4.3.2. Antifungal Activity Testing

The assessment of antimycotic activity of beta-glucan derivatives was performed using our own microdilution method in a liquid culture medium based on European Committee on Antimicrobial Susceptibility Testing methodology for antifungal susceptibility testing for yeasts and molds (EUCAST DEFINITIVE DOCUMENT E.DEF: 7.3.2. Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts; 9.3.2. Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia forming moulds) and the Hancock Lab [48] procedure for cationic antimicrobial peptides.

Stock solutions were prepared in sterile distilled water to obtain concentrations of 10 g/L. The working solutions were prepared as a 2-fold dilution series of stock solutions in sterile distilled water.

Wells 1 to 10 of each column of the flat-bottom polypropylene 96-well microdilution plates were filled with 20 µL of the corresponding concentration of CIGTMAC, SBBGTMAC, and SBBGTMAC2, while each well of columns 11 and 12 was filled with 20 µL of sterile distilled water.

The inocula were prepared from fresh cultures (24 h for yeasts, 2- to 7-day-old for filamentous fungi) on solid agar media—Sabouraud glucose agar with chloramphenicol for Candida species and Trichopyton mentagropytes and Czapek yeast extract agar for molds. Yeasts inocula were prepared by suspending a few representative colonies in sterile distilled water. Filamentous fungi colonies were covered with approximately 5 mL of sterile water, then the conidia were rubbed with a sterile cotton swab and transferred to a sterile tube. The suspensions were homogenized with a gyratory vortex mixer, and the cell density was adjusted to 0.5 McFarland. The inocula were used for testing within 30 min of preparation.

The working suspensions were prepared by dilution of the primary inocula in RPMI-1640 medium with L-glutamine, without sodium bicarbonate, with 2% glucose, buffered to pH 7 with MOPS (0.165 mol/L). Next, 20-fold dilutions were prepared, which gave the following inocula densities: 0.5–2.5 × 10⁵ CFU/mL for yeasts and 1–2.5 × 10⁵ CFU/mL for filamentous fungi.

The microdilution plates were inoculated with 180 µL of the working suspensions, except sterility control wells, which contained 180 µL of microbial-free RPMI-1640 with L-glutamine, without sodium bicarbonate, with 2% glucose, buffered to pH 7 with MOPS (0.165 mol/L). The range of final concentrations of beta-glucan derivatives on microplates after the addition of the fungal suspension was 1.95–1000 mg/L.

The microdilution plates were incubated without agitation at 37 °C (yeasts) or 27 °C (molds, dermatophyte) in ambient air for 24–48 h.

The antimicrobial activity was estimated visually. Minimal inhibitory concentration (MIC) values of tested compounds were defined as no visible growth of fungi by eye.