2.5. Determination of Biofilm Formation

Visual tube method (qualitative method): Assessment of biofilm formation by C. tropicalis was performed as previously described [25]. A volume of 5 mL Sabouraud dextrose broth (SDB), supplemented with 8% glucose, in screw-capped conical polystyrene tubes was inoculated with an overnight growth of C. tropicalis and incubated at 35 °C for 48 h without agitation. The tubes were then decanted, washed twice with distilled water, and dried. The dried tubes were stained with 1% crystal violet, and the excess stain was washed with distilled water. A development of a visible film, lining the walls and bottoms of the tubes, was considered as biofilm formation. Biofilm production was scored as negative (−), weak (+), moderate (++), or strong (+++). Staphylococcus epidermidis ATCC 35984 and C. albicans ATCC 90028 were used as positive and negative controls, respectively.

Spectrophotometric microplate method (semiquantitative method): In the beginning, 200 μL of a standardized cell suspension (10⁷ cells/mL in SDB) was transferred into each well of a microtiter plate with a pipette. After 48 h of incubation, the total biofilm biomass was quantified via crystal violet staining [26]. The medium was fully aspirated, and the non-adherent cells were removed by washing the biofilms once with 200 μL of PBS. Then, the biofilm was fixed with 200 μL of methanol (100% v/v), which was removed after 15 min of contact. Subsequently, the microplates were left to dry at room temperature, and 200 μL of crystal violet (0.1% v/v) was added to each well and incubated for additional 5 min. Afterwards, the wells were gently washed twice with 200 μL of sterile distilled water, and 200 μL of acetic acid (33% v/v) was added to release and dissolve the absorbed stain. Absorbance of the obtained solution was read in triplicate in a microplate reader at 620 nm (Tecan Infinite F50 Microplate Reader; Tecan Group Ltd., Mannedorf, Switzerland).

On the basis of the cut-off optical density (ODc), the biofilm formations of different strains were classified into groups. For test microplates, the ODc was defined as three standard deviations above the mean OD of the negative control. Isolates having an OD lower than the cut-off, less than 2 times the cut-off, and 2–4 times the cut-off were considered as non-adhered (non-biofilm forming), weakly adhered, moderately adhered, and strongly adhered, respectively [27]. ODc: = 0.06, 2× = 0.12, 4× = 0.24.