Scanning Electron Microscopy of Candida albicans

For SEM observations, C. albicans were incubated on sterile polyvinyl chloride coverslips (with a thickness of 0.13–17 mm and a diameter of 22 mm) in 12-well microtiter plates (Corning Costar; Sigma-Aldrich) with FEN (MIC value), fluconazol (MIC value) and untreated cells for 24 hr at 37℃. Thereafter, the coverslip were washed twice with sterile PBS (0.1M and pH 7.2) and placed in a primary fixative solution (glutaraldehyde 0.15 M 2.5% [vol/vol] in PBS) for 12 hr at 4℃. The samples were then washed with sterile PBS, then treated with the secondary fixative (osmium tetroxide [OsO₄ 1% w/v]) for 1 hr. The samples were subsequently washed with distilled water, dehydrated in an ethanol series (70% for 10 min, 95% for 10 min, and 100% for 20 min) and air dried overnight in a desiccator [20 (link)21 (link)]. The coverslip was coated twice with platinum vanadium using a sputter ion (Bal-Tec SCD 005; BAL-TEC, Balzers, Liechtenstein), followed by bonding to carbon double-side tape for examination by SEM (JEOL JED-2300; JEOL, Tokyo, Japan).