Scanning Electron Microscopy of C. albicans

Cell cultures prepared as described in “Preparation ofC. albicanscell culture with Venetin-1 and fluconazole” section of Materials and methods were centrifuged at room temperature at 2500×g for 10 min. The supernatant was discarded and the pellet was suspended in 70 µL of a fixing solution (10 ml phosphate buffer pH = 7, 10 ml glutaraldehyde 10%, 200 mg saccharose) and incubated for 2 h at room temperature. Then, the fixing solution was discarded and 0.1 M phosphate buffer pH = 7 was added. After centrifugation (2500×g, 30 min, room temperature), a 1.5% OsO₄ solution was added and incubated with the cells for 30 min at room temperature. After that, the fungal cells were centrifuged and the supernatant was removed. Then, the cultures were washed with phosphate buffer and centrifuged. The cells prepared in this way were dehydrated in a series of acetone dilutions (15%, 30%, 50%, 70%, 100%), transferred onto SEM stages with carbon discs, and dried in a desiccator for 24 h in the presence of roasted silicone gel (for 2 h at 180 °C). Afterwards, the stages with the probes were coated with a gold layer using a K550X sputtering machine (Quorum Technologies, United Kingdom) and observed with the use of a scanning electron microscope Tescan Vega 3 (Tescan Orsay Holding, Czech Republic)^{39 (link)}.