDB with 1 × 10⁶ Candida cells/ml for 4 h. The amphotericin B treatment severed as the control. 2.5 mM H₂O₂ was used as the positive control. After incubation at 37°C for 3 h, the Candida cells were stained with 5 μg/ml of PI at 37°C for 30 min in the dark. The Candida cell membrane permeabilization was directly analyzed by confocal microscopy (BD Pathways). The fluorescence was further measured using a spectrofluorophotometer (Shimadzu RF-5301PC, Shimadzu, Kyoto, Japan) at an excitation wavelength of 535 nm and an emission wavelength of 617 nm. The test was performed trice (Li et al., 2015).