For the antifungal bioassays, two strains of Aspergillus fumigatus were used: Aspergillus fumigatus (ATCC 204305), and Aspergillus fumigatus (human clinical isolate). The organisms are deposited at the Mycological Laboratory, Department of Plant Physiology, Institute for Biological Research “Siniša Stankovic,” Belgrade, Serbia.
The micromycetes were maintained on malt agar and the cultures were stored at 4°C and sub-cultured once a month. The antifungal assay was carried out by a modified microdilution technique [45 (link),46 (link)] in order to determine the minimum inhibitory (MIC) and minimum fungicidal concentrations (MFC) of the examined compounds. Briefly, the fungal spores were washed from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween 80 (v/v). The spore suspension was adjusted with sterile saline to a concentration of approximately 1.0×105 in a final volume of 100 μL per well. The examined compounds were dissolved in 5% of DMSO, serially diluted in broth malt medium after which fungal inoculum was added. The microplates were incubated for 72 h at 28°C. The lowest concentrations without visible growth (in a binocular microscope) were defined as MICs. The fungicidal concentrations (MFCs) were determined by serial sub cultivation of 2 μL of the wells content into microtiter plates containing 100 μL of broth per well and further incubation for 72 h at 28°C. The lowest concentration with no visible growth was defined as MFC indicating 99.5% killing of the original inoculum. The commercial antifungals econazole and ketoconazole were used as positive controls.
The micromycetes were maintained on malt agar and the cultures were stored at 4°C and sub-cultured once a month. The antifungal assay was carried out by a modified microdilution technique [45 (link),46 (link)] in order to determine the minimum inhibitory (MIC) and minimum fungicidal concentrations (MFC) of the examined compounds. Briefly, the fungal spores were washed from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween 80 (v/v). The spore suspension was adjusted with sterile saline to a concentration of approximately 1.0×105 in a final volume of 100 μL per well. The examined compounds were dissolved in 5% of DMSO, serially diluted in broth malt medium after which fungal inoculum was added. The microplates were incubated for 72 h at 28°C. The lowest concentrations without visible growth (in a binocular microscope) were defined as MICs. The fungicidal concentrations (MFCs) were determined by serial sub cultivation of 2 μL of the wells content into microtiter plates containing 100 μL of broth per well and further incubation for 72 h at 28°C. The lowest concentration with no visible growth was defined as MFC indicating 99.5% killing of the original inoculum. The commercial antifungals econazole and ketoconazole were used as positive controls.
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