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Influence of the fixation/permeabilization step on peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria

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  • معلومة اضافية
    • بيانات النشر:
      Public Library of Science
    • الموضوع:
      2018
    • Collection:
      Universidade of Minho: RepositóriUM
    • نبذة مختصرة :
      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). ; This work was the result of the projects: POCI-01-0145-FEDER-006939 (Laboratory for Process Engineering, Environment, Biotechnology and Energy - UID/EQU/00511/2013) and POCI-010145-FEDER-006684 (Centerof Biological Engineering - UID/EQU/00511/2013)funded by the European Regional Development Fund (ERDF), through COMPETE2020- Programa Operacional Competitividade e Internacionalização (POCI) and by national funds, throughFCT - Fundação para a Ciênciae a Tecnologia. NORTE-01-0145-FEDER000005 - LEPABE-2-ECO-INNOVATIONand NORTE-01-0145-FEDER-000004- BioTecNorte operation,supported by North Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 PartnershipAgreement, throughthe EuropeanRegional Development Fund (ERDF). PhD Fellowship SFRH/BDE/51910/2012 supported by ...
    • File Description:
      application/pdf
    • ISSN:
      1932-6203
    • Relation:
      info:eu-repo/grantAgreement/FCT/5876/147284/PT; info:eu-repo/grantAgreement/FCT/SFRH/SFRH%2FBDE%2F51910%2F2012/PT; info:eu-repo/grantAgreement/FCT/3599-PPCDT/138727/PT; PTDC/DTP-PIC/4562/2014; http://journals.plos.org/plosone/; 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; http://hdl.handle.net/1822/55190
    • الرقم المعرف:
      10.1371/journal.pone.0196522
    • Rights:
      info:eu-repo/semantics/openAccess
    • الرقم المعرف:
      edsbas.3AA0A9B1