A permanent cell viability assay using alcian blue.

The alcian blue dye exclusion method for glutaraldehyde-fixed cells has been utilized with "centrifugal cytology" to prepare permanent records of the viability of individual cells present in suspensions. The viability of spleen cell suspensions separated by linear bovine serum albumin density gradient centrifugation has been measured with this method. Combined light and scanning electron microscopy of nonviable and viable cells demonstrated membrane alterations in alcian blue-stained nonviable cells, while viable cells were spherical and displayed uniform surface features.     

Rapid Identification of Viable Bacterial Spores Using a Fluorescence Method

A method has been devised to differentiate viable and nonviable bacterial spores. “Germination-like” changes are initiated in spores with performic acid and lysozyme. The germinated spores are stained with aqueous acridine orange, a fluorescent dye. Nonviable spores fluoresce lemon-green and viable spores orange-red. It is proposed that with the use of a membrane filter resistant to performic acid and lysozyme, the method may be used for spore enumeration in foods in about 4 hr compared to conventional plating methods, which usually require up to 72 hr.     

The dye-exclusion test for cell viability: Persistence of differential staining following fixation

Summary A method is described whereby the differential staining of viable and nonviable unfixed cells, as observed by the dye-exclusion method, can be reproduced in glutaraldehyde-fixed preparations by staining with alcian blue. The results suggest that the differential staining is due, at least in part, to structural differneces that are retained following aldehyde fixation. This work was supported by grants from the National Cancer Institute of Canada and the National Research Council of Canada.     

The dye-exclusion test for cell viability: Persistence of differential staining following fixation

Summary A method is described whereby the differential staining of viable and nonviable unfixed cells, as observed by the dye-exclusion method, can be reproduced in glutaraldehyde-fixed preparations by staining with alcian blue. The results suggest that the differential staining is due, at least in part, to structural differences that are retained following aldehyde fixation. This work was supported by grants from the National Cancer Institute of Canada and the National Research Council of Canada. Recipient of a Research Studentship from the National Cancer Institute of Canada.     

The quantitation of bovine embryo viability using a bioluminescent assay for lactate dehydrogenase

Forty-seven bovine embryos, ranging from the four-cell to expanded blastocyst stage, with grades ranging from excellent to poor, were collected non-surgically from superovulated Holstein heifers. A viability assay based on the measurement of bioluminescent emission from the media surrounding an embryo was tested. This assay measured the activity of the enzyme lactate dehydrogenase (LDH) released into the media by the embryos. Lactate dehydrogenase has been reported to be released into media by nonviable embryos. The assay used is simple, rapid and nonsubjective, requiring approximately 5 min to complete. The LDH assay proved to be a practical method for distinguishing between nonviable and viable embryos. Viability was determined by the observation of embryo development in culture following the LDH assay. The activity of LDH in the media of nonviable embryos was consistently higher than for viable embryos (P<0.001), with no overlap between the two groups. Thus, the LDH assay was shown to be a reliable test of embryo viability.     

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.     

Inclined sedimentation for selective retention of viable hybridomas in a continuous suspension bioreactor.

The continuous separation of nonviable hybridoma cells from viable hybridoma cells by using a narrow rectangular channel that is inclined from the vertical has been investigated experimentally. The effectiveness of the settler in selectively retaining viable hybridomas in the bioreactor while permitting the removal of nonviable hybridomas has been shown to depend on the flow rate through the settler. Intermediate flow rates through the settler have been found to provide the highest removal of nonviable hybridomas relative to viable hybridoma retention. At high dilution rates through the chemostat, over 95% of the viable cells could be partitioned to the bottom of the settler while over 50% of the nonviable cells are removed through the top of the settler. This successful separation is due to the significantly larger size of the viable hybridomas than the nonviable ones. A continuous perfusion experiment was performed in which an external inclined settler was used to retain virtually all of the viable hybridomas in the culture, while selectively removing from the culture approximately 20% of the nonviable cells that entered the settler. A stable viable cell concentration of 1.0 x 10(7) cells/mL was achieved, as was an antibody productivity of over 50 micrograms/(mL.day). These represent 3- and 6-fold increases, respectively, over the values obtained from a chemostat culture without cell retention.     

