*2.9. Antifungal Susceptibility Testing*

The *in vitro* antifungal susceptibility was evaluated using extracts, rutin, and carvacrol (Sigma Aldrich, St. Louis, MO, USA). To evaluate the minimal inhibitory concentration (MIC) of the extracts and compounds, a broth microdilution method was performed according to a standardized protocol for yeasts [35].

The assay was carried out with *C. albicans* ATCC 24433, *C. albicans* ATCC 10261, and *C. albicans* ATCC 90028 coming from the American Type Culture Collection (ATCC, Rockville, MD, USA). *Candida* strains were grown on Sabouraud dextrose agar at 37 ◦C for 24 h. Then, cell suspensions of the strains were prepared in a RPMI 1640 medium (Sigma-Aldrich, Milan, Italy) buffered to pH 7.0 with 0.165 mmol L–1 3-(*N*-morpholino)propanesulfonic acid (MOPS). The final concentration of the inoculum was 1 <sup>×</sup> <sup>10</sup>3–5 <sup>×</sup> <sup>10</sup><sup>3</sup> cells mL–1. The extracts were dissolved in DMSO and diluted 100 times in RPMI 1640 broth. Ten concentrations ranging from 1000 to 1.9 μg mL–1 for the extracts and from 64 to 0.125 μg mL–1 for the compounds were tested against *Candida albicans* strains in 96-well round-bottom microtitration plates. The MIC50, MIC90, and MIC100—the lowest concentrations of extracts that caused growth inhibitions ≥50%, ≥90%, and 100%, respectively—were evaluated. Data was reported as the median of the MIC.
