A modified calorimetric assay for the antifungal susceptibility testing of filanientous fungi utilizing the tetrazolium salt MTT.



A modified assay for in vitro susceptibility testing of filamentous fungi in which the dye MTT is added during the incubation period was compared with the NCCLS method. The level of agreement was determined for clinical isolates of filamentous fungi including five strains each of Aspergillus fumigatus, A. flavus, Scedosporium prolificans, S. apiosperrnum, Fusarium solani and F. oxysporum. The activity of amphotericin B, miconazole, itraconazole, UR928, voriconazole, and terbinafine was tested by a broth microdilution method based on the NCCLS method. The MICs were determined on three different days by the NCCLS method and by the modified method using MTT. For the MTT method the inocula were made in RPMI 1640 containing MTT at a concentration of 0.2 mg/ml. The plates were incubated at 37`C and after 48 or 72h, the contents of the wells were removed and the formed formazan was extracted by isopropanol+5%HCI 1 M. The OD of each well was measured at 550 nm and the percentage of growth compared with that of the drug-free control was calculated. For the MTTassay the MIC-0 (the first optically clear well) and MIC-1 (the first well with slight growth of approximately 25%) were determined as the lowest concentration of drug showing growth .lower than 5% and 25% respectively and the results were compared with MIC-0 and MIC-1 obtained by NCCLS method. Possible toxic effects of MTT on the filamentous moulds was investigated but no toxic effect was observed at concentrations of

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5th Trends in Invasive and Fungal Infections
    • TIFI 5th