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.     

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.     

Flow cytometric assessment of cell viability: a multifaceted analysis

Flow cytometry offers the possibility to simultaneously analyze, on a cell by cell basis, different parameters related to cell viability i.e. cell size, morphology and incorporation of dyes. Different types of analysis: light absorption of unstained/stained cells, forward angle light scattering (FALS), right angle light scattering (RALS) or both, cell fluorescence based on dye retention or dye exclusion (due to erythrosin B, ethidium bromide, fluorescein diacetate, rhodamine 123) were tested and compared, with the classical Trypan blue exclusion test, for their effectiveness in the determination of cell viability. Two types of cells in monolayer cultures (L929, SIRC) and a freshly isolated suspension of mouse splenocytes were used. For each dye, the optimal dose, incubation time and conditions for analysis were determined. Viability indications by different techniques for the three type of cell line and their reliability as compared with Trypan blue were analyzed.     

The role of the cell cycle in determining gene expression and productivity in CHO cells

Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

A flow-cytometric method for determination of yeast viability and cell number in a brewery

A flow-cytometric assay, using the fluorescent dye, oxonol, for the simultaneous determination of yeast cell viability and cell number is described. The assay was optimised, and trialed at a brewery for 6 months. The flow-cytometry assay offered a substantially reduced error in viability determination, compared to methylene blue which is the industry standard for measuring viability. Further, by calculating yeast cell number at the same time, this assay provides a reliable method for determining pitching rate, allowing increased quality control of subsequent fermentations.     

Evaluation of flow cytometry as a method for quantification of circulating blood cell populations in salmonid fish

Flow cytometry was used to estimate the proportions of different blood cell types in brown and rainbow trout. On the basis of forward light scatter and 90° side scatter three populations were differentiated. The relative abundance of these cells correlated with that of erythrocytc (r²= 0.994), lymphocyte plus thrombocyte(r²= 0.676) and neutrophil populations (r²= 0.571) enumerated by direct microscopy. By density gradient separation of cells, cell sorting and acridine orange staining it was confirmed that these cell types could be assigned to the populations detected. Changes in blood cell populations were monitored by flow cytometry in a group of experimental fish placed under confinement stress. Flow cytometry proved to be a rapid and reliable method for monitoring cell population dynamics in fish blood.     

The effects of inulin,dried Jerusalem artichoke tuber and a multispecies probiotic preparation on microbiota ecology and immune status of the large intestine in young pigs

The study aimed at determining the effect of two types of prebiotics and a multispecies probiotic on microbiota activity and composition, as well as mucosal immunity in the large intestine of young pigs. In total 48 piglets were divided into 6 groups (n = 8), which received from day 10 of life probiotic-unsupplemented (PU) or probiotic-supplemented (PS) diets. Probiotics were added at 0.5 g/kg diet and contained: Lactococcus lactis, Carnobacterium divergens, Lactobacillus casei, Lactobacillus plantarum and Saccharomyces cerevisiae. The PU and PS diets were formulated without prebiotic addition (control) or with addition of 2% of inulin from chicory root (IN) or 4% of dried Jerusalem artichoke tubers (DJA). After 40 days of feeding, digesta and tissue samples were taken from the caecum and three sections of the colon for analyses of microbiota activity and composition, secretory immunoglobulin A (sIgA) and intraepithelial lymphocytes (IEL). IN diets decreased the caecal digesta pH and β-glucosidase activity but increased propionic, valeric and total short chain fatty acid (SCFA) concentrations compared to control diets. Feeding DJA diets increased caecal valeric acid level, decreased the concentration of isoacids in the colon, reduced β-glucosidase and β-glucuronidase activity in the middle colon and increased Bifidobacterium spp. populations in the proximal and distal colon. PS diets increased the caecal acetic acid and total SCFA level, and Clostridium spp. populations in the distal colon. Neither probiotic nor prebiotics affected sIgA level or IEL number in the large intestine. In conclusion, DJA modified the microbiota ecology in the large intestine of young pigs to a greater extent than IN and the applied probiotic did not enhance effects of prebiotics.     

Description and validation of a rapid (1 h) flow cytometry test for enumerating thermophilic bacteria in milk powders

AIMS: The aim of this study was to develop a rapid assay for enumerating thermophilic bacteria in milk powder. METHODS AND RESULTS: The BactiFlow flow cytometer was used to count bacteria based on esterase activity in viable bacterial cells. A protocol for total viable bacteria was modified by heat-treating the sample to selectively label thermophilic bacteria. Samples of milk powder dissolved in 0.1% peptone were treated with 0.8% ethylenediaminetetraacetic acid to reduce background interference because of denatured milk proteins. Either thermophilic bacteria were added to the dissolved milk powder or milk powder solutions were incubated at 55 degrees C for 2-3 h to enrich the natural thermophile population for testing. Results from the BactiFlow were compared with traditional plate count results. CONCLUSIONS: Thermophilic bacteria in milk powder can be enumerated within 1 h using the BactiFlow flow cytometer. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbiological test results obtained within 1 h can potentially be used to monitor manufacturing processes, effectively trace problems and provide confidence in the manufacture of product.     

