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Related Experiment Videos

Flow sorting of microorganisms for molecular analysis

G Wallner1, B Fuchs, S Spring

  • 1GSF-Forschungszentrum für Umwelt und Gesundheit, Durchflusszytometrie, Neuherberg, Germany.

Applied and Environmental Microbiology
|November 15, 1997
PubMed
Summary

Flow cytometry enables targeted isolation of specific microbial cells from complex environments. This method enriches rare bacteria for molecular analysis, revealing novel species in activated sludge and lake sediments.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Environmental Science

Background:

  • Traditional cultivation and molecular methods offer incomplete microbial diversity insights.
  • Environmental microbial communities are highly complex and diverse.
  • Targeted isolation of specific microbial populations is crucial for detailed analysis.

Purpose of the Study:

  • To demonstrate the utility of flow sorting for enriching specific microbial populations from environmental samples.
  • To assess the efficiency and purity of flow sorting for microbial cell selection.
  • To enable molecular analysis of rare or unculturable microorganisms.

Main Methods:

  • Flow cytometry was used to sort microorganisms based on light scattering, DNA content, and phylogenetic affiliation (using rRNA-targeted probes).

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  • Environmental samples included activated sludge, lake water, and lake sediment.
  • Sorted cells were analyzed using microscopy, PCR amplification of 16S ribosomal DNA (rDNA), dot blot hybridization, and sequencing.

    • Main Results:

    • Flow sorting achieved significant enrichment (up to 280-fold) of low-abundance microbial populations.
    • Purity of sorted cells could be improved by repeated sorting, though cell aggregation posed a limitation for some samples.
    • Almost full-length 16S rDNA fragments were successfully amplified from fixed, sorted cells.
    • Sequence analysis of amplified 16S rDNA identified previously unknown bacterial species.

    Conclusions:

    • Flow sorting is an effective technique for isolating and enriching specific microbial cells from diverse environmental samples.
    • This method facilitates molecular characterization of rare or previously uncharacterized microbial taxa.
    • Flow sorting significantly advances the study of microbial diversity and community structure.