scholarly journals Isolating and culturing of single microbial cells by laser ejection sorting technology

2021 ◽  
Author(s):  
Peng Liang ◽  
Bo Liu ◽  
Yun Wang ◽  
Kunxiang Liu ◽  
Yinping Zhao ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Cell ◽  
Single Cells ◽  
Lactobacillus Rhamnosus ◽  
Fluorescence Signal ◽  
Microbial Cells ◽  
E Coli ◽  
Average Survival ◽  
Forward Transfer ◽  
Single Cell Isolation

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.

scholarly journals Laser induced isolation and cultivation of single microbial cells

2020 ◽  
Author(s):  
Peng Liang ◽  
Huan Wang ◽  
Yun Wang ◽  
Yinping Zhao ◽  
Wei E. Huang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Cell ◽  
Cell Sorting ◽  
Single Cells ◽  
Lactobacillus Rhamnosus ◽  
Microbial Cells ◽  
E Coli ◽  
Eukaryotic Microorganisms ◽  
Cultivation Technique ◽  
Single Cell Isolation

AbstractSingle cell isolation and cultivation play an important role in studying physiology, gene expression and functions of microorganisms. Laser Induced Forward Transfer Technique (LIFT) has been applied to isolate single cells but the cell viability after sorting is unclear. We demonstrate that a three-layer LIFT system could be applied to isolate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae) and the sorted single cells were able to be cultured. The experiment results showed that the average cultivation recovery rate of the ejected single cells were 58% for Saccharomyces cerevisiae, 22% for E. coli, and 74% for Lactobacillus rhamnosus GG (LGG). The identities of the cultured cells from single cell sorting were confirmed by using colony PCR with 16S-rRNA for bacteria and large unit rRNA for yeast and subsequent sequencing. This precise sorting and cultivation technique of live single microbial cells can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture single microorganisms with specific functions, revealing their roles in the natural community.ImportanceSingle cell isolation and cultivation are crucial to recover microorganisms for the study of physiology, gene expression and functions. We developed a laser induced cell sorting technology to precisely isolate single microbial cells from a microscopic slide. More importantly, the isolated single microbial cells are still viable for cultivation. We demonstrate to apply the live sorting method to isolate and cultivate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae). This precise sorting and cultivation technique can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture specifically targeted single microorganisms from microbial community.Abstract Graphic

scholarly journals Separations-encoded microparticles for single-cell western blotting

2019 ◽  
Author(s):  
Burcu Gumuscu ◽  
Amy Elizabeth Herr
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Thin Sheet ◽  
Protein Electrophoresis ◽  
Microfluidic Channels ◽  
Fluorescence Signal ◽  
Analytical Sensitivity ◽  
Planar Array ◽  
Single Cell Isolation ◽  
Mechanical Release

Direct measurement of proteins from single cells has been realized at the microscale using microfluidic channels, capillaries, and semi-enclosed microwell arrays. Although powerful, these formats are constrained, with the enclosed geometries proving cumbersome for multistage assays, including electrophoresis followed by immunoprobing. We introduce a hybrid microfluidic format that toggles between a planar microwell array and a suspension of microparticles. The planar array is stippled in a thin sheet of polyacrylamide gel, for efficient single-cell isolation and protein electrophoresis of hundreds-to-thousands of cells. Upon mechanical release, array elements become a suspension of separations-encoded microparticles for more efficient immunoprobing due to enhanced mass transfer. Dehydrating microparticles offer improved analytical sensitivity owing to in-gel concentration of fluorescence signal for high-throughput single-cell targeted proteomics.

