A 96-well epifluorescence assay for rapid assessment of compounds inhibitory to Candida spp

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Abstract
A rapid method for the screening of potential antifungal compounds was developed. A variety of compounds were tested against regenerating protoplasts of Candida spp. in a microtiter format. The degree of cell wall formation was assessed by staining with Cellufluor (Polysciences, Inc. Warrintgon, Pa.), a fluorochrome with known affinity for chitin, followed by determination of fluorescence by using a Dynatech Microfluor reader (Dynatech Laboratories, Inc., Alexandria, Va.). Compounds with known activity against the cell wall or cytoplasmic membrane of fungi inhibited wall synthesis in a concentration-dependent fashion. Treatment with 5-fluorocytosine, however, resulted in no inhibition. In general, protoplasts of C. albicans regenerated more quickly and were more sensitive to the compounds tested than protoplasts of C. tropicalis and C. parapsilosis. While the described method is not specific for a given class of antifungal agents, it may prove useful for testing large numbers of compounds quickly.