K550x sputtering machine

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)}.

Scanning electron microscopy (SEM) was used to visualize the algal and fungal cells. The cells were washed with 0.1 M phosphate buffer, pH 7.0, followed by fixation in 4% glutaraldehyde. After 4 h of incubation at room temperature, the cells were washed again in the same buffer, suspended in a 1% OsO₄ solution, and incubated for 30 min. Dehydration was carried out using increasing concentrations of acetone (successively 30%, 50%, 70% and twice 100% for 30 min). The cell suspension was placed on microscope stages covered with carbon discs. The samples were then dried on silica gel in a desiccator for 24 h. The silica gel was previously prepared by incandescence in an oven at 121 °C for 2 h. The surface of the tables with algal cells was gold coated with a K550X sputtering machine (Quorum Technologies, United Kingdom). Samples with algal cells were then analyzed using a Tescan Vega 3 scanning electron microscope (Tescan Orsay Holding, Czech Republic)^{28 (link)}. The A. nordenskioeldii cells were not dried, but when samples immersed in acetone were applied to the carbon discs, they were directly inserted into the vacuum chamber of the Quanta 3D FEG high-resolution scanning electron–ion microscope (FEI Company, Hillsboro, USA) and immediately analyzed to minimize the negative effects of drying.