QUALITATIVE AND QUANTITATIVE-ANALYSIS OF AUTOFLUORESCENCE IN FUNGI

Fluorescence microscopy is an important method in mycology. It is a co mmon procedure used in immunology or histology and more recently in mo dern techniques of molecular biology like in-situ hybridization. Since several molds and yeasts show autofluorescence, an interference of th is phenomenon with the detection method cannot be excluded. Therefore, we studied autofluorescence in fungi in more detail, in particular wi th respect to the dependence of this phenomenon from growth conditions , fixing method or mounting medium used. Here we show that moulds cult ivated in a liquid medium are strongly autofluorescent which could be considerably reduced by repetitive washing. In moulds, we did not find important differences in autofluorescence levels with the three fixin g methods under study. However, this finding cannot be generalized. Th us, in the yeast Candida albicans we found the autofluorescence patter n being largely dependent from the fixing method and the excitation wa ve length, respectively. In particular, with green excitation we could show that aceton fixation resulted in strong fluorescence of individu al cells within a vast population of cells showing little or no autofl uorescence. In addition, we could demonstrate that mounting media are able to strongly modify autofluorescence in fungi. Using digital image acquisition with a cooled CCD camera we were able to quantify the inf luence of different mounting media on fluorescence intensities of Aspe rgillus fumigatus. Diagnostic methods in mycology based on fluorescenc e microscopy have to be thoroughly evaluated in order to prevent false positive results due to fungal autofluorescence.