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Related Concept Videos

Flow Cytometry01:23

Flow Cytometry

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The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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Improvement of Bacillus subtilis Spore Enumeration and Label Analysis in Flow Cytometry
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Absolute bacterial cell enumeration using flow cytometry.

F Ou1, C McGoverin1, S Swift2

  • 1Department of Physics, The Dodd-Walls Centre for Photonic and Quantum Technologies, The University of Auckland, Auckland, New Zealand.

Journal of Applied Microbiology
|June 11, 2017
PubMed
Summary
This summary is machine-generated.

This study presents a reliable flow cytometry method using reference beads for counting live and dead bacteria in mixtures. The method accurately enumerates bacteria, applicable to basic flow cytometers without specialized features.

Keywords:
bacteriabacterial cell enumerationcounting beadsdetectionflow cytometryfluorescence

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Area of Science:

  • Microbiology
  • Analytical Chemistry
  • Biotechnology

Background:

  • Accurate enumeration of live and dead bacteria is crucial for various applications.
  • Traditional methods can be labor-intensive and may not be suitable for complex mixtures.
  • Flow cytometry offers a rapid and sensitive approach for cell analysis.

Purpose of the Study:

  • To evaluate a flow cytometry protocol for enumerating live and dead bacteria in mixtures.
  • To establish a method using reference beads for accurate bacterial quantification.
  • To validate the protocol's applicability to basic flow cytometers.

Main Methods:

  • Prepared mixtures of live and dead Escherichia coli with varying live:dead ratios.
  • Stained bacteria with SYTO 9 and propidium iodide.
  • Added 6-μm reference beads for enumeration.
  • Validated results against agar plate counts.

Main Results:

  • Demonstrated a linear relationship between flow cytometry counts and agar plate counts for both live (R² = 0.99) and dead E. coli (R² = 0.93).
  • Achieved reliable enumeration of live E. coli at concentrations above 2.5% live.
  • Established a lower limit of approximately 20% dead for reliable enumeration of dead E. coli.

Conclusions:

  • The developed flow cytometry protocol provides accurate bacterial enumeration in mixtures.
  • The method is applicable to basic flow cytometers, enhancing accessibility.
  • This work lays the foundation for applying similar methods to diverse bacterial strains.