NucleoCounter—An efficient technique for the determination of cell number and viability in animal cell culture processes

The NucleoCounter is a novel, portable cell counting device based on the principle of fluorescence microscopy. The present work establishes its use with animal cells and checks its reliability, consistency and accuracy in comparison with other cytometric techniques. The main advantages of this technique are its ability to handle a large number of samples with a high degree of precision and its simplicity and specificity in detecting viable cells quantitatively in a heterogeneous culture. The work addresses and overcomes the problems of subjectivity, and some of the inherent sampling errors associated with using the traditional haemocytometer and Trypan Blue exclusion method. NucleoCounter offers reduced intra- and inter-observer variation as well as consistency in repetitive analysis that establishes it as an efficient and highly potential device for at-line monitoring of animal cell processes. Furthermore, since the only manual steps required are sample aspiration and mixing with two reagents, it is feasible that the whole method could be automated and brought on-line for process monitoring and control.     

A rapid method for evaluation of cell number and viability by flow cytometry

A simple, rapid and reliable method has been developed for assessing the number and viability of cells, as well as cell size, in suspension culture by the use of flow cytometry. Propidium iodide exclusion is used for viability determination and fluorescent beads serve as an internal standard for cell enumeration. The main advantages of this method are its ability to handle a large number of samples with a high degree of precision and its specificity in detecting viable cells quantitatively in a heterogeneous culture of living and dead cells and debris. The method shows only a fraction of the variation found in the haemacytometer/trypan blue counting method due to its very low operator dependence. CHO - Chinese hamster ovary; FCS - Foetal calf serum; FS - Forward scatter light; MTT - 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; NCS - newborn calf serum; PBS - Phosphate buffered saline; PI - Propidium iodide; SS - Side scatter light. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effectiveness of CSE to counterstain particles and dead bacterial cells with permeabilised membranes: application to viability assessment in waters

The CSE dye (Chemunex, Maisons-Alfort, France) was combined with an activity marker to improve bacterial activity assessment in natural waters. Its effectiveness to counterstain dead cells with permeabilised membranes was investigated on live and dead cells of a variety of strains from collections or isolated from the natural environment. Cells were killed by heat treatment. For all strains tested, the fluorescent dye showed an intense staining of killed cells having permeabilised membranes while no significant signal was detected when applied to live cells. Furthermore, the CSE dye had no toxicity on viable cells. Then, CSE was combined with the ChemChrome V6 dye (Chemunex) to assess the activity of bacterial cells in different waters. Both fluorescences were analysed simultaneously by solid-phase cytometry. The active cell counts were sometimes lower when both dyes were combined suggesting that CSE was able to counterstain cells having a residual esterase activity and compromised membranes. These cells were subtracted from the active cell counts determined with ChemChrome V6. In most samples, active cell counts were congruent with those determined by the direct viable count method.     

Vigour, vitality and viability of microorganisms

Abstract Assessment of the vigour, vitality or viability of microorganisms must be done on an individual basis and thus requires the non-invasive interrogation of single organisms. For suspended organisms, flow cytometry provides a powerful means of measurement of a wide range of characteristics. Similar information for microbes in aggregates or growing on surfaces may be obtained by use of confocal scanning laser microscopy. For instance, membrane potential-sensitive fluorophores can distinguish between vigorous, frail and dead cells.     

MTT方法评价微生物细胞活性的探讨

对MTT比色法用于评价微生物细胞活性进行了探讨。本文以大肠杆菌为模式菌株,研究了不同浓度MTT、不同用量、在不同时间对试验结果OD570值的影响,结果表明细菌数在4.9×107－4.9×108个/mL范围内测出的OD570值与细菌浓度呈良好的正相关,0.5 mg/mL MTT用量20 L,反应时间20 min时效果最佳,其相关回归方程为y = 0.1769x + 0.03,R2 = 0.9983。     

酵母及与藻类搭配对萼花臂尾轮虫饵料效果的研究

研究了２种酵母单独投喂萼花臂尾轮虫的最适密度、饵料效果及与藻类搭配的投喂效果．结果表明,在５种密度下,面包酵母和啤酒酵母的最适密度分别为１０和５×１０６个·ｍｌ－１．用面包酵母和啤酒酵母培养轮虫所得到的种群密度和瞬时增长率分别是用蛋白核小球藻培养的４０．２％、２６．０％和８５．５％、７６．６％．用面包酵母和蛋白核小球藻适当比例搭配投喂轮虫,其效果接近或超过单一用小球藻在最适密度下的培养效果．     

Multiparametric flow cytometry allows rapid assessment and comparison of lactic acid bacteria viability after freezing and during frozen storage

