Data di Pubblicazione:
2009
Citazione:
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.
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.
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
DAPI staining; FISH; Fungal conidia; Permeabilization; Spatial arrangement
Elenco autori:
F. Villa, F. Cappitelli, P. Principi, A. Polo, C. Sorlini
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