Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria

Pernthaler, Annelie; Pernthaler, Jakob; Amann, Rudolf

doi:10.1128/aem.68.6.3094-3101.2002

Cited by 931 publications

(810 citation statements)

References 52 publications

(53 reference statements)

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“…), then filtered onto 0.2 µm pore size, 47 mm diameter polycarbonate membranes and frozen at -20°C until analysis. Sections were subsequently cut from each filter and hybridized with the HRP probes (as described in Pernthaler et al 2002) specific to Archaea (Arch915), Bacteria (Eub338) and to 4 major phylogenetic lineages of this latter domain: the alpha (α; Alf968), beta (β; Bet42a) and gamma (γ; Gam42a) subclasses of the Proteobacteria and the CytophagaFlavobacter-Bacteroides cluster (CFB; CF319a). The sequences used for the probes are given in Table 1.…”

Section: Methodsmentioning

confidence: 99%

“…Microbial production was estimated with shipboard incubations of water samples with 3 H-leucine, and the offshore gradients in cell concentration of major taxonomic groups were determined by epifluorescence microscopy. Our approach included the application of a recently described molecular probe technique, the catalyzed reporter deposition for fluorescence in situ hybridization assay (CARD-FISH) (Pernthaler et al 2002, Schippers et al 2005. This method gives a much brighter detection of cells than the regular FISH approach, with values comparable to the application of polynucleotide probes (Pernthaler et al 2002).…”

Section: Resale or Republication Not Permitted Without Written Consenmentioning

confidence: 99%

“…Cell identification for the major prokaryotic groups was made by fluorescence in situ hybridization (FISH) using horseradish peroxidase (HRP)-labeled oligonucleotide probes combined with signal amplification by tyramide labeled with carboxyfluorescein. This catalyzed reporter deposition (CARD) approach offers advantages over the standard FISH assay by providing enhanced fluorescence intensities (Pernthaler et al 2002). Samples were fixed onboard the ship for 24 h at 4°C in 2% formalin (final conc.…”

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confidence: 99%

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Prokaryotic community structure and heterotrophic production in a river-influenced coastal arctic ecosystem

Garneau

Vincent²,

Alonso‐Sáez³

et al. 2006

Aquat. Microb. Ecol.

130

105

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Spatial patterns in prokaryotic biodiversity and production were assessed in the Mackenzie shelf region of the Beaufort Sea during open-water conditions. The sampling transect extended 350 km northwards, from upstream freshwater sites in the Mackenzie River to coastal and offshore sites, towards the edge of the perennial arctic ice pack. The analyses revealed strong gradients in community structure and prokaryotic cell concentrations, both of which correlated with salinity. Picocyanobacterial abundance was low (10 2 to 10 3 cells ml -1 ), particularly at the offshore stations that were least influenced by the river plume. Analysis by catalyzed reporter deposition for fluorescence in situ hybridization (CARD-FISH) showed that the dominant heterotrophic cell types were β-Proteobacteria at river sites, shifting to dominance by α-Proteobacteria offshore. Cells in the Cytophaga-Flavobacter-Bacteroides and γ-Proteobacteria groups each contributed < 5% of total counts in the river, but >10% of counts in the marine samples. Archaea were detected among the surface-water microbiota, contributing on average 1.3% of the total DAPI counts in marine samples, but 6.0% in turbid coastal and riverine waters. 3 H-leucine uptake rates were significantly higher at 2 stations influenced by the river (1.5 pmol l -1 h -1 ) than at other marine stations or in the river itself (≤0.5 pmol -1 h -1 ). Size-fractionation experiments at 2 coastal sites showed that > 65% of heterotrophic production was associated with particles > 3 µm. These results indicate the importance of particleattached prokaryotes, and imply a broad functional diversity of heterotrophic microbes that likely facilitates breakdown of the heterogeneous dissolved and particulate terrestrial materials discharged into arctic seas. KEY WORDS:Prokaryote diversity · Archaea · Proteobacteria · Cytophaga-FlavobacterBacteroides · Arctic Ocean · Mackenzie River estuary · Picocyanobacteria · CARD-FISH Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 42: [27][28][29][30][31][32][33][34][35][36][37][38][39][40] 2006 ments (Payette et al. 2004) may mobilize the large stocks of organic carbon contained within their soils and cause increased transfer of these materials to arctic rivers and ultimately to the ocean. Heterotrophic microbiota are likely to play a major role in the response of coastal arctic ecosystems to ongoing change, but prokaryotic diversity and production in these cold ocean environments have been little explored (Amon 2004).In the western Canadian Arctic, the Mackenzie River discharges large quantities of freshwater, solutes and sediments into a vast shelf region of the Beaufort Sea that extends >100 km offshore and encompasses a total area of 63 600 km 2 (Carmack et al. 2004). The annual discharge of the Mackenzie River (330 km 3 yr -1 ; Macdonald et al. 1998) is the 4th highest in the Arctic Basin after the Siberian rivers Yenisei, Lena and Ob, with concomitantly large inputs of freshwater biota, terrestria...

