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Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

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Isolation and Culture of Single Microbial Cells by Laser Ejection Sorting Technology.

Peng Liang^1,2, Bo Liu^1,2, Yun Wang³

¹State Key laboratory of Applied Optics, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun, People's Republic of China.

Applied and Environmental Microbiology

|November 24, 2021

Summary

We developed a laser-induced forward transfer (LIFT) system to isolate single live microbial cells, achieving high recovery rates for yeast and bacteria. This technology enables precise sorting of specific microbes from complex communities for functional studies.

Keywords:

fluorescence laser-induced forward transfer single bacterial cell isolation and culture single-cell culturing single-cell isolating three-layer LIFT chip

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Area of Science:

Microbiology
Biotechnology
Cell Biology

Background:

Single-cell isolation is crucial for understanding microbial physiology and gene expression.
Laser-induced forward transfer (LIFT) is a promising single-cell isolation technology, but cell viability post-sorting requires clarification.

Purpose of the Study:

To develop and evaluate a three-layer LIFT system for isolating and cultivating live single microbial cells.
To determine the viability and recovery rates of various microorganisms after LIFT-based isolation.
To demonstrate the capability of LIFT coupled with fluorescence microscopy for sorting specific microbial cells from complex environments.

Main Methods:

A three-layer LIFT system was designed using aluminum film, agar media, and a liquid medium containing microorganisms.
The system was tested for isolating single cells of Saccharomyces cerevisiae, Escherichia coli (Gram-negative), and Lactobacillus rhamnosus GG (Gram-positive).
Fluorescence-guided sorting was performed using green fluorescent protein (GFP)-expressing E. coli JM109 within a mixed soil bacterial community.

Main Results:

Average recovery rates for ejected single cells were 63% for S. cerevisiae, 22% for E. coli DH5α, and 74% for LGG.
A 25.3% average survival rate was achieved for GFP-expressing E. coli JM109 sorted from a complex soil bacterial community.
Isolated single colonies were confirmed via colony PCR and sequencing.

Conclusions:

The developed three-layer LIFT system effectively isolates and cultivates live single microbial cells with varying recovery rates.
This technology enables precise isolation of specific microbial cells, including those identified by fluorescence, from complex environmental samples.
LIFT-based single-cell sorting coupled with cultivation offers a powerful tool for functional studies of microorganisms within their natural communities.