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

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.