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Related Experiment Videos

Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting.

G Nebe-von-Caron1, P J Stephens, C J Hewitt

  • 1Unilever Research Colworth, Bedfordshire, MK44 1LQ, Sharnbrook, UK. gerhard.nebe-von-caron@unilever.com

Journal of Microbiological Methods
|September 23, 2000
PubMed
Summary

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Direct optical detection using flow cytometry offers a faster, more accurate way to analyze bacterial populations than traditional growth-dependent methods. This technique assesses cell viability and function, improving bacterial analysis and process control.

Area of Science:

  • Microbiology
  • Biotechnology
  • Analytical Chemistry

Background:

  • Traditional bacterial analysis relies on growth-dependent methods, which can be slow and prone to artifacts from post-sampling growth.
  • Direct optical detection methods, particularly flow cytometry, are gaining popularity for their speed and ability to analyze individual cells.
  • Assessing bacterial viability and physiological state is crucial for various applications, from fermentation control to understanding microbial ecology.

Purpose of the Study:

  • To highlight the advantages of direct optical detection, specifically flow cytometry, over traditional growth-dependent bacterial analysis.
  • To demonstrate the application of flow cytometry in assessing bacterial viability, physiological state, and culture heterogeneity.
  • To explore the use of flow cytometry in process control for bacterial fermentations and in understanding the viable but non-culturable (VBNC) state.

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Main Methods:

  • Bacterial discrimination using nucleic acid staining and sample disaggregation for enumeration.
  • Determination of bacterial cell functions including reproductive ability, metabolic activity, and membrane integrity.
  • Single-cell sorting coupled with fluorescent labeling and growth-based correlation for viability assessment.
  • Immunofluorescent labeling for species identification within complex populations.

Main Results:

  • Flow cytometry provides reliable enumeration, minimizing data artifacts from post-sampling growth.
  • Significant drops in membrane potential and integrity were observed in fed-batch fermentations, suggesting flow cytometry can improve process control.
  • New media protecting against oxidative stress improved recovery of heat-injured cells, with actively respiring cells showing the greatest improvement.
  • Multiparameter flow cytometry offers high precision in assessing cell function at the single-cell level, questioning the validity of classical viability methods.

Conclusions:

  • Flow cytometry offers a powerful alternative to growth-dependent methods for bacterial analysis, providing detailed insights into cell viability and function.
  • The technique has significant implications for optimizing industrial fermentations and understanding microbial stress responses, including the VBNC phenomenon.
  • Multiparameter flow cytometry challenges the reliance on traditional viability assessments, paving the way for more accurate and comprehensive microbial analysis.