Broth microdilution antifungal testing

For A. apis, the broth microdilution assay suggested by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) was applied. Stock compound solutions were prepared in dimethyl sulfoxide (DMSO) with 10 mg/mL concentration. The compound was serially two-fold diluted with Sabouraud dextrose (SD; TaKaRa, Japan), Sabouraud dextrose+0.2% yeast extract (SDYE), YPD (TaKaRa) or RPMI medium (RPMI 164, Sigma, USA) without sodium bicarbonate and with L-glutamine buffered to pH 7.0 with 0.165 M morpholinopropanesulfonic acid and added with 18 g of glucose per liter to make a final concentration of 2% (11 -15). One hundred microliter of the medium containing each compound ranging from 400-0.39 μg/mL were added to the 96-well flat-bottomed microtitration plates (SPL, Korea). The last well was used for sterility and growth controls. One hundred microliter of the mold inoculum suspension containing 1×10⁵ spores/mL were added to each well of microdilution plates. Plates were incubated at 35°C for 24 and 48 h. Minimum inhibitory concentration (MIC) values were defined as the lowest concentration of drug that completely inhibited cell growth.

For aspergillus species, the same broth microdilution assay described for A. apis antifungal testing was used, but with RPMI 1640 medium (14,16).

For Candida and Saccharomyces species, the broth microdilution assay recommended in the NCCLS document M27-A was used as described previously (15 -18). Each compound (concentration ranged from 200-0.2 μg/mL) was serially two-fold diluted with 100 μL of RPMI 1640 medium. The diluted drug was dispensed into 96 well round bottom microdilution plates (SPL) and 100 μL of yeast inoculum was added with a concentration of 1×10⁴ cells/mL to each well of microdilution plates. Plates were incubated at 35°C for 24 and 48 h. Tests were performed at least three times.