A simple colony‐formation assay in liquid medium,termed ‘tadpoling’, provides a sensitive measure of Saccharomyces cerevisiae culture viability

Here we describe the first high‐throughput amenable method of quantifying Saccharomyces cerevisiae culture viability. Current high‐throughput methods of assessing yeast cell viability, such as flow cytometry and SGA analysis, do not measure the percentage viability of a culture but instead measure cell vitality or colony fitness, respectively. We developed a method, called tadpoling, to quantify the percentage viability of a yeast culture, with the ability to detect as few as one viable cell amongst ~10⁸ dead cells. The most important feature of this assay is the exploitation of yeast colony formation in liquid medium. Utilizing a microtiter dish, we are able to observe a range of viability of 100% to 0.0001%. Comparison of tadpoling to the traditional plating method to measure yeast culture viability reveals that, for the majority of Saccharomyces species analyzed there is no significant difference between the two methods. In comparison to flow cytometry using propidium iodide, the high‐throughput method of measuring yeast culture viability, tadpoling is much more accurate at culture viabilities < 1%. Thus, we show that tadpoling provides an easy, inexpensive, space‐saving method, amenable to high‐throughput screens, for accurately measuring yeast cell viability. Copyright © 2013 John Wiley & Sons, Ltd.     

Monitoring of Brewing Yeast Propagation Under Aerobic and Anaerobic Conditions Employing Flow Cytometry

The vitality and viability of industrial strains of Saccharomyces cerevisiae was monitored during pilot plant experiments simulating yeast propagation under aerobic and anaerobic conditions. Industrial wort of 12°P original gravity was used as a growth substrate for yeast propagation. The work was carried out with three widely used Czech lager yeast industrial strains: strains 2, 7 and 95. Cell cycle, cell size, granularity, glycogen content, DNA and protein content were analyzed by flow cytometry. Significantly higher specific growth rates, higher content of yeast glycogen, earlier G2/M phase cells maximum, and faster cell protein creation was observed under aerobic conditions compared to anaerobic. Strains 7 and 95 showed losses in flocculation ability after aerobic propagation compared to anaerobic propagation. Under either aerobic or strictly anaerobic conditions, only strain 2 did not show a significant loss in flocculation ability.     

Screening for microorganisms with specific characteristics by flow cytometry and single-cell sorting

Flow cytometry used in combination with single-cell sorting is a powerful technique for the identification and isolation of microbial cells with particular characteristics, especially when such cells grow more slowly than other cells in a large heterogeneous population. Many applications of flow cytometry with cell sorting, originally used by specialists studying mammalian cells, have been modified so that microorganisms also can be evaluated. The methods can now be used more widely because of the increasing availability of the expensive equipment. There are means for the fluorescence detection of a wide variety of properties, such as amounts of various cell components, specific sequences of peptides and nucleotides, cell functions, and enzyme activities. From the extensive literature, representative reports of an assortment of uses of flow cytometry with cell sorting are reviewed in this article, intended to introduce the technique and its many advantages to microbiologists.     

RELATIONSHIP BETWEEN YEAST CELL PROLIFERATION AND INTRACELLULAR ESTERASE ACTIVITY DURING BREWING FERMENTATIONS

Flow cytometry was used for the determination of the intracellular esterase activity of unstressed and stressed Saccharomyces cerevisiae cells using fluorescein diacetate as substrate during brewing fermentations in EBC tubes. The determination of intracellular esterase activity by flow cytometry was compared with in vitro assays for determination of yeast esterase activity. The method was regarded valid with a high degree of reproducibility. Intracellular esterase activity during brewing fermentations was dependent on the yeast strain applied but independent of the wort compositions applied within this study. Further, the intracellular esterase activity during fermentation was correlated with cell proliferation determined by DNA staining and flow cytometry and by calculating the percentage of G1-phase cells. Yeast esterase activity in both unstressed and ethanol stressed cells followed a similar pattern during brewing fermentations. Furthermore, this pattern could be correlated with the percentage of G1-phase cells during fermentation indicating that the esterase activity was in some way related to cell cycle progression .     

Flow cytometry and cell sorting for yeast viability assessment and cell selection

Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities. Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable. The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h. We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re-hydrated high activity dried yeast (HADY). PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use. Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox. The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability. Sorting demonstrated that PI stained non-culturable cells whilst CY stained culturable cells. Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments. The Ox assay was in agreement with CY and PI for live/heat-killed mixtures. However, for re-hydrated HADY, Ox stained a significantly (P⩽0·05) higher proportion of cells than did PI. © 1998 John Wiley & Sons, Ltd.     

