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Bernd Eschweiler and Beate Kilb, Institute for Water Research, Schwerte, Germany
Introduction
Bacterial diversity in environmental samples is usually determined by
a characterization of isolated strains. A problem for the analysis and
characterization of microbial communities is the inability to culture
most of the bacteria species present in the sample. Therefore isolated
bacteria may account for only a minor portion of the total bacterial diversity
originally present in the sample. This problem is particularly severe
in oligotrophic habitats like ground water, where approximately only 0.1
to 1% of the bacterial species are culturable.1 A new approach
in microbial ecology is based on the analysis of bacterial genetic information
without cultivation. This culture-independent approach has greatly enhanced
the ability to assess bacterial diversity in ecosystems such as ground
and surface water environments. After isolation of total bacterial DNA,
variable regions of the 16S rRNA gene are amplified by PCR*. The similar
sized PCR-products are separated by subsequent DGGE, and the resulting
diversity pattern are analyzed and compared.2, 3
Materials and Methods
DNA from surface and ground water was isolated and purified as described
earlier.2, 3 Two universal bacterial 16S rDNA primers were used to amplify
a 527 bp fragment from total genomic DNA. PCR was performed in a total
volume of 100 l containing 1x PCR buffer (10 mM Tris-HCl, pH 8.3,
50 mM KCl, 1.5 mM MgCl2, 0.001% gelantine), 200 M each dNTP, 0.5
M each primer, 0.3 mg/ml bovine serum albumin (BSA), and 2.5 U
AmpliTaq DNA polymerase (Perkin
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Source:
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