Sabouraud agar plates

Colistin time-kill study was performed, as previously described [22 (link)]. 9 ml of the fungal suspension was adjusted to a 0.5 McFarland turbidity. One ml of the adjusted fungal suspension was added to 9 ml of either RPMI-1640 medium, as a control, or to a solution of growth medium supplemented with an appropriate concentration of antibiotic solution. The colistin concentrations in the resulting solutions were 0.5, 1, 2, 4, 8, 16, and 32 times the MICs for the tested isolates. Then, the tubes were incubated at 37 °C on an orbital shaker. At 0, 6, 12, 24, 36, and 48 h following the introduction of the tested isolate into the solutions tubes, 100 μl aliquots were taken from each test solution. Different serial dilutions were performed on these aliquots, and a 10 μl aliquot from each dilution was streaked on Sabouraud agar plates (Biomérieux, France) and incubated for approximately 24 h for colony count determination. Then, the Minimum fungicidal concentration (MFC) was determined as the lowest antibiotic concentration leading to no significant growth or less than three colonies on Sabouraud agar plates in comparison to the growth control. The experimental data was analyzed using the GraphPad Prism 5.3 software (GraphPad Inc., San Diego, CA, USA) to obtain time-kill curves.

Fungal adhesion tests were performed by incubating rectangular samples that measured 10 × 10 × 2 mm stored in 500 ± 20 mL of distilled water (Avantor, Gliwice, Poland) for 24 h and 30 days at 37 ± 1 °C. The water was changed every 3 days. After storage, all samples were placed for 18 h in 1 mL of C. albicans ATCC 10231 suspension ~1.5 × 10⁵ CFU/mL in tryptone water at 37 °C and the methodology described in the work [34 (link)] was then used with modifications regarding the substrate used to determine cell adherence. The samples were vortexed in 1 mL of sterile water, 100 µL of undiluted obtained suspensions were seeded onto Sabouraud agar plates (bioMerieux) (Marcy l’Etoille, Lyon, France) and incubated at 37 °C for 24 h. The number of cells was measured by counting the colonies (automatic colony counter ProtoCOL 3 PLUS, Synbiosis, Frederick, MD, USA).

Rectangular samples that measured 10 × 10 × 2 mm were stored in 500 ± 20 mL of distilled water (Avantor, Gliwice, Poland) for 24 h, 7 days and 30 days at 37 ± 1 °C. The water was changed every 3 days. After storage, the samples were dried at 37 ± 1 °C for 48 h in desiccators containing dried silica gel. The antifungal properties tests were carried out based on the previously described method [33 (link)]. Standard strains of Candida albicans ATCC 10231 (C. albicans) were used. Sterilized square samples were immersed individually in 1 mL of C. albicans suspensions containing approximately 1.5 × 10⁵ CFU/mL (CFU, colony-forming units) in tryptone water. C. albicans was tested as a positive control, pure tryptone water was tested as a negative control. The samples were incubated in a shaking incubator for 17 h at 35 °C for C. albicans and then 20 μL of suspension was seeded in Sabouraud agar plates (bioMerieux, (Marcy l’Etoille, Lyon, France). Cultured plates were incubated at 35 °C for 17 h, the colonies were counted and composite antifungal efficacy of the compounds was calculated: $$\mathrm{A}\mathrm{F}\mathrm{E}=\frac{{\mathrm{V}}_{\mathrm{c}}-{\mathrm{V}}_{\mathrm{t}}}{{\mathrm{V}}_{\mathrm{c}}}\times 100\%$$
where V_c is the number of viable colonies of the positive control (BLANK), CFU/mL, V_t is the number of viable colonies of the test specimen, CFU/mL.