Using propidium monoazide to distinguish between viable and nonviable bacteria, MS2 and murine norovirus

Aims: The ability to distinguish between viable and/or infectious micro-organisms and inactivated cells is extremely important for correctly performing microbial risk assessments. In this study, we evaluated whether propidium monoazide (PMA)-qPCR could distinguish between viable and nonviable bacteria and viruses. Methods and Results: A PMA-qPCR combined assay was applied to viable and inactivated bacteria (Escherichia coli and Bacillus subtilis) and viruses (MS2 and murine norovirus [MNV]). PMA, a DNA-intercalating agent, in combination with PCR was better able to distinguish between viable and nonviable bacteria and viruses than conventional PCR. Conclusions: These results suggest that a combined PMA-qPCR assay can be used to measure the viability of bacterial cells and bacteriophage MS2, but not MNV. Significance and Impact of the Study: PMA-qPCR could potentially be used to measure the viability of some micro-organisms, including virus. However, a thorough evaluation should be performed prior to measuring the viability of micro-organisms by PMA-qPCR in a quantitative way.     

Infrared spectroscopy of plant cell cultures : noninvasive measurement of viability

Infrared spectroscopy was used to examine suspension-cultured pear (Pyrus communis L.) and Spartina pectinata cells. Noninvasive measurements were made using internal reflectance sampling. Spectra of actively growing cells exhibited a pronounced absorbance at 2343 reciprocal centimeters. The absorbance peak was identified and verified as CO₂ dissolved in water. This peak was absent in nonviable cells. Peak height was directly proportional to percent viability in artificial mixtures of viable and nonviable cells, indicating that the level of intracellular CO₂ production could be used as a viability determinant for plant cells. Suspension-cultured cells were slowly cooled to subzero temperatures and analyzed for viability using infrared spectroscopy and tetrazolium staining. Both methods showed similar trends in viability assessment. Infrared spectroscopy could provide a more detailed understanding of cell viability and allow measurement on a noninvasive basis.     

An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide

A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.     

Comparative Analysis and Limitations of Ethidium Monoazide and Propidium Monoazide Treatments for the Differentiation of Viable and Nonviable Campylobacter Cells

The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (C_T) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R²) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 μg/ml) and PMA (51.10 μg/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>10⁴) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation.     

Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment

Propidium monoazide (PMA) or ethidium bromide monoazide (EMA) treatment has been used before nucleic acid detection methods, such as PCR, to distinguish between live and dead cells using membrane integrity as viability criterion. The performance of these DNA intercalating dyes was compared in many studies utilizing different microorganisms. These studies demonstrated that EMA and PMA differ in their abilities to identify nonviable cells from mixed cell populations, depending on the microorganism and the nature of the sample. Due to this heterogeneity, both dyes were used in the present study to specifically distinguish dead from live Candida albicans cells using viable quantitative PCR (qPCR). The viable qPCR was optimized, and the best results were obtained when pre-treating the cells for 10 min in the dark with 25 μM EMA followed by continuous photoactivation for 15 min. The suitability of this technique to distinguish clotrimazole- and fluconazole-treated C. albicans cells from untreated cells was then assessed. Furthermore, the antifungal properties of two commercial essential oils (Thymus vulgaris and Matricaria chamomilla) were evaluated. The viable qPCR method was determined to be a feasible technique for assessing the viability of C. albicans after drug treatment and may help to provide a rapid diagnostic and susceptibility testing method for fungal infections, especially for patients treated with antifungal therapies.     

