Scanning electron microscope imaging of bacteria based on DNA sequence

Abstract

Aims: To develop a scanning electron microscopic approach using in situ hybridization (SEM–ISH) for gaining both genetic and morphological information about target bacteria. Methods and Results: Target cells were hybridized with DNA‐targeted polynucleotide probes, and a tyramide signal amplification system was used to increase the sensitivity. The protocol of SEM–ISH enabled to detect low copy number target DNA sequences in individual cells. Conclusions: SEM–ISH allowed the in situ detection of bacteria carrying a specific gene. Significance and Impact of the Study: Combining morphological study with SEM and ISH techniques appears to be a valuable tool to understand the spatial distribution of target cells in complex microbial communities on various materials.

Micro-scale determinants of bacterial diversity in soil

“…1a) where X-rays are directed onto a polished, resin-embedded sample to obtain information on elemental ratios. The major downside of this technique is the inability to garner biological information after resin embedding, however, depending on the resolution and staining intensity requirements, bacterial staining with an electron-dense element could possibly allow the localization of bacterial cells (Kenzaka et al, 2005(Kenzaka et al, , 2009Ehrhardt et al, 2009) (Schmidt et al, 2012). 5).…”

CARD-FISH for Environmental Microorganisms: Technical Advancement and Future Applications

“…In combination with other techniques, Kenzaka et al (32) deposited biotinylated tyramide and subsequently applied nanogold-conjugated streptavidin. For further signal amplification, they employed gold enhancement and successfully detected the green fluorescent gene and ampicillin resistance gene on plasmids using a scanning electron microscope.…”

Scanning Electron Microscopy Imaging of Bacteria Based on Nucleic Acid Sequences

“…FISH analysis of bacterial communities with mRNA-or DNA-targeted probes presents a unique challenge because of its low sensitivity and resolution (Moraru et al, 2010;Pernthaler & Amann, 2004). The concepts of ISH can be incorporated into scanning electron microscopy imaging of microorganisms (Kenzaka et al, 2005a(Kenzaka et al, , 2009). …”

“…Consequently, hybridized cells contain gold up to tens of nm in size inside the hybridized cells. These hybridized cells release a strong backscatter electron signal (BSE) because of the accumulation of gold atoms inside cells (Kenzaka et al, 2005a(Kenzaka et al, , 2009. High-vacuum SEM images of a mixture of bacterial strains after ISH with an rRNA-targeted probe and labeling with gold are shown in Fig.…”

“…The signal in SEM-ISH can be amplified using a similar system to detect low copy number target DNA sequences in individual cells (Kenzaka et al, 2009). In the study, digoxigenin-labeled polynucleotide probes were hybridized with plasmid DNA.…”