2.9. Antifungal Drug Susceptibility Assays

The susceptibility to antifungal drugs was performed as described in the M27-A3 yeast microdilution test, standardized by the CLSI [44]. For the water-soluble antifungal drugs, a stock solution of 64 μg/mL was prepared using RPMI 1640 as diluent. For amphotericin B, a stock solution of 16 μg/mL was prepared using DMSO as diluent. In all cases, 10-fold dilutions series were prepared for incubation with fungal cells. A 5 h culture of C. albicans was washed with PBS and the O.D._{530 nm} adjusted to 0.5. Then, a 1:1000 dilution was prepared with RPMI 1640 medium (supplemented with glutamine and without sodium bicarbonate, buffered with 0.164 M morpholino propanesulfonic acid, adjusted to pH 7 and with 0.2% (w/v) glucose) and this dilution was used to inoculate 100 μL in flat-bottom 96-well plates. As controls, wells only with the medium were included. The antifungal dilutions were included in aliquots of 100 µL per well and plates were incubated at 37 °C for 24 h and gently shaken. Then, the absorbance of the plates was measured at 490 nm and 530 nm with a Multiskan™ FC microplate photometer (Thermo).