Gold-FISH: In Situ Hybridization of Microbial Cells for Combined Fluorescence and Scanning Electron Microscopy

Abstract

Understanding interactions of microorganisms with their habitat has recently become an important topic in (environmental) microbiology. In this context phylogenetic identification via in situ hybridization and specific detection and visualization of single microbial cells on resolutions beyond light microscopy is a promising approach. Here we describe a protocol which is based on rRNA-targeted in situ hybridization and catalyzed reporter deposition (CARD) enabling the detection of fluorescent signals and a specific nanogold deposition on the same target cells. The gold-stained microbial cells are analyzed via electron microscopy (EM) using backscattered electron detectors (BSD) or energy-dispersive X-ray spectroscopy (SEM-EDX). This Gold-FISH protocol has been successfully applied on pure and mixed bacterial cultures and environmental samples. The combined labeling allows reliable quantification of Gold-FISH-stained cells via fluorescence microscopy and analysis of microbe-surface interactions with techniques such as elemental map** via EDX or NanoSIMS on submicron scales. It is furthermore a sufficient proof of specific detection of single cells via nanogold and electron microscopy and can be combined with DNA-specific counterstains.