engleski

High-Throughput Antifungal Screening : Method Optimalization

The aim of this research was to establish high-throughput primary antifungal screening method and to define test conditions for screening, using currently available classes of antifungal agents. It was found that inoculum size of 50-100 CFU/mL, selection of two different media (chemically defined and complex) work well for testing primary panel of microorganisms comprising Candida albicans, Candida non-albicans and Saccharomyces cerevisiae strains. Test was automatized for TECAN Genesis 150 robotic system and the read out on both spectrophotometer and nephelometer after incubation at 37°C could give reliable positive hits. These hits could be further evaluated for the mode of action in a fast and automatized time-kill assay which is carried on the same platform. For this purpose optimal inoculum was 5000 CFU/mL. However, same plates used for MIC determinations inoculated with 50-100 CFU/mL, could also serve to distinguish between fungicidal, fungistatic and growth retarding acting substances but in that case the longer incubation time is needed. The next aim would be to use the newly established methods to identify novel antifungal agents with unknown/new mechanisms of action.

high-throughput antifungal screening; nephelometry; assay optimization

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