Fluorescent probes for wall porosity and membrane integrity in filamentous fungi

Abstract

To assess the viability of moulds, quick detection methods using fluorescent probes were evaluated. A major hurdle in the use of intracellular probes can be the wall of filamentous fungi. We studied the permeability of the wall of germinating conidiospores with anionic ellipsoid FITC-dextran molecules and confocal scanning laser microscopy. In contrast to S. cerevisiae, filamentous fungi internalized FITC-dextran molecules up to 150 kDa (Stokes radius of 8–9 nm). We concluded that the wall of germinating conidia forms no size exclusion barrier for fluorescent viability probes. To assess the viability of a suspension of germinating conidiospores, PI, CDFDA, and FUN1 were shown to be suitable. It was not possible to homogeneously stain fresh conidiospores with fluorescent viability probes. Nonetheless, fluorescence activated cell sorting was possible and allowed the sorting of different sub-populations. Whilst the presence of esterase activity (CDFDA staining) did not correlate with outgrowth potential, the uptake of PI did correlate with no outgrowth.