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

Abstract

Single cell isolation and cultivation play an important role in studying physiology, gene expression and functions of microorganisms. A series of single-cell isolation technologies have been developed, among which single-cell ejection technology is one of the most promising. Single cell ejection technology has applied Laser Induced Forward Transfer Technique (LIFT) to isolate bacteria but the viability (or recovery rate) of cells after sorting has not been clarified in the current research progress. In this work, to keep the cells alive as much as possible, we propose a three-layer LIFT system (top layer: 25-nm aluminum film; second layer: 3 μm agar media; third layer: liquid containing bacterial) for the isolation and cultivation of single Gram-negative ( E. coli ), Gram-positive ( Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms ( Saccharomyces cerevisiae ). The experiment results showed that the average survival rates for ejected pure single cells were 63% for Saccharomyces cerevisiae , 22% for E. coli DH5α, and 74% for LGG. In addition, we successfully isolated and cultured the GFP expressing E. coli JM109 from the mixture containing complex communities of soil bacteria by fluorescence signal. The average survival rate of E. coli JM109 was demonstrated to be 25.3%. In this study, the isolated and cultured single colonies were further confirmed by colony PCR and sequencing. Such precise sorting and cultivation technique of live single microbial cells could be coupled with other microscopic approaches to isolate single microorganisms with specific functions, revealing their roles in the natural community. Importance We developed a laser induced forward transfer (LIFT) technology to accurately isolate single live microbial cells. The cultivation recovery rates of the ejected single cells were 63% for Saccharomyces cerevisiae , 22% for E. coli DH5α, and 74% for Lactobacillus rhamnosus GG (LGG). Coupled LIFT with fluorescent microscope, we demonstrated that single cells of GFP expressing E. coli JM109 were sorted according to fluorescence signal from a complex community of soil bacteria, and subsequently cultured with 25% cultivation recovery rate. This single cell live sorting technology could isolate single microbes with specific functions, revealing their roles in the natural community.

High-throughput single-cell sorting by stimulated Raman-activated cell ejection

“…Successful cultivation after ejection for three microorganism species (S. cerevisiae, C. albicans, E. coli) was shown. For E. coli, a similar recovery rate as the previous report (~22%) (18) was achieved.…”

“…The “ejected” microparticle is then collected by the bottom collector, which is fabricated by coating a layer of polydimethylsiloxane (PDMS) on a standard coverslip. For live cell sorting, an additional thin layer of agarose gel is introduced on the TiO 2 coating, mitigating potential mechanical damage during the ejection process (18). In addition, a complementary layer of agarose is introduced at the bottom of the collector to safeguard the cells from any impact during the landing process (18, 44).…”

“…For live cell sorting, an additional thin layer of agarose gel is introduced on the TiO 2 coating, mitigating potential mechanical damage during the ejection process (18). In addition, a complementary layer of agarose is introduced at the bottom of the collector to safeguard the cells from any impact during the landing process (18, 44).…”

“…By detecting inelastic photon scattering (9), Raman spectroscopy could characterize the endogenous chemical content of single cells and is capable of probing cell metabolic activity (10). By integrating Raman spectroscopy with cell sorting methodologies, including flow (11, 12), optical tweezer (13, 14), dielectrophoresis (15, 16), and cell ejection (17, 18), a plurality of biomedical applications were achieved (19). Raman-activated cell sorting (RACS) has found extensive use in microbiology for isolating functional individuals from a community.…”

Visualization of a Limonene Synthesis Metabolon Inside Living Bacteria by Hyperspectral SRS Microscopy

“…Further investigations of localized limonene biosynthesis may include broadband coherent Raman imaging and time-lapse SRS imaging of live E. coli cells. With the live-cell imaging capacity, integration of our imaging platform with downstream cell sorting [58,59] is expected.…”