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

PloS One
Rui RochaNuno F Azevedo

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).

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Citations

Mar 29, 2020·PeerJ·Alexander P YoungRussell C Wyeth
Nov 1, 2020·BMC Molecular and Cell Biology·Yensi Flores BuesoMark Tangney
Oct 6, 2021·MSystems·Alba Boix-AmorósJose C Clemente
Oct 10, 2021·Communications Biology·Sreeparna MajeeSaptarshi Basu
Dec 7, 2021·Frontiers in Cellular and Infection Microbiology·Lúcia G V SousaNuno Cerca

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Methods Mentioned

BETA
flow cytometry
Fluorescence

Software Mentioned

Design Expert
GraphPad Prism
Sony analysis

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