A microfluidic device enabling deterministic single cell trapping and release

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Huichao Chai ◽  
Yongxiang Feng ◽  
Fei Liang ◽  
Wenhui Wang
Keyword(s):  
Chemical Analysis ◽  
Single Cell ◽  
Microfluidic Device ◽  
Single Cells ◽  
Cell Isolation ◽  
Cell Trapping ◽  
Analysis Techniques ◽  
Single Cell Trapping ◽  
Single Cell Isolation ◽  
Made In

Successful single-cell isolation is a pivotal technique for subsequent biological and chemical analysis of single cells. Although significant advances have been made in single-cell isolation and analysis techniques, most passive...

scholarly journals Connecting the Dots between Mechanosensitive Channel Abundance, Osmotic Shock, and Survival at Single-Cell Resolution

2018 ◽  
Vol 200 (23) ◽  
Author(s):  
Griffin Chure ◽  
Heun Jin Lee ◽  
Akiko Rasmussen ◽  
Rob Phillips
Keyword(s):  
Cell Survival ◽  
Single Cell ◽  
Osmotic Shock ◽  
Single Cells ◽  
Mechanosensitive Channel ◽  
Membrane Tension ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Structural Methods

ABSTRACTRapid changes in extracellular osmolarity are one of many insults microbial cells face on a daily basis. To protect against such shocks,Escherichia coliand other microbes express several types of transmembrane channels that open and close in response to changes in membrane tension. InE. coli, one of the most abundant channels is the mechanosensitive channel of large conductance (MscL). While this channel has been heavily characterized through structural methods, electrophysiology, and theoretical modeling, our understanding of its physiological role in preventing cell death by alleviating high membrane tension remains tenuous. In this work, we examine the contribution of MscL alone to cell survival after osmotic shock at single-cell resolution using quantitative fluorescence microscopy. We conducted these experiments in anE. colistrain which is lacking all mechanosensitive channel genes save for MscL, whose expression was tuned across 3 orders of magnitude through modifications of the Shine-Dalgarno sequence. While theoretical models suggest that only a few MscL channels would be needed to alleviate even large changes in osmotic pressure, we find that between 500 and 700 channels per cell are needed to convey upwards of 80% survival. This number agrees with the average MscL copy number measured in wild-typeE. colicells through proteomic studies and quantitative Western blotting. Furthermore, we observed zero survival events in cells with fewer than ∼100 channels per cell. This work opens new questions concerning the contribution of other mechanosensitive channels to survival, as well as regulation of their activity.IMPORTANCEMechanosensitive (MS) channels are transmembrane protein complexes which open and close in response to changes in membrane tension as a result of osmotic shock. Despite extensive biophysical characterization, the contribution of these channels to cell survival remains largely unknown. In this work, we used quantitative video microscopy to measure the abundance of a single species of MS channel in single cells, followed by their survival after a large osmotic shock. We observed total death of the population with fewer than ∼100 channels per cell and determined that approximately 500 to 700 channels were needed for 80% survival. The number of channels we found to confer nearly full survival is consistent with the counts of the numbers of channels in wild-type cells in several earlier studies. These results prompt further studies to dissect the contribution of other channel species to survival.

scholarly journals Single cell isolation process with laser induced forward transfer

2017 ◽  
Vol 11 (1) ◽  
Author(s):  
Yu Deng ◽  
Philippe Renaud ◽  
Zhongning Guo ◽  
Zhigang Huang ◽  
Ying Chen
Keyword(s):  
Single Cell ◽  
Cell Isolation ◽  
Forward Transfer ◽  
Single Cell Isolation

scholarly journals High-Throughput Single-Cell Cultivation on Microfluidic Streak Plates

2016 ◽  
Vol 82 (7) ◽  
pp. 2210-2218 ◽  
Author(s):  
Cheng-Ying Jiang ◽  
Libing Dong ◽  
Jian-Kang Zhao ◽  
Xiaofang Hu ◽  
Chaohua Shen ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Response Analysis ◽  
Bacterial Strains ◽  
Content Type ◽  
Chemical Gradients ◽  
Droplet Array ◽  
Polycyclic Aromatic ◽  
Single Cell Isolation