Yeast exo-β-glucanases can be used as efficient and readily detectable reporter genes in Saccharomyces cerevisiae

Yeast exo-1,3-β-glucanases are secretable proteins whose function is basically trophic and may also be involved in cell wall glucan hydrolytic processes. Since fluorescein di(β-D -glucopyranoside) is a fluorogenic substrate detectable and quantifiable by flow cytometry, it was used for testing the ability of the EXG1 gene product of Saccharomyces cerevisiae and its homologous gene in Candida albicans to function as reporter genes. These open reading frames were coupled to different promoters in multicopy plasmids, and exoglucanase activity quantified at flow cytometry. Exoglucanases were found to be useful tools for the study of promoter regions in S. cerevisiae. This technique has the advantage over other reporter gene systems—such as β-galactosidase fusions—that it does not require permeabilization of yeast cells and therefore it allows the recovery of viable cells—by sorting—after flow cytometry analysis.     

Flow Cytometry for Age Assessment of a Yeast Population and its Application in Beer Fermentations

An expeditious method of yeast age estimation was developed based on selective bud scar staining (Alexa Fluor 488‐labelled wheat‐germ agglutinin) and subsequent fluorescence intensity measurement by flow cytometry. The calibration curve resulting from the cytometric determination of average bud scar fluorescence intensities vs. microscopically counted average bud scar numbers of the same cell populations showed a good correlation and allowed routine cell age estimation by flow cytometry. The developed method was applied for yeast age control in traditional batch and continuous beer fermentations. At the pitching rates used in industrial beer fermentations, our results support former findings by locating a gradient of increasing yeast age from the top to the bottom zone of the fermenter cone. The results also indicate that in continuous beer fermentation, the increasing bud scar fluorescence of immobilized cells could help to schedule the replacement of aged biomass, prior to loss of viability or deterioration of process performance and product quality.     

Evaluation of Light Scattering and Autofluorescent Properties of Brewer's Worts for Flow Cytometric Analysis of Yeast Viability

Flow cytometric methods for determining yeast viability are currently available. For effective analysis of yeast in breweries it is important that the light scattering properties of the sample medium (wort) do not interfere with that of target yeast cells. For this reason, a number of wort samples were analysed for their light scattering and autofluorescent properties, as well as their ability to bind the yeast viability dye, oxonol. Worts were found to produce light scattering that was sufficiently different from yeast, such that the two were clearly distinguishable by flow cytometry. Although oxonol bound to wort particles, computer software techniques allowed determination of yeast viability in worts.     

Flow Cytometric Determinations of Glycogen Content of Yeast During Fermentation

Using flow cytometric analysis, the rapid detection of glycogen content of yeast cells in process is possible. Glycogen is a sensitive parameter which can express the physiological state of the yeast cells. A procedure was developed to stain and measure glycogen in yeast cells using flow cytometry. Glycogen content of lager yeast cells was tested after 1 and 15 h of nutrient limitation.     

Growth and enumeration of the meat spoilage bacterium Brochothrix thermosphacta

Brochothrix thermosphacta is a common meat spoilage bacterium. The morphology of this bacterium changes from coccobacilli and short rods to chains during growth, which may give a false estimation in numbers using some enumeration techniques. Methods for the quantification of this bacterium have been compared. Turbidimetric readings showed good agreement with cell dry weight indicating that the former provides a good measure of the change in cell mass during growth. The turbidimetric method also correlated well with bacterial numbers determined by plate counts, flow cytometry and manual counts (by microscope) over a limited range of 10(7)-10(9) cells/ml. Flow cytometry and manual counts gave a linear relationship over a wider range of 10(5)-10(9) cells/ml. The sensitivity of analysis, growth rates and lag time attained using these methods were also compared. As a consequence of changes in bacterial cell size during growth, turbidimetry over-estimated the growth rate. The plate count method proved unable to detect the difference between bacteria existing as chains or single cells. The sensitivity of analysis and the calculated growth related parameters were similar for flow cytometry and manual counts. This suggests that flow cytometry is capable of counting individual cells in a chain. Further investigation showed that passage of B. thermosphacta cells through the flow cytometer resulted in the breakage of chains into single cells. The reliability, low error and rapidity of this technique make it attractive for bacterial enumeration, something which has been demonstrated using B. thermosphacta, a bacterium which exhibits complex morphologies.     

