Automated quantification of budding <i>Saccharomyces cerevisiae</i> using a novel image cytometry method

Laverty, Daniel J.; Kury, Alexandria; Kuksin, Dmitry; Pirani, Alnoor; Flanagan, Kevin C.; Chan, Leo Li‐Ying

doi:10.1007/s10295-013-1263-9

Cited by 12 publications

(6 citation statements)

References 16 publications

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“…Other authors have used imaging microscopy [ 5 , 6 ] or image cytometry of shake flask cultures [ 7 , 8 ] for assessing the morphology of yeast cells. The acquisition of the images is conducted off line , but the particle recognition is usually automated.…”

Section: Introductionmentioning

confidence: 99%

Real-time monitoring of the budding index in Saccharomyces cerevisiae batch cultivations with in situ microscopy

Marbà-Ardébol

Emmerich²,

Muthig³

et al. 2018

Microb Cell Fact

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BackgroundThe morphology of yeast cells changes during budding, depending on the growth rate and cultivation conditions. A photo-optical microscope was adapted and used to observe such morphological changes of individual cells directly in the cell suspension. In order to obtain statistically representative samples of the population without the influence of sampling, in situ microscopy (ISM) was applied in the different phases of a Saccharomyces cerevisiae batch cultivation. The real-time measurement was performed by coupling a photo-optical probe to an automated image analysis based on a neural network approach.ResultsAutomatic cell recognition and classification of budding and non-budding cells was conducted successfully. Deviations between automated and manual counting were considerably low. A differentiation of growth activity across all process stages of a batch cultivation in complex media became feasible. An increased homogeneity among the population during the growth phase was well observable. At growth retardation, the portion of smaller cells increased due to a reduced bud formation. The maturation state of the cells was monitored by determining the budding index as a ratio between the number of cells, which were detected with buds and the total number of cells. A linear correlation between the budding index as monitored with ISM and the growth rate was found.ConclusionIt is shown that ISM is a meaningful analytical tool, as the budding index can provide valuable information about the growth activity of a yeast cell, e.g. in seed breeding or during any other cultivation process. The determination of the single-cell size and shape distributions provided information on the morphological heterogeneity among the populations. The ability to track changes in cell morphology directly on line enables new perspectives for monitoring and control, both in process development and on a production scale.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0922-y) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Real-time monitoring of the budding index in Saccharomyces cerevisiae batch cultivations with in situ microscopy

Marbà-Ardébol

Emmerich²,

Muthig³

et al. 2018

Microb Cell Fact

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“…Image cytometry technologies have made significant improvements over the last decade from single sample measurement to high‐throughput plate‐based analysis. Image cytometry has previously demonstrated improvements to cell‐based assays such as transduction efficiency (13,14), cell proliferation (15‐17), and cell killing assay (18‐21). In general, the system can image and analyze an entire 96‐well plate using bright field and fluorescence in less than 5 and 10 min, respectively.…”

mentioning

confidence: 99%

High‐Throughput Image Cytometry Detection Method for CAR‐T Transduction, Cell Proliferation, and Cytotoxicity Assays

Wang

Chan²,

Grimaud

et al. 2020

Cytometry Pt A

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Chimeric antigen receptor (CAR)‐T cell therapy has drawn much attention due to its recent clinical success in B‐cell malignancies. In general, the CAR‐T cell discovery process consists of CAR identification, T‐cell activation, transduction, and expansion, as well as assessment of CAR‐T cytotoxicity. The current evaluation methods for the CAR‐T discovery process can be time‐consuming, low‐throughput and requires the preparation of multiple sacrificial samples in order to produce kinetic data. In this study, we employed the use of a plate‐based image cytometer to monitor anti‐CAIX (carbonic anhydrase IX) G36 CAR‐T generation and assess its cytotoxic potency of direct and selective killing against CAIX+ SKRC‐59 human renal cell carcinoma cells. The transduction efficiency and cytotoxicity results were analyzed using image cytometry and compared directly to flow cytometry and Chromium 51 (51Cr) release assays, showing that image cytometry was comparable against these conventional methods. Image cytometry method streamlines the assays required during the CAR‐T cell discovery process by analyzing a plate of T cells from CAR‐T generation to in vitro functional assays with minimum disruption. The proposed method can reduce assay time and uses less cell samples by imaging and analyze the same plate over time without the need to sacrifice any cells. The ability to monitor kinetic data can allow additional insights into the behavior and interaction between CAR‐T and target tumor cells. © 2020 International Society for Advancement of Cytometry

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“…PI will stain the DNA of fixed cells providing a classic cell cycle to ensure the population is growing. Living cells, with intact plasma membranes, are generally impermeable to the stain; however, there is some evidence that PI may be capable of staining stressed yeast cells with damaged, but repairable, plasma membranes [9,11,[17][18][19][20][21][24][25][26]28,29]. As such, care should be taken in interpreting viability results when the strains used are chemically or environmentally stressed.…”

Section: Dyes and Fluorochromesmentioning

confidence: 99%

Flow Cytometry — A Primer for Modern Analytical Technology to Assess Yeast Culture Quality for Fermentation

Bird,

Danderson

2024

JDS

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Flow cytometry is an analytical technology that allows users to interrogate single cells at high speeds to provide a detailed data-rich analysis of complex cell populations. Flow cytometry can offer an effective means to quickly evaluate yeast culture integrity using a small sample to assess the complexity and underlying heterogeneity in a culture. It can assess cell viability, purity and complexity, with respect to potential contaminating species, of the culture providing a higher level of confidence in the population to be used for fermentation. This is an important problem to address in breweries where multiple domestic yeast strains are employed or where various wild strains of Saccharomyces cerevisiae as well as wild or cultured Brettanomyces species are used for fermentation. Such analysis can enhance confidence and reduce costs associated with new yeast culture purchase in commercial fermentations and promote a deeper understanding of the complexities of mixed fermentations. Lastly, flow cytometry can add new levels of analysis associated with fluorescent labeling of cell DNA content and selected cell proteins.

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Automated quantification of budding Saccharomyces cerevisiae using a novel image cytometry method

Cited by 12 publications

References 16 publications

Real-time monitoring of the budding index in Saccharomyces cerevisiae batch cultivations with in situ microscopy

Real-time monitoring of the budding index in Saccharomyces cerevisiae batch cultivations with in situ microscopy

High‐Throughput Image Cytometry Detection Method for CAR‐T Transduction, Cell Proliferation, and Cytotoxicity Assays

Flow Cytometry — A Primer for Modern Analytical Technology to Assess Yeast Culture Quality for Fermentation

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