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  Optimizing the hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) protocol for detection of microbes in sediments

Zeyu, J., Dong, Y., Xu, H., Wang, F. (2021 online): Optimizing the hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) protocol for detection of microbes in sediments. - Marine Life Science & Technology.
https://doi.org/10.1007/s42995-021-00098-8

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Zeyu, Jia1, Autor              
Dong, Yijing2, Autor
Xu, Heng2, Autor
Wang, Fengping2, Autor
Affiliations:
10 Pre-GFZ, Departments, GFZ Publication Database, Deutsches GeoForschungsZentrum, ou_146023              
2External Organizations, ou_persistent22              

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Schlagwörter: Fluorescence in situ hybridization Hybridization chain reaction HCR-FISH Microbial detection Sediment
 Zusammenfassung: Fluorescence in situ hybridization (FISH) is a canonical tool commonly used in environmental microbiology research to visualize targeted cells. However, the problems of low signal intensity and false-positive signals impede its widespread application. Alternatively, the signal intensity can be amplified by incorporating Hybridization Chain Reaction (HCR) with FISH, while the specificity can be improved through protocol modification and proper counterstaining. Here we optimized the HCR-FISH protocol for studying microbes in environmental samples, particularly marine sediments. Firstly, five sets of HCR initiator/amplifier pairs were tested on the laboratory-cultured bacterium Escherichia coli and the archaeon Methanococcoides methylutens, and two sets displayed high hybridization efficiency and specificity. Secondly, we tried to find the best combination of sample pretreatment methods and HCR-FISH protocol for environmental sample analysis with the aim of producing less false positive signals. Various detachment methods, extraction methods and formulas of hybridization buffer were tested using sediment samples. Thirdly, an image processing method was developed to enhance the DAPI signal of microbial cells against that of abiotic particles, providing a reliable reference for FISH imaging. In summary, our optimized HCR-FISH protocol showed promise to serve as an addendum to traditional FISH for research on environmental microbes.

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 Datum: 2021-04-30
 Publikationsstatus: Online veröffentlicht
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 Art der Begutachtung: -
 Identifikatoren: DOI: 10.1007/s42995-021-00098-8
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Titel: Marine Life Science & Technology
Genre der Quelle: Zeitschrift, other
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Seiten: - Band / Heft: - Artikelnummer: - Start- / Endseite: - Identifikator: CoNE: https://gfzpublic.gfz-potsdam.de/cone/journals/resource/2021-05-05
Publisher: Springer