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Updated: Aug 21, 2026
Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi
Published on: November 28, 2019
Methods to maximise the staining of fungal propagules with fluorescent dyes
Valeria Prigione1, Valeria Filipello Marchisio
1Dipartimento di Biologia Vegetale, Università degli Studi di Torino, Viale Mattioli 25, Turin I-10125, Italy.
Abstract:
The spores and conidia of most fungi have very thick and resistant cell walls that severely impede the staining with fluorescent dyes to allow epifluorescence microscopy to be employed for their direct detection and quantification in natural habitats. In this study, oxidation by sodium hypochlorite and microwave irradiation (MWI) were used to enhance the staining of Aspergillus fumigatus and Penicillium brevicompactum conidia with six fluorescent dyes. Sodium hypochlorite resulted in high percentages of stained conidia (up to 98.8% with 4',6-diamidino-2-phenylindole [DAPI]), but had to be removed prior to staining with consequent heavy conidia losses. By contrast, MWI gave very high percentages, while its enhancement of fluorescence intensity facilitated observation by epifluorescence microscopy.
Insights
Microwave irradiation effectively enhances fungal spore staining for microscopy. This method improves fluorescence, aiding direct detection and quantification of fungi like Aspergillus fumigatus in their natural environments.
Area of Science:
- Mycology
- Microscopy
- Biotechnology
Background:
- Fungal spores and conidia possess resistant cell walls hindering fluorescent dye staining.
- Direct detection and quantification in natural habitats require improved staining methods for epifluorescence microscopy.
Purpose of the Study:
- To evaluate methods for enhancing fluorescent staining of fungal conidia.
- To compare sodium hypochlorite oxidation and microwave irradiation (MWI) for improving epifluorescence microscopy of Aspergillus fumigatus and Penicillium brevicompactum.
Main Methods:
- Fungal conidia (Aspergillus fumigatus, Penicillium brevicompactum) were treated with sodium hypochlorite or microwave irradiation.
- Enhanced conidia were stained with six different fluorescent dyes.
- Staining efficiency and fluorescence intensity were assessed using epifluorescence microscopy.
Main Results:
- Sodium hypochlorite treatment yielded high staining percentages but caused significant conidia loss.
- Microwave irradiation (MWI) resulted in very high staining percentages.
- MWI significantly enhanced fluorescence intensity, facilitating epifluorescence microscopy.
Conclusions:
- Microwave irradiation is a superior method for enhancing fungal conidia staining for epifluorescence microscopy.
- MWI improves direct detection and quantification of fungal spores in environmental samples.
- This technique offers a valuable tool for mycological research in natural settings.
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