Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria

Abstract

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml(-1) at 37 degrees C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.

FIG. 1.

Map of the German Bight of the North Sea indicating the sampling locations along the three transects (numbers) and the site of sediment sampling (⊗). The arrow points to the location of the island of Helgoland.

FIG. 2.

Lines and symbols indicate the abundances of bacterioplankton cells on membrane filters during incubation with lysozyme (10 mg ml⁻¹ at 37°C) with or without embedding in low-gelling-point agarose. Bars indicate the percent FISH detection rates with an HRP-labeled probe and CARD in embedded samples. The error bars indicate either standard deviations (abundances) or total ranges (percentages) of triplicates. Symbols: ▧, EUB338-HRP; ○, agarose embedding (DAPI); •, not embedded (DAPI).

FIG. 3.

FISH detection rates with HRP-labeled probes and CARD at increasing dilution of tyramide substrate in plankton samples. The numbers on the x axis indicate parts of amplification diluent added to one part of tyramide-Cy3. The arrow indicates the range of tyramide concentrations recommended by the manufacturer. The error bars show the ranges of triplicate experiments.

FIG. 4.

Comparison of detection rates by FISH with a Cy3-monolabeled general bacterial probe (EUB338) and CARD-FISH with an HRP-labeled probe along three transects in North Sea surface water samples obtained in October 1999. Symbols: ○, EUB338-HRP, •, EUB338-mono.

FIG. 5.

Comparison of detection rates by FISH with a Cy3-monolabeled general bacterial probe (EUB338) and CARD-FISH with an HRP-labeled probe in Wadden Sea sediment samples obtained in February 2001. Symbols: ○, EUB338-HRP, •, EUB338-mono.

FIG. 6.

Photomicrographs of FISH-stained marine bacteria. Each double panel depicts DAPI staining in blue (left) and probe staining in red (right). Exposure times for images of FISH staining with Cy3-monolabeled probes (FISH-mono) were 10 times those for CARD-FISH staining. (a to d) FISH with the general bacterial probe EUB338: sediment, FISH-mono (a); sediment, CARD-FISH (b); plankton, FISH-mono (c); plankton, CARD-FISH (d). (e) Plankton, CARD-FISH with ROS537, specific for members of the Roseobacter lineage. (f) Plankton, CARD-FISH with SAR86-1249, specific for members of the SAR86 clade. Scale bar, 10 μm.

FIG. 7.

Abundances of members of the SAR86 (probe SAR86-1249) and Roseobacter (probe ROS537) clades, with specific HRP-labeled probes and CARD-FISH along three transects in surface water samples from the North Sea obtained in October 1999. Symbols: ▵, ROS537-HRP; ▪, SAR86-1249-HRP.