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Section: Methodsmentioning

confidence: 99%

Section: Resale or Republication Not Permitted Without Written Consenmentioning

confidence: 99%

mentioning

confidence: 99%

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Prokaryotic community structure and heterotrophic production in a river-influenced coastal arctic ecosystem

Garneau

Vincent²,

Alonso‐Sáez³

et al. 2006

Aquat. Microb. Ecol.

130

105

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“…All cells were stained with a nucleic acid dye [4 0 ,6-diamidino-2-phenylindole (DAPI) (17)]. Specific phylogenetic staining was performed by fluorescence in situ hybridization (FISH) (18,19). On some of the preparations, microautoradiography (MAR) was additionally performed for assessment of bacterial activity (20).…”

Section: Biological Samples Microscopic System and Imagesmentioning

confidence: 99%

Automated quality assessment of autonomously acquired microscopic images of fluorescently stained bacteria

Zeder

Kohler

2009

Cytometry Pt A

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Quality assessment of autonomously acquired microscopic images is an important issue in high-throughput imaging systems. For example, the presence of low quality images (!10%) in a dataset significantly influences the counting precision of fluorescently stained bacterial cells. We present an approach based on an artificial neural network (ANN) to assess the quality of such images. Spatially invariant estimators were extracted as ANN input data from subdivided images by low level image processing. Different ANN designs were compared and [400 ANNs were trained and tested on a set of 25,000 manually classified images. The optimal ANN featured a correct identification rate of 94% (3% false positives, 3% false negatives) and could process about 10 images per second. We compared its performance with the image quality assessment by different humans and discuss the difficulties in assigning images to the correct quality class. The computer program and the documented source code (VB

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“…CARD-FISH was performed according to a previously published protocol (Pernthaler et al 2002) with slight modifications, as described below. Microbial cells on polycarbonate filters were permeabilized with lysozyme (10 mg/ ml in 0.05 M EDTA and 0.1 M Tris-HCl buffer [pH 7.5]) for 70 min at 37 C. The filters were then treated with 1 % H 2 O 2 in methanol for 30 min at room temperature to deactivate the endogenous peroxidase.…”

Section: Catalyzed Reporter Depositionfluorescence In Situ Hybridizationmentioning

confidence: 99%

Quantification of Microbial Communities in Hydrothermal Vent Habitats of the Southern Mariana Trough and the Mid-Okinawa Trough

Yanagawa

Ishibashi

Arai

et al. 2014

Subseafloor Biosphere Linked to Hydrothermal Systems

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The structure of microbial populations near chemosynthetic faunal communities of two geographically and geologically distinct deep-sea hydrothermal vent fields were quantitatively evaluated using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The hydrothermal vent of the Southern Mariana Trough (SMT) was dominated by colonization of gastropods in the low-temperature diffuse hydrothermal fluid, whereas macrofauna in mixing zones of the Mid-Okinawa Trough (MOT) consisted of polychaetes, galatheid crabs, and bivalves. A quantitative comparison revealed that the microbial community of the SMT hydrothermal vent field is significantly different from that of the MOT and is strongly influenced by mixing conditions between reduced hydrothermal fluid and oxygenated seawater. In particular, a high proportion of Epsilonproteobacteria was found in the SMT hydrothermal fluid, which is composed of approximately 88 % seawater. In contrast, sulfur oxidizers in Gammaproteobacteria were most abundant near vent fauna habitats in the MOT. Our results suggest that the SMT hydrothermal environment is distinct from that of the MOT and affects the community structure of macrofauna and microbial flora.

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Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria

Cited by 931 publications

References 52 publications

Prokaryotic community structure and heterotrophic production in a river-influenced coastal arctic ecosystem

Prokaryotic community structure and heterotrophic production in a river-influenced coastal arctic ecosystem

Automated quality assessment of autonomously acquired microscopic images of fluorescently stained bacteria

Quantification of Microbial Communities in Hydrothermal Vent Habitats of the Southern Mariana Trough and the Mid-Okinawa Trough

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