Stable Isotope Probing

Among the more exciting advances in molecular ecology is the use of stable isotope probing (SIP). This approach allows microbial identity to be linked to functional activity through the use of substrates labeled with stable isotopes. It has been used to its best advantage by labeling substrates that are used almost exclusively by the population of interest (Radajewski et at., 2000; Wellington et at., 2003; Dumont and Murrell, 2005). In this method, a selected substrate is first highly enriched with a stable isotope, frequently 13C or 15N, and then incorporated into the soil. After a brief incubation, cellular components of interest are recovered from the soil sample and analyzed for the incorporated stable isotope label. In this way, microbes that are actively using the added substrate can be identified. DNA and rRNA are the bio-markers used most frequently (Radajewski et at., 2003), although PLFAs have also been used successfully (Treonis et at., 2004). The isotopically labeled nucleic acids are purified from unlabeled nucleic acids by density-gradient centrifugation. Once separated, labeled nucleic acids can be amplified using PCR and universal primers to Bacteria, Archaea, or Eucarya. Analysis of the PCR products, through

C-subtrate, pure culture control c

13C-labelled substrate

Rubber stopper —Serum vial

13C-labelled substrate

Rubber stopper —Serum vial

Environmental sample

13C-subtrate, pure culture control

Environmental sample

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