Absolute bacterial cell enumeration using flow cytometry

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.