Approximately 10 μl of the suspensions were then mounted on glass slides and cells were visualized by LM. Chitin assembly analysis To discriminate between hyphae and pseudohyphae cell wall chitin assembly was assessed with CFW staining. Cultures were diluted to 1 × 107 cells/ml and to 1 ml of cells suspension

was added 100 μl of CFW (300 μg/ml). Samples were incubated at room temperature for 5 min and 5 μl of each suspension placed on glass slide for microscopic inspection. The dye fluoresces when bound to chitin, primarily, and to glucans, staining cell wall and septa. Representative images were obtained by LM. Adherence to agar and invasion capacities Equal volumes of young cultures of each strain were diluted to 1 × 107 cells/ml, and 1 ml of cells suspension was spotted onto YPD medium agar plates. Solid cultures Caspase activity assay were allowed to grow at 37°C for 5 days. The cells on click here the surface were removed by washing under

running water [45, 46] and then visualized by LM. Inspection of agar invasion was performed by visualization of longitudinal cuts displaying the aerial and internal agar/growth boundaries by LM. Light microscopy Microscopy assessments were done in a Leica Microsystems DM-5000B epifluorescence microscope, with appropriate filter settings. Images were acquired through a Leica DCF350FX digital camera and processed with LAS AF Leica Microsystems software. Cell wall hydrophobicity MATH test was utilized to evaluate cell wall hydrophobicity as described by Rosenberg [77]. Yeast cells were harvested in stationary phase and washed twice with PBS pH 7.0. A yeast cell suspension displaying an optical density at 600 nm (OD600 nm) between 0.4-0.5 was prepared in PBS (A0). In an acid washed spectrophotometer glass tubes, 3 ml of the prepared yeast suspension was spread and overlaid by 0.4 ml of a hydrophobic Thymidylate synthase hydrocarbon, hexadecane. After vigorous vortexing,

phases were allowed to separate for 10 min at 30°C and OD600 nm of the aqueous phase was measured (A1). The percentage of hydrophobicity was calculated as follows: hydrophobicity (%) = [1-(A1/A0)] × 100. Assays were performed in triplicate and statistical analysis (T-test, p < 0.05) of the results was carried out. Adhesion and biofilm formation Adhesion and biofilm formation ability was assessed through quantification of total biomass by crystal violet (CV) staining as described before [47–49]. For this, standardized cell suspensions (1 ml containing 1 × 107 cells/ml in YPD) from young cultures were placed into selected wells on polystyrene plates (Orange Scientific, Braine-l’Alleud, Belgium) and incubated at 37°C in a shaker at 120 rev/min. Adhesion ability was measured after 2 h of incubation and biofilm formation ability was inspected after 24 h and 48 h. Regarding the 48 h sample, an extra step was performed, at half period, i.e.