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Author(s): Rui Rocha
Carina Almeida
Nuno F. Azevedo
Title: Influence of the fixation/permeabilization step on peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria
Issue Date: 2018
Abstract: Fluorescence in situ Hybridization (FISH) is a versatile, widespread and widely-used technique in microbiology. The first step of FISH fixation/permeabilization is 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).
Related Information: info:eu-repo/grantAgreement/FCT - Fundação para a Ciência e Tecnologia/Projetos Estratégicos/UID/EQU/00511/2013 - POCI-01-0145-FEDER-006939/Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia/LEPABE
info:eu-repo/grantAgreement/Comissão de Coordenação e Desenvolvimento Regional do Norte/P2020|Norte2020-Projetos Integrados ICDT/NORTE-01-0145-FEDER-000005/LEPABE-2-ECO-INNOVATION/LEPABE-2-ECO-INNOVATION
info:eu-repo/grantAgreement/FCT - Fundação para a Ciência e Tecnologia/P2020|COMPETE - Projetos em Todos os Domínios Científicos/PTDC/DTP-PIC/4562/2014 - POCI-01-0145-FEDER-016678/Desenvolvimento de um novo método de RING FISH codificado por cores: uma ferramenta inovadora para a deteção de patogénios resistentes a antibióticos/Coded-FISH
Document Type: Artigo em Revista Científica Internacional
Rights: openAccess
Appears in Collections:FEUP - Artigo em Revista Científica Internacional

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