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).
Highlights
Fluorescence in situ Hybridization (FISH) is a widely used technique in the field of microbiology [1]
response surface methodology (RSM) was applied to the hybridization data obtained from 3 Gram-positive (S. epidermidis, L. innocua and B. cereus) and 2 Gram-negative species (P. fluorescens and E. coli)
It should be noticed that paraformaldehyde at a concentration of 4% is a common step to most of the procedures and the main procedural differences are related to the type of permeabilization agent used, as well as the concentration and exposure periods [1,6]
Summary
Fluorescence in situ Hybridization (FISH) is a widely used technique in the field of microbiology [1]. A standard FISH protocol targeting the rRNA, involves 4 different steps: fixation/permeabilization, hybridization, washing and visualization/detection [4,5]. Influence of the fixation/permeabilization step on PNA-FISH for the detection of bacteria. Competitividade e Internacionalizacão (POCI) and by national funds, through FCT - Fundacão para a Ciência e a Tecnologia. PhD Fellowship SFRH/BDE/51910/2012 supported by national funds through FCT - Fundacão para a Ciência e a Tecnologia. ENMed/0003/2014 NanoDiaBac and PTDC/DTP-PIC/4562/2014 Coded-FISH funded by national funds through FCT - Fundacão para a Ciência e a Tecnologia. Rui Rocha works for and receives salary from Biomode S.A, a company that develops and commercializes in vitro diagnostic kits for microorganism detection, using PNA-FISH technology. The specific roles of these authors are articulated in the ‘author contributions’ section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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