An improved fluorescence in situ hybridization protocol for the identification of bacteria and archaea in marine sediments

Kousuke Ishii, Marc Mußmann, Barbara J. MacGregor, Rudolf Amann

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126 Scopus citations


In situ identification of prokaryotic cells in subsurface sediments is hampered by the low cellular rRNA contents of the target organisms. Fluorescence in situ hybridization with catalyzed reporter deposition (CARD-FISH) has the potential to overcome this limitation, and was therefore optimized for a 40 cm deep sediment core sampled from a tidal sandy flat of the German Wadden Sea. Treatment with methanol and H2O2 inactivated endogenous peroxidases and effectively reduced the background signal. Percentage of DAPI stained cells detected with the probe combination EUB(I-III), targeting nearly all the Bacteria, were comparable for CARD-FISH with a horseradish peroxidase (HRP)-labeled probe and FISH with a fluorescently monolabeled probe in the 2-3 cm depth interval (92% and 82%, respectively), but significantly higher with the HRP-labeled probe at 35-40 cm, the deepest layer sampled (63% with HRP vs. 26% with monolabeled probe). With CARD-FISH Alphaproteobacteria and the Desulfobulbaceae group of sulfate-reducing bacteria were detected only in the upper layers. In contrast, Desulfosarcinales, the Bacteroidetes group, Planctomycetes, Betaproteobacteria, and Gammaproteobacteria were found at all depths. Archaea were detectable with ARCH915-HRP after achromopeptidase treatment. Surprisingly, aggregates of Bacteria and Archaea were found, below 12 cm depth, that strongly resemble consortia involved in anoxic oxidation of methane that have previously been found in sediments near methane hydrate deposits. With the optimized CARD-FISH protocol, microbial populations could also be detected in deeper sediment horizons. Furthermore, the intensity of the CARD-FISH signals improved detection of rare organisms such as Archaea.

Original languageEnglish (US)
Pages (from-to)203-213
Number of pages11
JournalFEMS microbiology ecology
Issue number3
StatePublished - Nov 22 2004

Bibliographical note

Funding Information:
We would like to thank Enric Llobet-Brossa for his support in the early stages of this work; Michael E. Böttcher for organization and Johanna Wesnigk, Eva Walpersdorf, Ursula Werner, Katja Bosselmann and Marcus Billerbeck for their help during the sampling. Annelie Pernthaler is acknowledged for useful suggestions. This research was funded by the Max Planck Society.


  • Archaea
  • Black spots
  • Catalyzed reporter deposition
  • Endogenous peroxidase activity
  • Fluorescence in situ hybridization
  • Marine sediment
  • Planctomycetes

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