FLUORESCENT-PROBES FOR WALL POROSITY AND MEMBRANE INTEGRITY IN FILAMENTOUS FUNGI

To assess the viability of moulds, quick detection methods using fluor escent probes were evaluated. A major hurdle in the use of intracellul ar probes can be the wall of filamentous fungi. We studied the permeab ility of the wall of germinating conidiospores with anionic ellipsoid FITC-dextran molecules and confocal scanning laser microscopy. In cont rast to S. cerevisiae, filamentous fungi internalized FITC-dextran mol ecules up to 150 kDa (Stokes radius of 8-9 nm). We concluded that the wall of germinating conidia forms no size exclusion barrier for fluore scent viability probes. To assess the viability of a suspension of ger minating conidiospores, PI, CDFDA, and FUN1 were shown to be suitable. It was not possible to homogeneously stain fresh conidiospores with f luorescent viability probes. Nonetheless, fluorescence activated cell sorting was possible and allowed the sorting of different sub-populati ons. Whilst the presence of esterase activity (CDFDA staining) did not correlate with outgrowth potential, the uptake of PI did correlate wi th no outgrowth. (C) 1997 Elsevier Science B.V.