Single-cell entrapment and microcolony development within uniform microspheres amenable to flow cytometry

Area of Science:

• Microbiology
• Biotechnology
• Analytical Chemistry

Background:

• Traditional methods for microbial analysis often require cell lysis or culturing, limiting direct colony-based analysis.
• Flow cytometry is a powerful tool for cell analysis but is typically limited to single-cell applications.
• There is a need for methods that combine microbial growth with high-throughput analysis techniques.

Purpose of the Study:

• To develop a method for encapsulating viable microbial cells within uniform microspheres.
• To enable the growth of microbial colonies within these microspheres.
• To facilitate the analysis and sorting of microbial colonies using flow cytometry.

Main Methods:

• Microbial cells suspended in a gellable liquid (low-melting-temperature agarose) were ejected through an ultrasonically vibrated orifice.
• The cell-containing jet was sheared into uniform droplets.
• Droplets were gelled into solid spheres using directed cooling air streams, causing dehydration and size reduction.

Main Results:

• Uniform microbeads with precisely controlled diameters (10-40 microns) were produced.
• Encapsulated bacterial and yeast cells successfully multiplied into microcolonies within the beads.
• The developed system demonstrated the ability to analyze and sort entire microbial colonies via flow cytometry.

Conclusions:

• The described method successfully encapsulates viable microbial cells in uniform agarose beads.
• The encapsulated cells can proliferate into microcolonies, expanding the scope of flow cytometry applications.
• This technology offers a promising approach for high-throughput analysis and sorting of microbial colonies in various microbiological fields.