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 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.

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 Using single-cell transcriptomics to understand functional states and interactions in microbial eukaryotes

2019 ◽  
Vol 374 (1786) ◽  
pp. 20190098 ◽  
Author(s):  
Chuan Ku ◽  
Arnau Sebé-Pedrós
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Interspecific Interactions ◽  
Single Cells ◽  
Life Cycles ◽  
Modern Biology ◽  
Microbial Eukaryotes ◽  
Eukaryotic Microorganisms

Understanding the diversity and evolution of eukaryotic microorganisms remains one of the major challenges of modern biology. In recent years, we have advanced in the discovery and phylogenetic placement of new eukaryotic species and lineages, which in turn completely transformed our view on the eukaryotic tree of life. But we remain ignorant of the life cycles, physiology and cellular states of most of these microbial eukaryotes, as well as of their interactions with other organisms. Here, we discuss how high-throughput genome-wide gene expression analysis of eukaryotic single cells can shed light on protist biology. First, we review different single-cell transcriptomics methodologies with particular focus on microbial eukaryote applications. Then, we discuss single-cell gene expression analysis of protists in culture and what can be learnt from these approaches. Finally, we envision the application of single-cell transcriptomics to protist communities to interrogate not only community components, but also the gene expression signatures of distinct cellular and physiological states, as well as the transcriptional dynamics of interspecific interactions. Overall, we argue that single-cell transcriptomics can significantly contribute to our understanding of the biology of microbial eukaryotes. This article is part of a discussion meeting issue ‘Single cell ecology’.

scholarly journals Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis

2020 ◽  
Author(s):  
Ayushi Agrawal ◽  
Chandra Kanth Bandi ◽  
Tucker Burgin ◽  
Youngwoo Woo ◽  
Heather B. Mayes ◽  
...  
Keyword(s):  
Single Cell ◽  
Click Chemistry ◽  
Directed Evolution ◽  
Cell Sorting ◽  
Screening Methods ◽  
Carbohydrate Active Enzymes ◽  
E Coli ◽  
Highly Sensitive ◽  
Increased Activity

AbstractEngineering of carbohydrate-active enzymes like glycosynthases for chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable directed evolution based protein engineering methods. Currently there are no ultrahigh-throughput screening methods available for rapid and highly sensitive single cell-based screening of evolved glycosynthase enzymes employing azido sugars as substrates. Here, we report a fluorescence-based approach employing click-chemistry for the selective detection of glycosyl azides (versus free inorganic azides) that facilitated ultrahigh-throughput in-vivo single cell-based assay of glycosynthase activity. This discovery has led to the development of a directed evolution methodology for screening and sorting glycosynthase mutants for synthesis of desired fucosylated oligosaccharides. Our screening technique facilitated rapid fluorescence activated cell sorting of a large library of glycosynthase variants (>106 mutants) expressed in E. coli to identify several novel mutants with increased activity for β-fucosyl-azide activated donor sugars towards desired acceptor sugars, demonstrating the broader applicability of this methodology.

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 The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors

Blood ◽  
1995 ◽  
Vol 85 (9) ◽  
pp. 2422-2435 ◽  
Author(s):  
EK Waller ◽  
J Olweus ◽  
F Lund-Johansen ◽  
S Huang ◽  
M Nguyen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Progenitor Cells ◽  
Cell Sorting ◽  
Single Cells ◽  
Light Scatter ◽  
Hematopoietic Progenitor ◽  
Individual Culture ◽  
Hla Dr ◽  
Growth Properties

There is a long-standing controversy as to whether a single bone marrow (BM)-derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence- activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38- , HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM.

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.

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