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6352651 
Journal Article 
Influence of the fixation/permeabilization step on peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria 
Rocha, R; Almeida, C; Azevedo, NF 
2018 
1 
PLoS ONE
EISSN: 1932-6203 
13 
5 
e0196522-e0196522 
English 
29851961 
WOS:000433893200007 
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). 
Laboratories; Bacteria; Listeria; Listeria monocytogenes; Response surface methodology; Gram-positive bacteria; Lysozyme; Flow cytometry; Peptides; Ethanol; Fluorescence; Structural integrity; Peptide nucleic acids; Optimization; Microbiology; Process engineering; Methods; Enzymes; Biotechnology; Conjugation; Listeria innocua; Hybridization; Permeability; Staphylococcus aureus; Species; Escherichia coli; Bioengineering; Fluorescence in situ hybridization; Fixation; Microorganisms; Chemical engineering; Pseudomonas fluorescens/