M. Oryzae Lab Report

Radioactively Labeled Azole Import by M. oryzae
Radioactively labeled FLC (3H-FLC), (481 GBa/mmol, 13 Ci/mmol, 1 µCi/µL; 77 µM FLC) was custom synthesized by Amersham Biosciences. The drug concentration used during the import assay was well below the Minimum Inhibitory Concentration (MIC) for the strain (M. oryzae FLC MIC >32 µg/ml).
To directly measure azole intracellular accumulation in the fungal cell, we used 3H-FLC in our drug uptake assay designed for M. oryzae. Unless specifically noted, 5 ml YAD medium with 2% glucose in 50 mL conical tubes was inoculated with M. oryzae conidia and grown at 27° C, 180 rpm shaking, for 48 hr at which point they were mycelial masses or fungal balls, approximately 3 mm in diameter. The mycelial masses were transferred to 2 mL microcentrifuge tubes and washed by centrifugation and resuspension in fresh media three times using YNB complete (1.7 g yeast nitrogen base without amino acids or ammonium sulfate, 5 g ammonium sulfate per liter, pH 5.0) without glucose unless

The samples were filtered by vacuum over pre-weighed and wetted glass fiber filters (24 mm GF/C; Whatman; Kent, UK). The samples on the filters were rinsed with 5 ml stop solution to remove non-specific, cell surface binding of 3H-FLC. The filters with fungal balls were either allowed to dry for 24-48 hr or were baked in a drying oven for 15 minutes at 95° C. Each dried filter containing fungal balls was then re-weighed to obtain the dry mass of each fungal sample. The filters were finally transferred to 5 mL scintillation vials containing 3 ml of scintillation cocktail (Ecoscint XR, National Diagnostics, Atlanta GA). Radioactivity associated with the fungal sample on each filter was measured in a liquid scintillation analyzer (Beckman Coulter, LS 6500 multipurpose scintillation