Nunclon delta surface 96 wells microtiter plates for adherent cells

European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines for antifungal susceptibility testing of yeasts and molds with modifications for broth microdilution checkerboard procedures were used in this study [33 ,34 ]. Nunclon^TM delta surface 96-wells microtiter plates for adherent cells (Thermo Fisher Scientific, Darmstadt, Germany) were used. Drugs tested in combination were amphotericin B (Merck), and colistin (Merck). Final concentrations tested ranged from 0.03 to 16 µg/mL, and from 1 to 64 µg/mL for amphotericin B and for colistin, respectively. Before the addition of the inoculum, each well contained 100 µL of double strength RMPI medium with 1% (v/v) of DMSO.

Combination studies were performed using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines for antifungal susceptibility testing of molds with modifications for a broth microdilution checkerboard procedure. Experiments were carried out in Nunclon^TM delta surface 96-wells microtiter plates for adherent cells (Thermo Fisher Scientific, Darmstadt, Germany). The included drugs were isavuconazole (Pfizer, Berlin, Germany) and colistin (Merck). The stock solutions of isavuconazole (3200 µg/mL) and colistin (12,800 µg/mL) were prepared in dimethyl sulfoxide (DMSO) and sterile, distilled water, respectively. Drug dilutions were performed at four times the final concentration in double-strength RPMI medium. The combination was studied on a two-dimensional checkerboard with two-fold dilution. The final concentration for isavuconazole ranged from 0.03 to 16 µg/mL. The final concentration for colistin was 1 to 64 µg/mL. Fifty microliters of each concentration were distributed from row 1 to 8 for colistin and from column 1 to 11 for isavuconazole. Column 12 was used as growth control and contained 100 µL of double-strength RMPI medium with 1% DMSO.

The combination was tested using the EUCAST guidelines for antifungal susceptibility testing of yeasts³⁶ , with modifications for a broth microdilution checkerboard procedure. Experiments were performed using Nunclon delta surface 96-wells microtiter plates for adherent cells (Thermo Fisher Scientific, Darmstadt, Germany). The included drugs were isavuconazole (Pfizer, Berlin, Germany) and colistin (Merck). The stock solutions of isavuconazole (3200 µg/mL) and colistin (12,800 µg/mL) were prepared in DMSO and sterile, distilled water, respectively. The drug dilutions were performed at four times the final concentration in double strength RPMI medium. The combination was studied on a two-dimensional checkerboard with two-fold dilutions. The final concentrations were 0.002 to 0.5 µg/mL and 1 to 64 µg/mL for isavuconazole and colistin, respectively. Fifty microliters of each concentration were distributed from row 1 to 8 for colistin and from column 1 to 11 for isavuconazole. Column 12 was used a as growth control and contained 100 µL of double strength RPMI medium with 1% of DMSO.