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Related Experiment Videos

An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy.

Mirko Lunau1, Andreas Lemke, Katja Walther

  • 1Institute for Chemistry and Biology of the Marine Environment, University of Oldenburg, D-26111 Oldenburg, Germany. lunau@icbm.de

Environmental Microbiology
|June 11, 2005
PubMed
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This study introduces a novel, faster, and less toxic method for detaching and staining particle-associated bacteria using methanol, sonication, and SybrGreen I. The optimized protocol enhances bacterial enumeration in marine samples, offering an improved alternative to existing techniques.

Area of Science:

  • Microbiology
  • Environmental Science
  • Analytical Chemistry

Background:

  • Accurate enumeration of particle-associated bacteria is crucial for understanding aquatic ecosystems.
  • Existing methods for detaching and staining bacteria can be time-consuming, toxic, or less effective for high-particle-load samples.

Purpose of the Study:

  • To develop and optimize a rapid, efficient, and less toxic protocol for detaching particle-associated bacteria.
  • To improve the accuracy and reproducibility of bacterial enumeration in marine samples, particularly sediments.
  • To present SybrGreen I staining as a superior alternative to DAPI for aquatic bacteria.

Main Methods:

  • Optimized a detachment procedure using 10% methanol and sonication at 35°C, with specific centrifugation parameters.

Related Experiment Videos

  • Developed a one-step sample preparation combining the dsDNA-specific stain SybrGreen I with polyvinylalcohol (moviol) mounting medium.
  • Compared the new method with pyrophosphate and Tween-80 detachment procedures using various marine samples.

    • Main Results:

    • The new methanol-sonication method consistently yielded higher numbers and/or fractions of particle-associated bacteria compared to pyrophosphate and Tween-80.
    • The moviol-SybrGreen I staining provided intense fluorescence with low background, enabling accurate enumeration even in samples with high suspended particulate matter (SPM).
    • Glutardialdehyde fixation resulted in brighter fluorescence than formalin fixation.

    Conclusions:

    • The developed protocol offers a faster, easier, and less toxic approach for detaching and enumerating particle-associated bacteria.
    • SybrGreen I staining with moviol is highly effective for accurate and reproducible bacterial counts in diverse marine samples.
    • The method is a viable and attractive alternative to DAPI staining, with the added benefit of sample storage stability.