ABSTRACTThis paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation ofEscherichia coliand antimicrobial susceptibility testing ofPseudomonas aeruginosaPAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including fourMycobacteriumisolates and a previously unknown fluoranthene-degradingBlastococcusspecies.

dMSCC: a microfluidic platform for microbial single-cell cultivation of Corynebacterium glutamicum under dynamic environmental medium conditions

Lab on a Chip ◽  
2020 ◽  
Vol 20 (23) ◽  
pp. 4442-4455
Author(s):  
Sarah Täuber ◽  
Corinna Golze ◽  
Phuong Ho ◽  
Eric von Lieres ◽  
Alexander Grünberger
Keyword(s):  
Single Cell ◽  
Corynebacterium Glutamicum ◽  
Environmental Conditions ◽  
Single Cells ◽  
Dynamic Environment ◽  
Microfluidic System ◽  
Small Cell ◽  
Cell Cultivation ◽  
Microbial Cells ◽  
Cell Clusters

Microbial cells are often exposed to rapidly fluctuating environmental conditions. A novel microfluidic system for the cultivation of single cells and small cell clusters is presented under dynamic environment conditions.

scholarly journals A practical solution for preserving single cells for RNA sequencing

2017 ◽  
Author(s):  
Moustafa Attar ◽  
Eshita Sharma ◽  
Shuqiang Li ◽  
Claire Bryer ◽  
Laura Cubitt ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Rna Seq ◽  
Rna Integrity ◽  
Tissue Integrity ◽  
Free Amine ◽  
Gene Level ◽  
Single Cell Isolation ◽  
Amine Groups ◽  
Cell Preservation

AbstractThe design and implementation of single-cell experiments is often limited by their requirement for fresh starting material. We have adapted a method for histological tissue fixation using dithio-bis(succinimidyl propionate) (DSP), or Lomant’s Reagent, to stabilise cell samples for single-cell transcriptomic applications. DSP is a reversible cross-linker of free amine groups that has previously been shown to preserve tissue integrity for histology while maintaining RNA integrity and yield in bulk RNA extractions. Although RNA-seq data from DSP-fixed single cells appears to be prone to characteristic artefacts, such as slightly reduced yield of cDNA and a detectable 3’ bias in comparison with fresh cells, cell preservation using DSP does not appear to substantially reduce RNA complexity at the gene level. In addition, there is evidence that instantaneous fixation of cells can reduce inter-cell technical variability. The ability of DSP-fixed cells to retain commonly used dyes, such as propidium iodide, enables the tracking of experimental sub-populations and the recording of cell viability at the point of fixation. Preserving cells using DSP will remove several barriers in the staging of single-cell experiments, including the transport of samples and the scheduling of shared equipment for downstream single-cell isolation and processing.

scholarly journals Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging

2018 ◽  
Vol 115 (5) ◽  
pp. 962-967 ◽  
Author(s):  
Carola Gregor ◽  
Klaus C. Gwosch ◽  
Steffen J. Sahl ◽  
Stefan W. Hell
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Flavin Mononucleotide ◽  
Background Signal ◽  
Cell Level ◽  
Spatiotemporal Resolution ◽  
High Background ◽  
E Coli ◽  
Bacterial Bioluminescence ◽  
Single Cell Imaging

Bioluminescence imaging of single cells is often complicated by the requirement of exogenous luciferins that can be poorly cell-permeable or produce high background signal. Bacterial bioluminescence is unique in that it uses reduced flavin mononucleotide as a luciferin, which is abundant in all cells, making this system purely genetically encodable by the lux operon. Unfortunately, the use of bacterial bioluminescence has been limited by its low brightness compared with other luciferases. Here, we report the generation of an improved lux operon named ilux with an approximately sevenfold increased brightness when expressed in Escherichia coli; ilux can be used to image single E. coli cells with enhanced spatiotemporal resolution over several days. In addition, since only metabolically active cells produce bioluminescent signal, we show that ilux can be used to observe the effect of different antibiotics on cell viability on the single-cell level.

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