An improved method for nanogold in situ hybridization visualized with environmental scanning electron microscopy
Ehrhardt, C. J.
Haymon, R. M.
Sievert, Stefan M.
Holden, P. A.
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An important goal in geomicrobiology is the identification of microbes associated with specific mineral surfaces. Yet, simultaneously collecting phylogenetic and mineral information remains methodologically challenging. Recently, whole-cell in situ hybridization techniques using oligonucleotide rRNA probes bound to nanogold particles have been used to detect microbes with scanning electron microscopy (SEM) for geomicrobiological applications (Gerard et al., 2005; Kenzaka et al., 2005). These techniques rely on backscattered electron images or energy dispersive X-ray spectroscopy to map the presence and distribution of nanogold, and to identify areas of rRNA hybridization within cells and on mineral surfaces. Although these nanogold hybridization techniques have been successful for pure cultures of Bacteria and Archaea (Gerard et al., 2005) and for natural microbial communities associated with river sediment particles (Kenzaka et al., 2005) and basalt surfaces (Menez et al., 2007), their application to other metal-rich geomicrobiological systems is problematic. First, metallic substrates and surfaces can obscure detection of nanogold-labelled cells imaged with backscattered electron microscopy (Richards et al., 2001). Second, metallic surfaces can interfere with the hybridization reaction by causing non-specific precipitation of nanogold (Humbel et al., 1995; Weipoltshammer et al., 2000). Because many geomicrobiological systems of interest have a high concentration of metal substrates (i.e. hydrothermal vents, acid mine drainage) a new technique is needed to identify microbes found in these types of environments. In this work, we present a new nanogold in situ hybridization method that increases the concentration of nanogold probes bound to rRNA targets within the cell and makes individual hybridization events directly visible with secondary electron SEM imaging.
This paper is not subject to U.S. copyright. The definitive version was published in Journal of Microscopy 236 (2009): 5-10, doi:10.1111/j.1365-2818.2009.03207.x.