期刊
PLOS ONE
卷 13, 期 5, 页码 -出版社
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0196522
关键词
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资金
- European Regional Development Fund (ERDF), through COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI) [POCI-01-0145-FEDER-006684, UID/EQU/00511/2013, POCI-01-0145-FEDER-006939]
- national funds, through FCT - Fundacao para a Ciencia e a Tecnologia
- North Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) [NORTE-01-0145-FEDER-000005 - LEPABE-2-ECO-INNOVATION NORTE-01-0145-FEDER-000004 - BioTecNorte operation]
- national funds through FCT - Fundacao para a Ciencia e a Tecnologia [SFRH/BDE/51910/2012, ENMed/0003/2014 - NanoDiaBac, PTDC/DTP-PIC/4562/2014 - Coded-FISH]
- Biomode S.A
- Fundação para a Ciência e a Tecnologia [ENMed/0003/2014] Funding Source: FCT
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).
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