Bacterial viability assessment by flow cytometry analysis in soil

Soil microhabitats and their heterogeneity are often considered to be among the most important factors affecting soil biotic communities. The microbial commu-nity has become one of the most important links in soil nutrient cycles and trophic components due to its role in biological processes, spatial and temporal dynamics, and physiological adaptation. Sandy-soil desert systems are characterized by fast water infiltration during the rainy season, high salinity, and low moisture availability in the upper soil layers. Plants have developed different ecophy-siological adaptations in order to cope with this harsh environment. The Tamarix aphylla is known to be one of the most commonly adapted plants, exhibiting a mechan-ism for secretion of excess salts as aggregates through its leaves. These leaves aggregate beneath the plant, creating 'islands of salinity'. Soil biotic components are, therefore, exposed to extreme abiotic stress conditions in this niche. The goal of this study was to examine the effect of T. aphylla on the live/dead bacterial population ratio on a spatial and temporal scale. The results emphasize the effect of abiotic factors, which changed on temporal as well as spatial scales, and also on the size of the active soil bacterial community, which fluctuated between 1.44% and 25.4% in summer and winter, respectively. The results of this study elucidate the importance of moisture availability and the 'island-of-salinity' effect on the active microbial community in a sandy desert system.     

Eukaryotic picophytoplankton community response to copper enrichment in a metal‐perturbed coastal environment

Copper is an essential micronutrient, especially for photosynthetic organisms, but can be toxic at high concentrations. In the past years, coastal waters have been exposed to an increase in copper concentration due to anthropogenic inputs. One well known case is the Chañaral area (Easter South Pacific coast), where a long term coastal copper enrichment event has occurred. That event strongly affected benthic marine diversity, including microbial communities. In this work, microcosm experiments were carried out to address the changes on picophytoplankton community composition of the disturbed area, when challenged to copper additions. Eukaryotic picophytoplankton communities from two areas were analyzed: one in the most copper‐perturbed area and another at the north edge of the perturbed area. Flow cytometry data showed that 25 μg L^?1 of copper addition exerted a positive effect in the growth kinetics on part of the eukaryotic picophytoplankton communities, independently of the site. 16S‐plastid terminal restriction fragment length polymorphisms analysis suggested that eukaryotic picophytoplankton display a short and directional response to high copper levels. Members of the Prasinophyceae class, a Coscinodiscophyceae diatom, as well as Phaeocystis, respond in a short time to the environmental disturbance, making them excellent candidates for further studies to evaluate phytoplanktonic species as sentinels for copper disturbances in coastal marine ecosystems.     

Rational optimization of culture conditions for the most efficient ethanol production in Scheffersomyces stipitis using design of experiments

Optimization of culture parameters for achieving the most efficient ethanol fermentation is challenging due to multiple variables involved. Here we presented a rationalized methodology for multi‐variables optimization through the design of experiments DoE approach. Three critical parameters, pH, temperature, and agitation speed, affecting ethanol fermentation in S. stipitis was investigated. A predictive model showed that agitation speed significantly affected ethanol synthesis. Reducing pH and temperature also improved ethanol production. The model identified the optimum culture conditions for the most efficient ethanol production with the yield and productivity of 0.46 g/g and 0.28 g/l h, respectively, which is consistent with experimental observation. The results also indicated the scalability of the model from shake flask to bioreactor. Thus, DoE is a promising tool permitting the rapid establishment of culture conditions for the most efficient ethanol fermentation in S. stipitis. The approach could be useful to reduce process development time in lignocellulosic ethanol industry. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

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.     

Induction of acid resistance in Bifidobacterium: a mechanism for improving desirable traits of potentially probiotic strains

AIMS: To generate stable acid-resistant Bifidobacterium strains isolated from human subjects and characterize the phenotypic changes of the acid-resistant derivatives. METHODS AND RESULTS: The ability of 20 Bifidobacterium strains isolated from human faeces to survive to simulated gastrointestinal transit was evaluated, showing major reductions in viability (0.25-5.8 logarithmic units) because of gastric stress conditions. Six acid-sensitive strains belonging to the species Bifidobacterium longum and Bifidobacterium catenulatum were submitted to prolonged incubation at pH 2.0 to generate acid-resistant strains. The acid-sensitive and acid-resistant derivative strains were characterized to determine the changes associated with the acquisition of an acid-tolerant phenotype. The acid-resistant derivatives showed better ability to grow in the presence of bile salt (1-3%) and NaCl (6-10%) and higher resistance at elevated temperatures (60-70 degrees C, 10 min) than the parental strains. The acid-resistant derivatives displayed higher fermentative ability, and enzymatic activities. These strains also showed higher sensitivity to most of the tested antibiotics than the parental strains. CONCLUSIONS: The stress tolerance of B. longum and B. catenulatum strains was improved by prolonged exposure to acid stress conditions. Some of the generated strains also seemed to have enhanced metabolic properties of relevance for probiotic applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The successful use of prolonged exposures to acid stress to improve the stability of human bifidobacteria indicates that this strategy could be useful for the production of robust probiotic strains, but involves other phenotypic changes that required an individual characterization.     

