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


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 beta-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.


CONCLUSIONS
The method described is reproducible, simple and inexpensive and might be attractive for other direct visualization techniques.
Villa
F
F
Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Università degli Studi di Milano, Italy.
Cappitelli
F
F
Principi
P
P
Polo
A
A
Sorlini
C
C
D002463:Cell Membrane Permeability / Q000187:drug effects; D005456:Fluorescent Dyes / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007211:Indoles / Q000378:metabolism; D009997:Osmotic Pressure; D013172:Spores, Fungal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013501:Surface-Active Agents / Q000494:pharmacology
D005456:Fluorescent Dyes; D007211:Indoles; D013501:Surface-Active Agents; C007293:DAPI