title: Optimization of a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the detection of bacteria and disclosure of a formamide effect.
abstract: Despite the fact that fluorescence in situ hybridization (FISH) is a well-established technique to identify microorganisms, there is a lack of understanding concerning the interaction of the different factors affecting the obtained fluorescence. In here, we used flow cytometry to study the influence of three essential factors in hybridization - temperature, time and formamide concentration - in an effort to optimize the performance of a Peptide Nucleic Acid (PNA) probe targeting bacteria (EUB338). The PNA-FISH optimization was performed with bacteria representing different families employing response surface methodology. Surprisingly, the optimum concentration of formamide varied according to the bacterium tested. While hybridization on the bacteria possessing the thickest peptidoglycan was more successful at nearly 50% (v/v) formamide, hybridization on all other microorganisms appeared to improve with much lower formamide concentrations. Gram staining and transmission electron microscopy allowed us to confirm that the overall effect of formamide concentration on the fluorescence intensity is a balance between a harmful effect on the bacterial cell envelope, affecting cellular integrity, and the beneficial denaturant effect in the hybridization process. We also conclude that microorganisms belonging to different families will require different hybridization parameters for the same FISH probe, meaning that an optimum universal PNA-FISH procedure is non-existent for these situations.
authors.affiliation: Faculty of Engineering of the University of Porto, LEPABE, Department of Chemical Engineering, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal.
authors.forename: Rita S
authors.initials: RS
authors.lastname: Santos
authors.affiliation: Faculty of Engineering of the University of Porto, LEPABE, Department of Chemical Engineering, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal; Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) , Rua Dr. Roberto Frias, 4200-465 Porto, Portugal.
authors.forename: Nuno
authors.initials: N
authors.lastname: Guimarães
authors.affiliation: Abel Salazar Institute of Biomedical Sciences (ICBAS), University of Porto, Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal; Institute for molecular and cell biology (IBMC), University of Porto, Rua do campo alegre no. 823, 4150-180 Porto, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Madureira
authors.affiliation: Faculty of Engineering of the University of Porto, LEPABE, Department of Chemical Engineering, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal. Electronic address: nazevedo@fe.up.pt.
authors.forename: Nuno F
authors.initials: NF
authors.lastname: Azevedo
mesh_terms: D001419:Bacteria / Q000737:chemistry* / Q000145:classification* / Q000378:metabolism; D015373:Bacterial Typing Techniques / Q000379:methods*; D005559:Formamides / Q000737:chemistry*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D046529:Microscopy, Electron, Transmission; D020135:Peptide Nucleic Acids / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005559:Formamides; D020135:Peptide Nucleic Acids; C031066:formamide
keywords: Bacteria; Formamide; PNA EUB338; PNA-FISH; Peptidoglycan