To monitor growth under cell wall stress, serial dilutions of conidia were spotted onto agar plates that were supplemented with varying concentrations of caffeine (CAFF), calcofluor white (CFW), congo red (CR), anidulafungin, (AF), caspofungin (CASP), sodium dodecyl sulfate (SDS), fluconazole (FLUC), and ethylenediaminetetraacetic acid (EDTA), and grown for 48 hr. Alternatively, to assess their sensitivity to nikkomycin Z, 1 × 105 conidia of each strain were grown on 1 ml of solid media in 24-well plates. Endoplasmic reticulum (ER) stress in the presence of DTT (dithiothreitol) was likewise tested in liquid YG culture, and brefeldin A (BFA) and tunicamycin (TM) were tested in solid media, as previously described for A. fumigatus (Richie et al. 2009 (link)). The plates were incubated for 2–3 d at 37°, and the extent of vegetative growth was used as a relative indicator of sensitivity. For the experiments on solid MM supplemented with 1.2% sorbitol, serial dilutions of conidia ranging from 1 × 106 to 1 × 103 were spotted onto agar plates. To evaluate the oxidative stress tolerance, 1 × 105 conidia were inoculated into 24-well plates containing 1 ml of liquid MM and varying concentrations of menadione, paraquat, or T-butyl hydroperoxide. Sensitivity to the oxidative damage generated by H2O2 was tested by inhibition zone assay on MM agar plates, as described by (Valiante et al. 2008) (link). Antifungal susceptibility by E-test diffusion assay was performed essentially as described by Richie et al. (2009) (link).
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