Novel image cytometric method for detection of physiological and metabolic changes in <i>Saccharomyces cerevisiae</i>

Chan, Leo Li‐Ying; Kury, Alexandria; Wilkinson, Alisha R.; Berkes, Charlotte A.; Pirani, Alnoor

doi:10.1007/s10295-012-1177-y

Cited by 18 publications

(11 citation statements)

References 26 publications

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“…46 Monitoring additional parameters using the currently developed platform can be easily performed by adding adequate fluorescence markers to the dilution buffer and recording the fluorescence signal in different wavelength bands. Besides viability, other physiological and metabolic parameters, such as apoptosis, hypoxia and other stress related conditions, can be quantified and used to enhance the efficiency of bioreactors.…”

Section: Discussionmentioning

confidence: 99%

Real-time direct cell concentration and viability determination using a fully automated microfluidic platform for standalone process monitoring

Nunes

Kjærulff²,

Dufva

et al. 2015

Analyst

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The industrial production of cells has a large unmet need for greater process monitoring, in addition to the standard temperature, pH and oxygen concentration determination. Monitoring the cell health by a vast range of fluorescence cell-based assays can greatly improve the feedback control and thereby ensure optimal cell production, by prolonging the fermentation cycle and increasing the bioreactor output. In this work, we report on the development of a fully automated microfluidic system capable of extracting samples directly from a bioreactor, diluting the sample, staining the cells, and determining the total cell and dead cells concentrations, within a time frame of 10.3 min. The platform consists of custom made stepper motor actuated peristaltic pumps and valves, fluidic interconnections, sample to waste liquid management and image cytometry-based detection. The total concentration of cells is determined by brightfield microscopy, while fluorescence detection is used to detect propidium iodide stained non-viable cells. This method can be incorporated into facilities with bioreactors to monitor the cell concentration and viability during the cultivation process. Here, we demonstrate the microfluidic system performance by monitoring in real time the cell concentration and viability of yeast extracted directly from an in-house made bioreactor. This is the first demonstration of using the Dean drag force, generated due to the implementation of a curved microchannel geometry in conjunction with high flow rates, to promote passive mixing of cell samples and thus homogenization of the diluted cell plug. The autonomous operation of the fluidics furthermore allows implementation of intelligent protocols for administering air bubbles from the bioreactor in the microfluidic system, so that these will be guided away from the imaging region, thereby significantly improving both the robustness of the system and the quality of the data.

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Section: Discussionmentioning

confidence: 99%

Real-time direct cell concentration and viability determination using a fully automated microfluidic platform for standalone process monitoring

Nunes

Kjærulff²,

Dufva

et al. 2015

Analyst

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“…The obtained results are in agreement with the results obtained by other authors using the manual method [11,17,30,31]. A comparison of the obtained linear interval and CV of Easycounter YC with other similar image cytometers showed that they have a similar CV, but the optimal interval is different for each different device: 1 Â 10 5 -2 Â 10 6 cells/mL with CV 3-10% for NucleoCounter YC-100 (company ChemoMetec) and 4 Â 10 6 -6.6 Â 10 7 cells/mL with CV 4-13% for Cellometer Auto X4 (10Â) (Nexcelom Bioscence) [11,23,28,31].…”

Section: Cell Count Linearity and Reliabilitymentioning

confidence: 99%

“…Currently, with the emergence of automated cell counters, researchers can analyze a large number of samples faster [15,16] and with less variability stemming from human-error. The development of computers, automation software, optics, fluorescent dyes and precision manufacturing techniques together with modern technologies such as flow cytometry and image cytometry, has helped to overcome many of the drawbacks associated with the hemacytometer [17][18][19][20][21][22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Brewing yeast viability measured using a novel fluorescent dye and image cytometer

Atanasova

Yordanova

Nenkova

et al. 2019

Biotechnology & Biotechnological Equipment

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This study presents an automated, image-based cytometry method for determination of total counts and viability of yeast cells by using a newly synthesized DNA fluorescent dye PO-TEDM-1 and a new Easycounter YC instrument. The synthesized polycationic asymmetric monomethine cyanine dye PO-TEDM-1 penetrates only into dead cells. The new fluorescent dye has high nucleic acid sensitivity and rapid interaction kinetics. The optimal concentration of the fluorescent dye for staining dead cells was 1 mg mL À1. The Easycounter YC system was used to determine the total cell count and viability of Saccharomyces carlsbergensis. The actual viability measured using the proposed method significantly correlated with the theoretical viability (R 2 of 0.9988). The optimal linear interval was from 1 Â 10 5 to 1 Â 10 7 cells mL À1. The coefficient of variation with Easycounter YC in the optimal range was 2.5-4%, whereas that of the manual hemocytometry method, in the same range was higher, 15-23%. We tested the procedure in a study of the total cell count and viability of yeast cells from the propagators in beer production as a function of the dilution. The proposed method can be used in assays involving simple cell counting and quality assurance in sample bioprocessing.

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“…The DNA content of yeast cells provides information about the cell cycle and is thus a powerful tool to assess the effects of propagation parameters on the yeast cell cycle and yeast physiology. FCM assays were developed for assessing the physiological status of the yeast cells during fermentation process [24,25]), for determination of yeast viability and cell number in a brewery [26][27][28][29][30][31][32], predictions of flocculation ability of brewer yeasts, separation of the prototroph yeast fusants [33], assessment of Saccharomyces cerevisiae vitality [34,35,36], detection of spoilage microorganisms [24,37], the influence of beer process conditions on beer stability [38], identification of haploid strains of industrial brewer's yeast, fermentation process control [20,39,40]. The use of FCM for age assessment of a yeast population and its application in beer fermentation was reviewed in the publication [41].…”

Section: Brewingmentioning

confidence: 99%

Updates on the applications of flow cytometry in microbiology

2019

Lett Appl NanoBioSci

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Flow cytometry (FCM) was first developed for medical and clinical applications such as hematology and oncology. Although these areas still account for the vast majority of publications on this technique, during the past few years it has been also introduced in other areas, such as optimization and monitoring of biotechnological and environmental processes, pharmacology, toxicology, bacteriology and virology. In the food and drinks industries, the time required for conventional microbiology tests can lead to substantial delays in product release to the market. FCM has been used in conjunction with viability markers for rapid counting of yeast, molds and bacterial cells, including foodborne pathogens and microbial contaminants, in food products as well as for monitoring and improving the final products quality. FCM is an excellent tool, still unexplored in clinical microbiology, allowing for detection of cellular and non-cellular components in different clinical specimens, the study of antimicrobial activity, allowing for rapid and direct antimicrobial susceptibility testing and for the investigation of resistance mechanisms. Recent FCM developments important for addressing questions in environmental microbiology include the study of microbial physiology under environmentally relevant conditions, the development of new methods to identify active microbial populations and to isolate previously uncultured microorganisms and of high-throughput autofluorescence bioreporter assays. Moreover, the technological advancements will make possible the miniaturisation and automation of FCM devices, allowing to revolutionize their applications in the near future. The purpose of this minireview is to update the current applications of FCM in different fields of applied microbiology, and to highlight the main advantages and pitfalls for each of them.

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Novel image cytometric method for detection of physiological and metabolic changes in Saccharomyces cerevisiae

Cited by 18 publications

References 26 publications

Real-time direct cell concentration and viability determination using a fully automated microfluidic platform for standalone process monitoring

Real-time direct cell concentration and viability determination using a fully automated microfluidic platform for standalone process monitoring

Brewing yeast viability measured using a novel fluorescent dye and image cytometer

Updates on the applications of flow cytometry in microbiology

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