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https://hdl.handle.net/1822/55190
Título: | Influence of the fixation/permeabilization step on peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria |
Autor(es): | Rocha, Rui Almeida, Carina Azevedo, Nuno F. |
Palavras-chave: | Ethanol fixation Gram positive bacteria Bacillus cereus Gram negative bacteria Staphylococcus epidermidis Fluorescent in situ hybridization Pseudomonas fluorescens Cell hybridization |
Data: | Mai-2018 |
Editora: | Public Library of Science |
Revista: | PLoS One |
Citação: | Rocha, Rui; Almeida, Carina; Azevedo, Nuno F., Influence of the fixation/permeabilization step on peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria. PLoS One, 13(5), e0196522, 2018 |
Resumo(s): | Fluorescence in situ Hybridization (FISH) is a versatile, widespread and widely- used technique in microbiology. The first step of FISHfixation/permeabilizationis crucial to the outcome of the method. This work aimed to systematically evaluate fixation/permeabilization protocols employing ethanol, triton X-100 and lysozyme in conjugation with paraformaldehyde for Peptide Nucleic Acid (PNA)-FISH. Response surface methodology was used to optimize these protocols for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). In general, the optimal PNA-FISH fluorescent outcome in Gram-positive bacteria was obtained employing harsher permeabilization conditions when compared to Gram-negative optimal protocols. The observed differences arise from the intrinsic cell envelope properties of each species and the ability of the fixation/permeabilization compounds to effectively increase the permeability of these structures while maintaining structural integrity. Ultimately, the combination of paraformaldehyde and ethanol proved to have significantly superior performance for all tested bacteria, especially for Gram-positive species (p<0.05). |
Tipo: | Artigo |
URI: | https://hdl.handle.net/1822/55190 |
DOI: | 10.1371/journal.pone.0196522 |
ISSN: | 1932-6203 |
Versão da editora: | http://journals.plos.org/plosone/ |
Arbitragem científica: | yes |
Acesso: | Acesso aberto |
Aparece nas coleções: | CEB - Publicações em Revistas/Séries Internacionais / Publications in International Journals/Series |
Ficheiros deste registo:
Ficheiro | Descrição | Tamanho | Formato | |
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document_47740_1.pdf | 3,2 MB | Adobe PDF | Ver/Abrir |