Susceptibility of the Two Aspergillus Species to Cell Wall Perturbing Agents and Antifungals

(i) Cell wall perturbing agents: Congo red (CR) and Calcofluor white (CFW) were added to malt-agar medium (1 ml/well into a 6-well plate, at 0–250 µg/ml concentrations). Conidia (5,000/well) were seeded on this agar medium and incubated at 37°C for 24 h. Following, the radial growths were measured. For Menadione (starting at 160 µM with two-fold dilutions) and H₂O₂ (starting at 6 µM with two-fold dilutions) assays, resazurin method was followed (Clavaud et al., 2012). (ii) Antifungal drug susceptibility assay: Performed by resazurin method as described earlier (Clavaud et al., 2012). Briefly, yeast-extract medium (1%; 9 ml of the medium containing 9 μl Tween-20) was mixed with 3 ml of resazurin (1 mg/ml dissolved in Milli-Q water) and 1 ml of conidial suspension (containing 1x10⁴ conidia/ml); 130 μl of this mixture was seeded into each well in 96-well culture plate. Two-fold dilution of caspofungin (starting at 0.72 μg/well) or nikkomycin (starting at 1.92 μg/well) were added to these well, the culture-plates were incubated at 37°C for 30 h and the optical densities were measure at 569 nm using Tecan plate-reader Infinite 200 PRO (Tecan Group Ltc., Switzerland).