Viability stains

To ensure that a cell culture is growing exponentially it is useful to know the percentage viability and percentage of dead cells and hence the stage of growth of the cells. This can be estimated by their appearance under the microscope, because live healthy cells are usually round, retractile and relatively small in comparison to dead cells, which can appear larger, crenated and non-refractile when in suspension. The use of viability stains such as Trypan blue ensures a more quantitative analysis of the condition of the culture. Trypan blue is a stain that will only enter across the membranes of dead/non-viable cells. 

Cells in trypan blue viability stain were enumerated microscopically. The spleen cells were mixed with an equal number of SP/2.0 myeloma cells in the semi-log growth phase in RPMI medium. The cell mixture was centrifuged (200 x g) for 10 minutes and the cell pellet was suspended in 1 mL of polyethylene glycol (3 000 - 4 000 mol. wt. range) at 37 °C. The suspension was mixed continuously for one minute, followed by the addition of 1 mL of RPMI medium and another one minute of continuous mixing. An additional 9 mL of RPMI medium... 

Culture uninfected cells and virus-infected cells, under optimal conditions for the particular cell type to be tested, in the presence and absence of increasing concentrations of the potential antiviral compound. Harvest cell samples daily and perform viability staining. 

Recently, we have developed a novel approach for the rapid enumeration of airborne bacteria and fungi based on SPC. Air samples are collected by impaction on a water soluble polymer that is subsequently dissolved. For labelling of the airborne microorganisms, the viability stain ChemChrome V6 was used (Vanhee et al. 2008, 2009a). 

D Haese et al. (2005) studied the potential of SPC for the rapid enumeration of the extremely slow-growing Mycobacterium paratuberculosis in milk. However, as the viability stain ChemChrome V6 was used for labelling, no specific detection of the bacteria was obtained and only spiked samples were analysed. 

De Vos MM, Nelis HJ (2006) An improved method for the selective detection of fungi in hospital water by solid phase cytometry. J Microbiol Meth 67 557-565 D Haese E, Nelis HJ (2000) Effect of antibiotics on viability staining of Escherichia coli in solid phase cytometry. J Appl Microbiol 89 778-784... 

C.4 Microscopic (Direct) Examination Using Viability Stains... 

After incubation for 30 min, the cell lines which immersed GNPs suspension were exposed to 532 nm laser at various power densities for 500 times. The cell images were taken using Motic AE21 microscope under 40 x to test cell viability stained with 0.4% trypan blue (Sigma). 

Caruso, G. Mancuso, M. Crisafi, E. Combined fluorescent antibody assay and viability staining for the assessment of the physiological states of Escherichia coli in seawaters. J. Appl. Microbiol. 2003, 95, 225-233. 

Bouchez, J. C. Cornu, M. Danzart, M. Leveau, J. Y. Duchiron, K Bouix, M. Physiological sigiuficance of the cytometric distribution of fluorescent yeasts after viability staining. Biotechnol. Bioeng. 2004,86, 520-530. 

Nexmann J., C. Rasmussen, J. Jakobsen, M. Viability staining and flow cytometric detection of... 

Fig. 15. Effects of turbulent shear stress level and exposure time on cell viability measured by trypan blue staining. Cells were sheared in a concentric cylinder viscometer [1]...

A dual isotope labeling technique [85] has been used to measure membrane permeability in plant cells, based on the selective permeabiHty of the membranes of living cells to tritiated water and carbon-14 labeled mannitol. Kieran [29] showed that the results of the dual isotope labeling and Evan s Blue staining methods correlated well as indicators of cell viability however, the latter was preferable in terms of reagent cost and ease of analysis. 

Isolated hepatocytes incubated with ionic iron rapidly undergo lipid peroxidation. Some studies have not shown a consequent decrease in viability (as indicated by uptake of trypan blue or release of enzymes). This is probably a result of short incubation times, as changes in viability lag behind increases in lipid peroxidation, and may not occur for more than 2 h after lipid peroxidation begins (Bacon and Britton, 1990). Recent studies have shown strong correlations between increased lipid peroxidation [production of thiobarbituric acid (TBA) reactants] and loss of cell viability (trypan blue staining) (Bacon and Britton, 1989). The significance of the lag between lipid peroxidation and decreases in cell viability is as yet uncertain. 

Imbert D, Cullander C (1999) Buccal mucosa in vitro experiments I. Confocal imaging of vital staining and MTT assays for the determination of tissue viability. J Control Release 58 39-50... 

Figure 22. Human embryonic kidney cells (A), rat vascular smooth muscle cells (B, C) and human osteoblast-like MG 63 cells (D) in cultures on micropattemed surfaces. A, B PTFE irradiated with UV light produced by a Xe2 -excimer lamp for 30 min in an ammonia atmosphere through a mask with holes 100 pm in diameter and center-to-center distance 300 pm C PE irradiated with Ar ions (energy 150 keV, ion dose lO ions/cm ) through a mask with holes 100 pm in diameter and center-to-center distance 200 pm fullerenes Qo deposited through a mask with rectangular holes with an average size of 128 3 pm per 98 8 pm on glass coverslips. Day 7 after seeding. A native cells in an inverted phase-contrast microscope B, C cells stained with hematoxylin and eosin, Olympus microscope IX 50 D cells stained with fluorescence-based LIVE/DEAD viability/cytotoxicity kit (Invitrogen), Olympus microscope IX 50. Bars 300 pm (A), 200 pm (B, D), Imm (C) [10,11].

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