Aims: 4',6-diamidino-2-phenylindole (DAPI) staining and fluorescent in-situ hybridization (FISH) show great potential for the detection of fungal conidia, also conserving the spatial architecture of their colonization. These investigations are often greatly hampered by the complicated wall structure of many fungal taxa. The aim of the present study was to develop an efficient permeabilization strategy for both DAPI staining and the FISH technique, applicable to various fungal species and maintaining their relationships with surfaces. Methods and Results: We compared different DAPI staining permeabilization strategies based on alcohol dehydration, surfactants and osmotic shock, tested with Aspergillus niger conidia. Among four permeabilization methods leading to a strong DAPI signal, only one, based on Triton X-100, EDTA and b-mercaptoethanol followed by hyperosmotic stress, appeared suitable for FISH investigation and was successfully applied to an additional 10 fungal taxa and three environmental samples. Conclusions: The effective permeabilization method, which employed a combination of surfactant and osmotic strategies, was successfully applied as preliminary step in both DAPI staining and the FISH protocol. Significance and Impact of the Study: The method described is reproducible, simple and inexpensive and might be attractive for other direct visualization techniques.

Permeabilization method for in-situ investigation of fungal conidia on surfaces / F. Villa, F. Cappitelli, P. Principi, A. Polo, C. Sorlini. - In: LETTERS IN APPLIED MICROBIOLOGY. - ISSN 0266-8254. - 48:2(2009 Feb), pp. 234-240.

Permeabilization method for in-situ investigation of fungal conidia on surfaces

F. Villa;F. Cappitelli;P. Principi;A. Polo;C. Sorlini
2009

Abstract

Aims: 4',6-diamidino-2-phenylindole (DAPI) staining and fluorescent in-situ hybridization (FISH) show great potential for the detection of fungal conidia, also conserving the spatial architecture of their colonization. These investigations are often greatly hampered by the complicated wall structure of many fungal taxa. The aim of the present study was to develop an efficient permeabilization strategy for both DAPI staining and the FISH technique, applicable to various fungal species and maintaining their relationships with surfaces. Methods and Results: We compared different DAPI staining permeabilization strategies based on alcohol dehydration, surfactants and osmotic shock, tested with Aspergillus niger conidia. Among four permeabilization methods leading to a strong DAPI signal, only one, based on Triton X-100, EDTA and b-mercaptoethanol followed by hyperosmotic stress, appeared suitable for FISH investigation and was successfully applied to an additional 10 fungal taxa and three environmental samples. Conclusions: The effective permeabilization method, which employed a combination of surfactant and osmotic strategies, was successfully applied as preliminary step in both DAPI staining and the FISH protocol. Significance and Impact of the Study: The method described is reproducible, simple and inexpensive and might be attractive for other direct visualization techniques.
DAPI staining; FISH; Fungal conidia; Permeabilization; Spatial arrangement
Settore AGR/16 - Microbiologia Agraria
feb-2009
Article (author)
File in questo prodotto:
Non ci sono file associati a questo prodotto.
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/48948
Citazioni
  • ???jsp.display-item.citation.pmc??? 1
  • Scopus 14
  • ???jsp.display-item.citation.isi??? 12
social impact