Separation of viable and nonviable animal cell using dielectrophoretic filter

Selective separation of cells using dielectrophoresis (DEP) has recently been studied and methods have been proposed. However, these methods are not applicable to large‐scale separation because they cannot be performed efficiently. In DEP separation, the DEP force is effective only when it is applied close to the electrodes. Utilizing a DEP filter is a solution for large‐scale separation. In this article, the separation efficiency for viable and nonviable cells in a DEP filter was examined. The effects of an applied AC electric field frequency and the gradient of the squared electric field intensity on a DEP velocity for the viable and nonviable animal cells (3‐2H3 cell) were discussed. The frequency response of the DEP velocity differed between the viable and the nonviable cells. We deducted an empirical equation that can be used as guiding principle for the DEP separation. The results indicate that the viable and the nonviable cells were separated using the DEP filter, and the best operating conditions such as the applied voltage and the flow rate were discussed. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Determination of viable yeast cells by gravitational field-flow fractionation with fluorescence detection

Vinification processing is largely related to yeast performance and depends on the initial cell viability. To optimize the quality of wine fermentation, control of the yeast quality is mandatory. The present paper describes a new method using gravitational field flow fractionation (GrFFF) with fluorescence detection for the determination of yeast cell viability before the fermentation process. A GrFFF calibration procedure was developed using commercial yeast to prepare standards of viable cells and propidium iodide (PI) as fluorescent probe for nonviable cells. The suitability of the new method was tested with several commercial yeast strains with a g/L content ranging from 1 to 3. The validation of the method was performed by comparing GrFFF viability values with those obtained using Coulter counter and flow cytometry techniques.     

Fluoro-Gold: An alternative viability stain for multicolor flow cytometric analysis.

BACKGROUND: The viability stains propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) are excited at 488 nm, as are the commonly used antibody conjugates fluorescein isothiocyanate (FITC), phycoerythrin (PE), and cyanine 5 dye covalently coupled to R-phycoerythrin (RPE-Cy5). When excited by a single laser, spectral overlap in the emission of PI and 7-AAD with RPE-Cy5 precludes the use of these viability stains for three-color immunophenotyping, particularly when evaluating low levels of marker expression in viable target cells. The ultraviolet excitable dye hydroxystilbamidine methanesulfonate (Fluoro-Gold, or FG) binds to DNA at the A-T-rich regions of the minor groove in permeabilized or dead cells. We assessed the suitability of this dye as a viability stain. METHODS: The ability of FG to detect nonviable cells in fresh and cryopreserved human apheresed peripheral blood cells was compared with that of PI and 7-AAD. The stability of FG staining and the effects of dye and cell concentration on the discrimination of nonviable cells was determined by measuring changes in the median fluorescence of viable and nonviable cells. RESULTS: FG labeling at dye concentrations of 2-8 microM is stable for at least 3 h over a wide range of cell concentrations (4 x 10(5) to 4 x 10(7) cells/ml). Costaining studies and linear regression analysis show that cell viability as determined by FG is strongly correlated with estimates using PI (r = 0.9636) and 7-AAD (r = 0.9879). CONCLUSIONS: FG is a reliable, alternative viability stain that can be used in conjunction with fluorochromes including FITC, PE, and RPE-Cy5 for multicolor analysis using dual-laser instruments.     

Long-term nutrient starvation of continuously cultured (glucose-limited) Selenomonas ruminantium.

Selenomonas ruminantium, a strictly anaerobic ruminal bacterium, was grown at various dilution rates (D = 0.05, 0.25, and 0.35 h-1) under glucose-limited continuous culture conditions. Suspensions of washed cells prepared anaerobically in mineral buffer were subjected to nutrient starvation (24 to 36 h; 39 degrees C; N2 atmosphere). Regardless of growth rate, viability declined logarithmically, and within about 2.5 h, about 50% of the populations were nonviable. After 24 h of starvation, the numbers of viable cells appeared to be inversely related to growth rate, the highest levels occurring with the slowest grown population. Cell dry weight, carbohydrate, protein, ribonucleic acid (RNA), and deoxyribonucleic acid declined logarithmically during starvation, and the decline rates of each were generally greater with cells grown at higher D values. Both cellular carbohydrate and RNA declined substantially during the first 12 h of starvation. Most of the cellular RNA that disappeared was found in the suspending buffer as low-molecular-weight, orcinol-positive materials. During growth, S. ruminantium made a variety of fermentation acids from glucose, but during starvation, acetate was the only acid made from catabolism of cellular material. Addition of glucose or vitamins to starving cell suspensions did not decrease loss of viability, whereas a starvation in the spent culture medium resulted in a slight decrease in the rate of viability loss. Overall, the data indicate that S. ruminantium strain D has very little survival capacity under the conditions tested compared with other bacterial species that have been studied.     

