Fluorescence microscopic investigation of Aspergillus awamori growing on synthetic and complex media and producing xylanase

Abstract

Several fluorescence techniques were used to detect cellular components within the hyphae of Aspergillus awamori. Single-stranded RNA (ss RNA) and double-stranded nucleic acids (ds RNA and DNA) were localized in mycelial hyphae and pellets of Aspergillus awamori by means of acridine orange (AO) staining. Pyronine Y/methyl green (PY/MG) staining allowed the localization of ds RNA and DNA, but no ss RNA in the cells. The replicating DNA was localized by the detection of the incorporated thymidine analogue (BrdU) via immunofluorescence. The ss RNA marks the local protein production in the cells. It indicates that the main protein production occurs in subapical and branching zones of the hyphae. In pellets no protein synthesis was observed in the corpus, but only in the hyphae on the pellet periphery. The replicating DNA observed in subapical and branching zones and nucleus indicates the cell propagation. On account of the intensive yellow fluorescence of the wheat bran, it was not possible to identify the cells with replicating DNA growing on a complex medium. By means of the vitality staining by fluorescein diacetate (PDA) and propidium iodide (PI) no internal hyphal structures were observed. The ethidium bromide (EB) is more suitable for structural investigations. The protein staining with fluorescein isothiocyanate (FITC) does not give useful information on the protein production, because of low fluorescence intensity, rapid fading and low selectivity.