流式细胞术检测单增李斯特菌与酿酒酵母

为探讨流式细胞术对单增李斯特菌和酿酒酵母的活菌与热灭活菌的检出效果,本文采用荧光染色试剂SYTO-9和碘化乙锭(PI)对单增李斯特菌和酿酒酵母的活菌与热灭活菌的细胞悬液进行染色,采用流式细胞仪同时测量红色荧光与绿色荧光从而得出细胞悬液中的细菌和酵母的含量。结果表明经核酸荧光染料染色后,再结合流式细胞术对细菌与酵母菌进行检测,步骤简单、耗时短。该法不仅简化了测量步骤且分辨率高,对单增李斯特菌和酿酒酵母均具有良好的检出结果,能分辨同一体系中同一菌种的活细胞与热灭活细胞和同一体系中的细菌与酵母活细胞;该法检出限低,将单增李斯特菌稀释后,最低检出限可达1.2×104 cells/mL,将酿酒酵母稀释后,最低检出限可达6×103 cells/mL,因此能大大缩短增菌时间或者避免繁复的增菌步骤。     

产酯酵母和耐高温酵母原生质体形成与再生条件研究

选取脱壁预处理时间、蜗牛酶浓度、酶解时间三因素设计三因素三水平正交试验来研究产酯酵母和耐高温酵母原生质体形成与再生条件。结果表明,产酯酵母原生质体形成与再生最佳条件为：预处理20min、1%蜗牛酶酶解30min;耐高温酵母为：预处理10min,1.5%蜗牛酶酶解60min。     

Physiological analysis of yeast cells by flow cytometry during serial-repitching of low-malt beer fermentation

At the end of beer brewing fermentation, yeast cells are collected and repitched for economical reasons. Although it is generally accepted that the physiological state of inoculated yeast cells affects their subsequent fermentation performance, the effect of serial-repitching on the physiological state of such yeast cells has not been well clarified. In this study, the fermentation performance of yeast cells during serial-repitching was investigated. After multiple repitchings, the specific growth rate and maximum optical density (OD(660)) decreased, and increases in isoamyl alcohol, which causes an undesirable flavor, and residual free amino acid nitrogen (FAN) concentrations were observed. The physiological state of individual cells before inoculation was characterized by flow cytometry using the fluorescent dyes dehydrorhodamine 123 (DHR) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (OXN). The fluorescence intensities of DHR, an indicator of reactive oxygen species (ROSs), and OXN, which indicates membrane potential, gradually increased as the number of serial-repitching cycles increased. Fluorescence intensity correlated strongly with cell growth. The subsequent fermentation performance can be predicted from this correlation.     

YEAST PROTEIN MEASUREMENT USING NEAR INFRARED REFLECTANCE SPECTROSCOPY

The measurement of total protein in yeast cells is useful for fermentation and yeast research as well as for byproduct improvement studies. The protein content of the cell changes with alterations in several fermentation and medium parameters and during the course of the cell cycle. In general, the protein content of the harvested cells is maximised under conditions ideal for yeast growth. Brewers often sell excess yeast as a byproduct and the quality of the yeast, as well as the associated price per unit weight, are often determined by the protein content. Many methods to measure the protein content in yeast cells exist but can be impractical for various reasons or prone to error. In this research, a method was developed to measure the protein content of yeast cells by near infrared reflectance spectroscopy (NIR) - a technique which is finding many applications in brewing and is simple, rapid and amenable to on-line control systems. With this latest application for NIR, it is now possible to measure yeast glycogen, trehalose and protein contents on the same sample in less than 30 minutes including a sample preparation time of 25 minutes .     

Growth and by‐product profiles of Kluyveromyces marxianus cells immobilized in foamed alginate

The aim of this research was to study how the yeast cell immobilization technique influences the growth and fermentation profiles of Kluyveromyces marxianus cultivated on apple/chokeberry and apple/cranberry pomaces. Encapsulation of the cells was performed by droplet formation from a foamed alginate solution. The growth and metabolic profiles were evaluated for both free and immobilized cells. Culture media with fruit waste produced good growth of free as well as immobilized yeast cells. The fermentation profiles of K. marxianus were different with each waste material. The most varied aroma profiles were noted for immobilized yeast cultivated on apple/chokeberry pomace. Copyright © 2014 John Wiley & Sons, Ltd.     