Freezing is widely used for the long-term preservation of lactic acid bacteria, but often affects their viability and technological properties. Different methods are currently employed to determine bacterial cryotolerance, but they all require several hours or days before achieving results. The aim of this study was to establish the advantages of multiparametric flow cytometry by using two specific fluorescent probes to provide rapid assessment of the viability of four strains of Lactobacillus delbrueckii after freezing and during frozen storage. The relevance of carboxyfluorescein diacetate and propidium iodide to quantify bacterial viability was proven. When bacterial suspensions were simultaneously stained with these two fluorescent probes, three major subpopulations were identified: viable, dead and injured cells. The cryotolerance of four L. delbrueckii strains was evaluated by quantifying the relative percentages of each subpopulation before and after freezing, and throughout one month of storage at -80 degrees C. Results displayed significant differences in the resistance to freezing and frozen storage of the four strains when they were submitted to the same freezing and storage procedures. Whereas resistant strains displayed less than 10% of dead cells after one month of storage, one sensitive strain exhibited more than 50% of dead cells, together with 14% of stressed cells after freezing. Finally, this study proved that multiparametric flow cytometry was a convenient and rapid tool to evaluate the viability of lactic acid bacteria, and was well correlated with plate count results. Moreover, it made it possible to differentiate strains according to their susceptibility to freezing and frozen storage.     

Sequence of occurring damages in yeast plasma membrane during dehydration and rehydration: Mechanisms of cell death

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Although the involvement of the plasma membrane is strongly suspected, the mechanism remains unclear. Here, the integrity and functionality of the yeast plasma membrane at different levels of dehydration and rehydration during an osmotic treatment were assessed using various fluorescent dyes. Flow cytometry and confocal microscopy of cells stained with oxonol, propidium iodide, and lucifer yellow were used to study changes in membrane polarization, permeabilization, and endocytosis, respectively. Cell volume contraction, reversible depolarization, permeabilization, and endovesicle formation were successively observed with increasing levels of osmotic pressure during dehydration. The maximum survival rate was also detected at a specific rehydration level, of 20 MPa, above which cells were strongly permeabilized. Thus, we show that the two steps of an osmotic treatment, dehydration and rehydration, are both involved in the induction of cell death. Permeabilization of the plasma membranes is the critical event related to cell death. It may result from lipidic phase transitions in the membrane and from variations in the area-to-volume ratio during the osmotic treatment.     

New at-line flow cytometric protocols for determining carotenoid content and cell viability during Rhodosporidium toruloides NCYC 921 batch growth

Rhodosporidium toruloides NCYC 921 batch growth was monitored as a means to evaluate the yeast biomass potential as a source for the production of carotenoids and other lipids.Carotenoid content, cell viability and size were assessed by multiparameter flow cytometry. The saponifiable lipid fraction was assayed by gas–liquid chromatography.The carotenoid production increased during the stationary phase, reaching 78 μg/g while the total fatty acid content attained 32% (w/w) at the end of the fermentation. The fatty acid profile was suitable for biodiesel purposes.As the yeast cells entered the stationary phase, the proportion of cells with depolarised mitochondrial membrane and cells with permeabilised cytoplasmic membrane increased, attaining 65% and 14%, respectively. Nevertheless, a high proportion of cells (82%) showed esterase activity.These results demonstrated that flow cytometry can be a powerful at-line technique to monitor the total carotenoids and cell viability during the yeast growth, being useful for the yeast process optimisation at lab and pilot scales.     

Concomitant wastewater treatment with lipid and carotenoid production by the oleaginous yeast Rhodosporidium toruloides grown on brewery effluent enriched with sugarcane molasses and urea

In this study, secondary brewery wastewater (SBWW) supplemented with sugarcane molasses (SCM) was used for SBWW treatment with concomitant lipid and carotenoid production by the yeast Rhodosporidium toruloides NCYC 921. In order to improve the biomass production, ammonium sulfate, yeast extract and urea were tested as nitrogen sources. Urea was chosen as the best low-cost nitrogen source. A fed-batch cultivation was carried out with SBWW supplemented with 10 g L⁻¹ of sugarcane molasses as carbon source, and 2 g L⁻¹ of urea as nitrogen source. A maximum biomass concentration of 42.5 g L⁻¹ was obtained at t = 126.5 h and the maximum biomass productivity was 0.55 g L⁻¹ h⁻¹ at t = 48.25 h. The maximum lipid content was 29.9 % w/w (DCW) at t = 94 h of cultivation and the maximum carotenoid content was 0.23 mg g⁻¹ at 120 h of cultivation. Relatively to the SBWW treatment, after the batch phase, 45.8 % of total Kjeldahl nitrogen removal, 81.7 % of COD removal and 100 % of sugar consumption were observed. Flow cytometry analysis revealed that 27.27 % of the cells had injured membrane after the inoculation. This proportion was reduced to 10.37 % at the end of the cultivation, indicating that cells adapted to the growth conditions.     

