A Simple Antifungal assay for Testing Actinomycetes and Other Microbial Extracts.

Abstract

Antifungal assay in vitro is a useful tool for the characterization of biological activity of microbial extracts. Here we describe a simple in vitro test at two final extract concentrations that allows long-term storage of the plates containing dry extracts before using. The assay protocol is described for two fungal strains, a unicellular yeast, with clinical interest (Candida albicans), and a sporulated and phytopathogenic filamentous fungus (Botrytis cinerea). They could serve as models for adapting other filamentous/yeast-like fungi.Plates are prepared by placing 100 and 10μg, respectively, of the organic extracts in microtiter 96-well plates, where the test will be performed. The assay develops by adding 200μL of a spore suspension 104spores/mL for B. cinerea and 106cells/mL for C. albicans in Sabouraud medium.After the incubation of the plates at 25°C, for 2days for C. albicans and 5days for B. cinerea , the growth of the fungal targets is evaluated in a plate reader for unicellular yeast , or visually under the microscope for filamentous fungi. If visually evaluated, observed growth can be assigned to different categories by comparison with growth control and inhibition control. Inhibition effect on C. albicans at eight concentrations of amphotericin B (8-4-2-1-0.5-0.25-0.125-0.00625μg/mL) or B. cinerea exposed of eight concentrations of iprodione (100-50-10-5-1-0.5-0.1-0.05μg/mL) are used as inhibition controls, respectively.