Effects of cholesterol and docosahexaenoic acid on cell viability and (Ca2+)i levels in acutely isolated mouse thymocytes

We investigated the effects of lipids on thymocyte function. The effects of application of cholesterol or docosahexaenoic acid (DHA), a C22, omega‐3 (n‐3) polyunsaturated fatty acid (PUFA), on viability and intracellular calcium concentrations of acutely isolated mouse thymocytes were investigated using flow cytometry. Cholesterol (100 µM) caused significant cell death after 30–60 min whether or not calcium was present in the medium. Cell death was associated with an elevation of intracellular calcium whether or not calcium was present in the extracellular medium. However, the elevation of calcium concentration was not responsible for the cell death since calcium levels in the presence of ionomycin rose higher without significant cell death. DHA had similar actions but was more potent, causing significant cell death and elevation of calcium concentration within 5 min at 1 µM. In the absence of extracellular calcium 1 µM DHA caused 100% cell death within 15 min. Linolenic acid, a C18 omega‐3 fatty acid also caused cytotoxicity at low concentrations whether or not albumin was present, but omega‐6 or saturated C22 fatty acids were much less effective. These observations demonstrate that thymocyte viability is very sensitive to acute exposure to low concentrations of omega‐3 fatty acids. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and optimization of a metabolomic method for analysis of adherent cell cultures

In this investigation, a gas chromatography/mass spectrometry (GC/MS)-based metabolomic protocol for adherent cell cultures was developed using statistical design of experiments. Cell disruption, metabolite extraction, and the GC/MS settings were optimized aiming at a gentle, unbiased, sensitive, and high-throughput metabolomic protocol. Due to the heterogeneity of the metabolome and the inherent selectivity of all analytical techniques, development of unbiased protocols is highly complex. Changing one parameter of the protocol may change the response of many groups of metabolites. In this investigation, statistical design of experiments and multivariate analysis also allowed such interaction effects to be taken into account. The protocol was validated with respect to linear range, precision, and limit of detection in a clonal rat insulinoma cell line (INS-1 832/13). The protocol allowed high-throughput profiling of metabolites covering the major metabolic pathways. The majority of metabolites displayed a linear range from a single well in a 96-well plate up to a 10 cm culture dish. The method allowed a total of 47 analyses to be performed in 24 h.     

The Taguchi methodology as a statistical tool for biotechnological applications: a critical appraisal

Success in experiments and/or technology mainly depends on a properly designed process or product. The traditional method of process optimization involves the study of one variable at a time, which requires a number of combinations of experiments that are time, cost and labor intensive. The Taguchi method of design of experiments is a simple statistical tool involving a system of tabulated designs (arrays) that allows a maximum number of main effects to be estimated in an unbiased (orthogonal) fashion with a minimum number of experimental runs. It has been applied to predict the significant contribution of the design variable(s) and the optimum combination of each variable by conducting experiments on a real-time basis. The modeling that is performed essentially relates signal-to-noise ratio to the control variables in a 'main effect only' approach. This approach enables both multiple response and dynamic problems to be studied by handling noise factors. Taguchi principles and concepts have made extensive contributions to industry by bringing focused awareness to robustness, noise and quality. This methodology has been widely applied in many industrial sectors; however, its application in biological sciences has been limited. In the present review, the application and comparison of the Taguchi methodology has been emphasized with specific case studies in the field of biotechnology, particularly in diverse areas like fermentation, food processing, molecular biology, wastewater treatment and bioremediation.     

Y1 receptors are critical for the proliferation of adult mouse precursor cells in the olfactory neuroepithelium

While the regenerative capacity of the olfactory neuroepithelium has been well studied less is known about the molecular events controlling precursor cell activity. Neuropeptide Y (NPY) is expressed at high levels in the olfactory system, and NPY has been shown to play a role in neuroregeneration of the brain. In this study, we show that the numbers of olfactory neurospheres derived from NPY, NPY/peptide YY, and Y1 receptor knockout mice are decreased compared with wild type (WT) controls. Furthermore, flow cytometric analysis of isolated horizontal basal cells, globose basal cells, and glandular cells showed that only glandular cells derived from WT mice, but not from NPY and Y1 receptor knockout mice, formed secondary neurospheres suggesting a critical role for NPY signaling in this process. Interestingly, olfactory function tests revealed that olfaction in Y1 knockout mice is impaired compared with those of WT mice, probably because of the reduced number of olfactory neurons formed. Together these results indicate that NPY and the Y1 receptor are required for the normal proliferation of adult olfactory precursors and olfactory function.