Simple monitoring of cell leakiness and viability in Escherichia coli bioprocesses—A case study

In a recently published study, we developed a simple methodology to monitor Escherichia coli cell integrity and lysis during bioreactor cultivations, where we intentionally triggered leakiness. In this follow‐up study, we used this methodology, comprising the measurement of extracellular alkaline phosphatase to monitor leakiness and flow cytometry to follow viability, to investigate the effect of process parameters on a recombinant E. coli strain producing the highly valuable vascular endothelial growth factor A165 (VEGF‐A165) in the periplasm. Since the amount of soluble product was very little (<500 μg/g dry cell weight), we directly linked the effect of the three process parameters temperature, specific uptake rate of the inducer arabinose and specific growth rate (μ) to cell integrity and viability. We found that a low temperature and a high μ were beneficial for cell integrity and that an elevated temperature resulted in reduced viability. We concluded that the recombinant E. coli cells producing VEGF‐A165 in the periplasm should be cultivated at low temperature and high μ to reduce leakiness and guarantee high viability. Summarizing, in this follow‐up study we demonstrate the usefulness of our simple methodology to monitor leakiness and viability of recombinant E. coli cells during bioreactor cultivations.     

Flow cytometric determination of cell cycle phase-specific changes in cellular phosphatase and glycosidase activities

The activities of two phosphatases (E.C. 3.1.3.1 and 3.1.4.1) and four glycosidases (E.C. 3.2.1.21, 3.2.1.30, 3.2.1.31 and 3.2.1.51) were measured by fluorescence spectrophotometry, and flow cytometry, in mitogen-stimulated lymphocytes, and in cultures of Molt-4-F and F-89 cell lines, synchronized by hydroxyurea or thymidine. All enzymes were active throughout the cycle but the activities of three enzymes were elevated at specific points in the cycle, alkaline phosphatase activity increased at G2 + M/G1 boundary and in early S-phase, the activity of beta-L fucosidase was elevated in G1 and late S-phase. Orthophosphate diesterase activity was elevated at the G1/S boundary, and during G2 + M. The increase in beta-L fucosidase activity was due to an increased number of cells showing activity, whilst the increase in orthophosphate diesterase activity was attributable to an increase in cellular enzyme activity. Only the activities of orthophosphate diesterase and beta-L fucosidase were measurable by flow cytometry, alkaline phosphatase activity was mainly extracellular, and therefore not detectable by flow cytometric methods employed.     

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.     

A micromethod for the quantitative determination of the viability of Candida albicans hyphae

A micromethod for the quantitative determination of the viability of Candida albicans hypae was devised which takes advantage of the dimorphic nature of C. albicans which grows exclusively in the yeast form when incubated aerobically on Sabouraud dextrose agar at 30°C. When tested by thisd method, all viable, C. albicans hyphae were recognized as microcolonies consisting of one hypha surrounded by several yeast form progeny. In contrast to this, no yeast form progeny emerged from nonviable hypae. By counting appropriate total numbers (200–400) of microcolony-forming hypae and infertile hyphae, it was possible to determine the ratio of viable to nonviable cells in a given hyphal suspension. This micromethod may be used for quantitative assessment of the candidacidal effects of various antimycotic agents or phagocytes C. albicans hyphae whose viability could not have been determined by the conventional plating technique because of the species' high propensity to clump.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

A new methodology for plant cell viability assessment using intracellular esterase activity

 A plant cell suspension culture of Alfalfa (Medicago sativa L.) was grown in a bioreactor using a batch procedure. The cytoplasmic esterase activity (EC 3.1) was extracted from the cells and measured during cultivation using fluorescein diacetate as the fluorogenic substrate. This enzymatic activity was conclusively found to be correlated to cell viability assessed with the membrane integrity test using the trypan blue dye. This new viability determination method is convenient, simple and can be reproduced because: (1) the difficult step of counting the cells when using the trypan blue exclusion method is avoided and (2) the esterase activity level per viable cell constituted of numerous enzymes depends on cell viability but is independent of cellular metabolism. Received: 28 January 1999 / Accepted: 1 April 1999     

Optimization of temperature,sugar concentration,and inoculum size to maximize ethanol production without significant decrease in yeast cell viability

Aiming to obtain rapid fermentations with high ethanol yields and a retention of high final viabilities (responses), a 2³ full-factorial central composite design combined with response surface methodology was employed using inoculum size, sucrose concentration, and temperature as independent variables. From this statistical treatment, two well-fitted regression equations having coefficients significant at the 5% level were obtained to predict the viability and ethanol production responses. Three-dimensional response surfaces showed that increasing temperatures had greater negative effects on viability than on ethanol production. Increasing sucrose concentrations improved both ethanol production and viability. The interactions between the inoculum size and the sucrose concentrations had no significant effect on viability. Thus, the lowering of the process temperature is recommended in order to minimize cell mortality and maintain high levels of ethanol production when the temperature is on the increase in the industrial reactor. Optimized conditions (200 g/l initial sucrose, 40 g/l of dry cell mass, 30 °C) were experimentally confirmed and the optimal responses are 80.8 ± 2.0 g/l of maximal ethanol plus a viability retention of 99.0 ± 3.0% for a 4-h fermentation period. During consecutive fermentations with cell reuse, the yeast cell viability has to be kept at a high level in order to prevent the collapse of the process.