Gold-FISH: A new approach for the in situ detection of single microbial cells combining fluorescence and scanning electron microscopy

A novel fluorescence in situ hybridisation (FISH) method is presented that allows the combination of epifluorescence and scanning electron microscopy (SEM) to identify single microbial cells. First, the rRNA of whole cells is hybridised with horseradish peroxidase-labelled oligonucleotide probes and...

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Vydáno v:	Systematic and applied microbiology Ročník 35; číslo 8; s. 518 - 525
Hlavní autoři:	Schmidt, Hannes, Eickhorst, Thilo, Mußmann, Marc
Médium:	Journal Article Konferenční příspěvek
Jazyk:	angličtina
Vydáno:	München Elsevier GmbH 01.12.2012 Elsevier
Témata:	Bacteriological methods and techniques used in bacteriology Bacteriology binding sites Biological and medical sciences CARD–FISH energy-dispersive X-ray analysis Environmental Microbiology Epifluorescence microscopy fluorescence fluorescence in situ hybridization fluorescence microscopy fluorescent dyes Fluorescent Dyes - metabolism Fundamental and applied biological sciences. Psychology Geologic Sediments - microbiology gold Gold - metabolism Gold-FISH goldfish horseradish In situ hybridisation In Situ Hybridization, Fluorescence - methods light microscopy marine sediments Microbiology microorganisms Microscopy, Electron, Scanning - methods Nanogold Nanoparticles oligonucleotide probes physicochemical properties Plant Roots - microbiology ribosomal RNA rice roots Scanning electron microscopy Staining and Labeling - methods streptavidin Gold-FISH CARD–FISH Scanning electron microscopy Epifluorescence microscopy In situ hybridisation Nanogold Method CARD-FISH Vertebrata Pisces Fluorescence in situ hybridization Isolated cell Detection Fluorescence microscopy
ISSN:	0723-2020, 1618-0984, 1618-0984
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Shrnutí:	A novel fluorescence in situ hybridisation (FISH) method is presented that allows the combination of epifluorescence and scanning electron microscopy (SEM) to identify single microbial cells. First, the rRNA of whole cells is hybridised with horseradish peroxidase-labelled oligonucleotide probes and this is followed by catalysed reporter deposition (CARD) of biotinylated tyramides. This facilitates an amplification of binding sites for streptavidin conjugates covalently labelled with both fluorophores and nanogold particles. The deposition of Alexa Fluor 488 fluoro-nanogold–streptavidin conjugates was confirmed via epifluorescence microscopy and cells could be quantified in a similar way to standard CARD–FISH approaches. To detect cells by SEM, an autometallographic enhancement of the nanogold particles was essential, and allowed the in situ localisation of the target organisms at resolutions beyond light microscopy. Energy dispersive X-ray spectroscopy (EDS) was used to verify the effects of CARD and autometallography on gold deposition in target cells. The gold-FISH protocol was developed and optimised using pure cultures and environmental samples, such as rice roots and marine sediments. The combination of epifluorescence and scanning electron microscopy provides a promising tool for investigating microorganisms at levels of high resolution. Correlative characterisation of physicochemical properties by EDS will allow for the analysis of microbe-surface interactions.
Bibliografie:	http://dx.doi.org/10.1016/j.syapm.2012.04.006 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0723-2020 1618-0984 1618-0984
DOI:	10.1016/j.syapm.2012.04.006