Responses of Ruminococcus flavefaciens, a Ruminal Cellulolytic Species, to Nutrient Starvation

Ruminococcus flavefaciens strain C94, a strictly anaerobic, cellulolytic ruminal bacterial species, was grown either in batch or continuous cultures (cellobiose limited or nitrogen limited) at various dilution rates. Washed cell suspensions were incubated anaerobically at 39°C without nutrients for various times up to 24 h. The effects of starvation on direct and viable cell counts, cell composition (DNA, RNA, protein, and carbohydrate), and endogenous production of volatile fatty acids by the cell suspensions were determined. In addition, the effect of the pH of the starvation buffer on direct and viable cell counts was determined. Survival of batch-grown cells during starvation was variable, with an average time for one-half the cells to lose viability (ST₅₀) of 10.9 h. We found with continuous cultures that viable cell counts declined faster when the initial cell suspensions had been grown at faster dilution rates; this effect was more pronounced for suspensions that had been limited by cellobiose (ST₅₀ = 6.6 h at a dilution rate of 0.33 h⁻¹) than for suspensions that had been limited by nitrogen (ST₅₀ = 9.5 h at a dilution rate of 0.33 h⁻¹). With continuous cultures, viable cell counts in all cases declined faster than direct cell counts did. The rates of disappearance of specific cell components during starvation varied with the initial growth conditions, but could not be correlated with the loss of viability. Volatile fatty acid production by starving cells was very low, and acetate was the main product. Starved cells survived longer at pH 7.0 than they did at pH 5.5, and this effect of pH was greater for cellobiose-limited cells (mean ST₅₀ = 7.1 h) than for nitrogen-limited cells (mean ST₅₀ = 12 h). Although it has relatively low ST₅₀ values, R. flavefaciens has sufficient survival abilities to maintain reasonable numbers in domestic animals having maintenance or greater feed intake.     

Study of the viability of cultured human cells in suspensions

The success of cell therapy is directly related to the viability of cells used for transplantation. The cells used for transplantation are in some cases injected in suspension. However, the optimal conditions for the preservation of cell viability upon the preparation and storage of cell suspensions for transplantation have not been defined yet. The aim of the present work consisted in the selection of optimal conditions for the storage of suspensions of human submandibular salivary gland cells, differentiated cells of the submandibular salivary gland, and dermal fibroblasts in biocompatible solutions. Standard procedures of cell isolation and cultivation were used in the study. An automatic cell counter from BioRad was used to count the cells, and viability of the cells was assessed using staining with 4% Trypan Blue. The biocompatible solutions tested included phosphate-buffered saline, physiological saline for injections, and a 2% solution of human albumin in phosphate-buffered saline. The study showed that the human cells under investigation remained viable in suspension at both +4°С and +25°С for at least 24 hours, regardless of the carrier solution used. The highest content of viable cells of the salivary gland (more than 50%) at both temperatures examined was observed when cells were suspended in phosphate-buffered saline. However, the adhesive and proliferative properties of the salivary gland cells were better preserved at +4°С in case of 24 hours of incubation under the conditions described above. Fibroblasts maintained in physiological saline formed a homogeneous single-cell suspension that remained stable for 30 hours at +4°С; virtually no loss of cell viability was observed. The addition of 2% albumin resulted in a decrease of the viability of fibroblasts. Thus, storage and transportation in phosphate- buffered saline at +4°С can be recommended for suspensions of cells of the human submandibular salivary gland, whereas human fibroblast suspensions should be maintained at +4°С in physiological saline.     

Rapid method for enumeration of viable Legionella pneumophila and other Legionella spp. in water

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.     

Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry.

Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non-vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7-amino-actinomycin D (7-AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7-AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7-AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)-fluorescence dual-color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7-AAD was used on fluoresceinisothiocyanate (FITC) and PE surface-labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.