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Single-cell techniques

The Center for Geomicrobiology is currently developing methods for the genomic analysis of single microbial cells isolated from environmental samples. Methods currently used for single cell isolation include micromanipulation and laser microdissection.

Many of the microorganisms important in environmental processes are resistant to growth in laboratories. We are developing methods to work on single cells rather than whole cultures, in order to circumvent traditional culturing techniques and discover the genetic potential of these (non-culturable) microorganisms.

This will allow us to determine not only the identity, but to also predict the environmental function of previously unknown microorganisms. While other labs have developed similar techniques for isolating cells from seawater and other fluids, our focus on marine sediments and oceanic crust represents a new technical challenge. Sediment particles can make it difficult to pick out single microbial cells, and often humic substances are present which interfere with subsequent amplification of genetic material.

Laser Microdissection (LMD) microscopy and micromanipulation

Two methods currently used for isolating single cells from environmental samples at the center, are laser microdissection and micromanipulation. The center has a Leica LMD 7000 laser microdissection microscope. With this microscope, cells can be excised directly from filter- or membrane-mounted samples. For multi-cellular microbial assemblages, we additionally employ micromanipulation techniques for cell isolation. Here multi-cellular filamentous or aggregated microorganisms are being separated from the sample using micrometer thin glass instruments. After isolation, single cells are subjected to downstream analyses such as whole genome amplification (where the whole genomic material of a single cell is replicated) and PCR (where single target genes are replicated). The amplified genetic material is then sequenced using Sanger or next generation sequencing (e.g. 454 pyrosequencing or ion torrent sequencing) techniques. Finally, we analyze the sequences using bioinformatics. Results from the single cell analyses complement the knowledge gained by using other powerful geomicrobiological techniques at the center, such as metagenomics, qPCR, FISH, or isotope analysis, and will help us to better understand the microbial ecology of marine sediments.

Fluorescence-activated cell sorting (FACS)

In addition to developing single cell techniques in-house, we also maintain a collaboration with the Single Cell Genomics Center at the Bigelow Laboratory for Ocean Sciences, USA. Together with this partner, we work on establishing the isolation of single bacterial cells from marine sediments using fluorescence-activated cell sorting.

Nano-Secondary Ion Mass Spectrometry (Nano-SIMS)

The center collaborates with the Max Plank Institute in Bremen to use Nano-SIMS to measure substrate uptake by microorganisms obtained from the environment. When incubated with stable isotope labeled substrates, the activity of sedimentary microorganisms can be determined. Combining stable isotope labeling with halogenated in situ hybridization of isolated cells, analysis of the elemental composition using NanoSIMS allows us to directly link the activity and identity of single cells in the environment.

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Revised 2014.03.03

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