USE OF FLOW CYTOMETRY FOR RAPID ESTIMATION OF INTRACELLULAR EVENTS IN BREWING YEASTS

A flow cytometric method based on staining with fluorescein diacetate (FDA) for rapid measurement of the fluorescence intensity (FI) of individual brewing yeast cells is reported. Distinct changes in the FI of yeast populations during the course of both laboratory scale and brewery fermentations were observed. The changes followed a characteristic pattern defined as the FI-profile. The FI-profile was related to the rate of attenuation as well as changes in yeast glycogen and ergosterol contents. The results obtained suggest that flow cytometry can be applied for estimation of intracellular events in brewing yeasts and prediction of their performance.     

肉桂醛诱导酿酒酵母Persisters细胞的形成

本文研究了肉桂醛对酿酒酵母persister细胞形成的影响。通过96孔板微量法测定肉桂醛对酿酒酵母生长的最小抑制浓度(MIC)为0.4 m M;采用流式细胞仪以及梯度稀释滴平板计数法研究了肉桂醛处理后酿酒酵母persister细胞的形成情况。结果表明,肉桂醛可以抑制酿酒酵母生长,且能够诱导酿酒酵母细胞形成persister状态,该状态下的细胞对两性霉素B产生耐药性。进一步研究发现肉桂醛处理后可以使酿酒酵母细胞停滞在细胞周期的任何阶段,而雷帕霉素诱导的细胞自噬只能停留在G1期,所以酿酒酵母perisister与自噬状态存在区别。目前,对于Persister的研究集中在原核微生物,对真核生物persister的研究非常有限。由于persister群体通常占总群体极小一部分,这就给基因水平上研究persister的形成机制带来很大的挑战。本研究发现肉桂醛处理酿酒酵母细胞后,可以促使其大部分细胞形成persister。这就为从基因水平上认识真核生物persister的形成机制提供了方法,实验结果表明YGL基因也与酿酒酵母persister的形成有很大关系。     

巴豆醛诱导人支气管上皮细胞凋亡研究

为了评价卷烟烟气的危害性,研究了巴豆醛导致人支气管上皮细胞BEAS-2B死亡机制.即采用MTT法检测细胞存活率,化学发光法检测胞内ATP含量,流式细胞仪检测细胞凋亡率,流式细胞仪结合免疫化学检测线粒体细胞色素c,化学发光法检测caspase-9,caspase-3/7.结果显示,巴豆醛诱导细胞凋亡,且随着剂量增大,细胞由凋亡转向坏死.巴豆醛导致胞内ATP水平迅速下降,导致细胞线粒体膜电位下降,导致细胞色素c由线粒体释放至胞浆,激活caspase-9和caspase-3/7.结论:巴豆醛诱导细胞凋亡,且caspase级联酶参与巴豆醛诱导的细胞凋亡.     

Microfluidics device for single cell gene expression analysis in Saccharomyces cerevisiae

We have measured single-cell gene expression over time using a microfluidics-based flow cell which physically traps individual yeast using microm-sized structures (yeast jails). Our goal was to determine variability of gene expression within a cell over time, as well as variability between individual cells. In our flow cell system, yeast jails are fabricated out of PDMS and gene expression is visualized using fluorescently-tagged proteins of interest. Previously, single-cell yeast work has been done using micromanipulation on agar, or FACS. In the present device agar is eliminated, resulting in a superior optical system. The flow of media through the flow cell washes daughter cells away, eliminating the need for micromanipulation. Unlike FACS, the described device can track individual yeast over a time course of many hours. The flow cells are compatible with the needs of quantitative fluorescence microscopy, and allow simultaneous measurements to be done on a large number of individual yeast. We used these flow cells to determine the expression of HSP104-GFPand RAS2-YFP, genes known to affect yeast life span. The results demonstrate inter-cell variation in expression of both genes that could not have been detected without this single-cell analysis.     

THE BASIS OF THE DETERMINATION OF CELL VIABILITY WITH THE FLUOROCHROME PRIMULINE

The fluorochrome primuline has been shown, by the micromanipulation of individual stained yeast cells, to be a useful stain for the determination of cell viability. A high correlation (96%) between non-fluorescence and viability was demonstrated for cells taken from young cultures, but the correlation decreased with increasing age of the cell suspension. The correlation between fluorescence and non-viability was 100% for yeast cells taken from any environment. Cells which were stained green after primuline treatment were inferred to be non-viable but an absolute correlation between non-viability and the “green” reaction could not be demonstrated. The staining technique was tested with animal cells (Paramecia, Amoebae) and the correlation between fluorescence and non-viability was confirmed. The staining result with a mammalian cell line was not confirmed by cell culture. The primuline technique for viable cell counts was primarily dependent on the loss of the selective permeability of the plasma membrane.