A new method for determining the metabolic activity of specific bacterial populations in soil using tritiated leucine and immunomagnetic separation |
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Authors: | Gitte Sengeløv Søren J Sørensen Lone Frette N Kroer |
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Institution: | National Environmental Research Institute, Frederiksborgvej 399, 4000 Roskilde, Denmark e-mail: nk@dmu.dk Tel.: +45-46-301388 Fax: +45-46-301216, DK Department of General Microbiology, University of Copenhagen, S?lvgade 83H, 1307 Copenhagen K, Denmark, DK
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Abstract: | A new assay, using immunomagnetic separation and uptake of tritiated leucine (3H]-Leu), was developed for measuring the in situ metabolic activity of specific bacterial populations in soil. Such assays
are needed to assess the role individual species play in diverse microbial soil communities. The method was optimized using
Pseudomonas putida KT2440 : :Tc+/TOL::gfp inoculated into soil microcosms. Inoculated soil samples were incubated with 3H]-Leu followed by an immunomagnetic separation to recover the target bacteria. Radiolabel incorporated by the target bacteria
was then measured. Incubation time with immunomagnetic beads was not critical for optimal target cell recovery, but samples
needed to be washed at least 5 times during the immunomagnetic separation to reduce unspecific binding of the indigenous soil
bacteria to the magnetic beads. Soil absorption of the polyclonal antibody further reduced this unspecific binding, resulting
in <1% contamination by indigenous soil bacteria relative to numbers of recovered target cells. The assay was tested by investigating
the effect of different incubation temperatures on the metabolic activity of the target cells. As expected, a linear relationship
between activity and temperature was observed, demonstrating the sensitivity of the assay. The method was applied to compare
activities of the target strain in bulk soil and in the rhizosphere of barley. Contrary to what was anticipated, no significant
difference in metabolic activity was observed.
Received: 4 November 1999 |
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Keywords: | Immunomagnetic separation Tritiated leucine Metabolic activity Microbial soil communities Pseudomonas putida |
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