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

TLDR

FISH using HRP‑labeled probes and tyramide signal amplification (CARD) is limited for heterotrophic marine bacteria because permeabilization required for HRP entry causes species‑selective cell loss. The study presents an improved CARD‑FISH protocol for marine planktonic and benthic microbial assemblages. The protocol concentrates samples onto membrane filters, embeds them in low‑gelling‑point agarose, and incubates with lysozyme (10 mg ml⁻¹ at 37 °C) for 90 min, preventing cell loss. CARD‑FISH achieved high detection rates (≈94 % in North Sea bacterioplankton, 81 % in Wadden Sea sediments) and visualized SAR86 clade, whereas monolabeled probes yielded only 48 % and 44 % respectively, with virtually no unspecific staining, demonstrating CARD‑FISH’s superiority for low‑rRNA marine bacteria.

Abstract

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°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.