Quantitative viability of seaweed tissues assessed with 2,3,5-triphenyltetrazolium chloride

A spectrophotometric quantification method was optimized to evaluate its utility in seaweed tissue viability tests using the enzymatic reduction of colorless 2,3,5-triphenyltetrazolium chloride (TTC) to a colored triphenylformazan (TPF). To allow accurate determination of TPF in the seaweed Porphyra thallus and conchocelis, 0.2 g of tissues are incubated with 4 mL of 0.8% TTC reagent in the dark at 20°C for 1 h under a mineral oil layer. The TPF formed in tissues was extracted for 15 min at 60°C with 2 mL of 0.2 N KOH in 25% ethanol. Then TPF is partitioned away by prompt addition of hexane and vortexing. By this procedure, we have observed nearly complete separation of TPF, and observed good spectrophotometric discrimination between TPF and other hexane-soluble pigments at 545 nm. This procedure has proved applicable to a wide range of seaweed taxa; 1 species of Chlorophyta, 4 species of Phaeophyta and 7 species of Rhodophyta tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Osmotic induced stimulation of the reduction of the viability dye 2,3,5-triphenyltetrazolium chloride by maize roots and callus cultures

Live cells can reduce colorless 2,3,5-triphenyltetrazolium chloride (TTC) to a red insoluble compound, formazan. Maize (Zea mays) callus, when osmotically stressed by 0.53 mol/L mannitol, produced 7-times or more formazan than untreated control callus. This result was seen with all osmotica tested and could not be attributed to differences in TTC uptake rate or accumulation, increased respiration rate as measured by O2 uptake, or to de novo protein synthesis. Increased formazan production could be detected after 2.5 h of exposure to osmotic stress and leveled off after 48 h of exposure. The increased formazan production was only detected when callus was moved from high osmotic medium to low osmotic, TTC-containing medium. Abscisic acid increased TTC reduction only when added in combination with 0.53 mol/L mannitol. Incubation of maize seedling roots with 0.53 mol/L mannitol also increased formazan production as seen visually. Further studies are needed to determine the cause of the increased formazan production. These results show that TTC viability measurements must be carefully evaluated with appropriate controls to confirm their validity.     

A modified Kelsey-Sykes method for testing disinfectants with 2,3,5-triphenyltetrazolium chloride reduction as an indicator of bacterial growth

Different dilutions of chlorhexidine gluconate were tested by the Kelsey-Sykes procedure. The method was further modified on microtitration plates using 2,3,5-triphenyltetrazolium chloride (TTC) reduction as an indicator of bacterial growth. There was a good correlation with the results based on TTC reduction and the conventional method based on turbidity changes caused by bacterial growth. Furthermore, the modified method using TTC reduction is more rapid and can be read by the naked eye because of the red colour.     

A modified Kelsey-Sykes method for testing disinfectants with 2,3,5-triphenyltetrazolium chloride reduction as an indicator of bacterial growth

Different dilutions of chlorhexidine gluconate were tested by the Kelsey-Sykes procedure. The method was further modified on microtitration plates using 2,3,5-triphenyltetrazolium chloride (TTC) reduction as an indicator of bacterial growth. There was a good correlation with the results based on TTC reduction and the conventional method based on turbidity changes caused by bacterial growth. Furthermore, the modified method using TTC reduction is more rapid and can be read by the naked eye because of the red colour.     

Reduction of 2,3,5-triphenyltetrazolium chloride by the KCN-insensitive,salicylhydroxamic acid-sensitive alternative respiratory pathway of mitochondria from cultured grapevine cells

The reduction of 2,3,5-triphenyltetrazolium chloride (TTC) by grapevine cells cultured in suspension was studied in order to assess the reliability of using TTC reduction as a measure of cell viability. Similar to the reduction observed in animals cells, TTC can be reduced in grapevine cells by the cytochrome respiratory path of the mitochondria, although it is mostly reduced (about 72 %) by the alternative respiratory path sensitive to salicylhydroxamic acid. Engagement of the alternative path in TTC reduction was calculated through the V_alt plot, and was established to be 89 %.Abbreviations SHAM salicylhydroxamic acid - TTC 2,3,5-triphenyltetrazolium chloride     

Distinguishing between metabolically active and inactive roots by combined staining with 2,3,5-triphenyltetrazolium chloride and image colour analysis

The objective of the present work was to develop a method to distinguish between metabolically inactive and active parts of plant roots. White clover (Trifolium repens L.) roots were stained with 2,3,5-triphenyltetrazolium chloride (TTC) followed by root colour classification with an interactive scanner-based image analysis programme (WinRHIZO). Roots inactivated by boiling were unstained and pale brown, whereas fresh samples with predominantly metabolically active roots turned dark red, red or pale red after staining. A small amount of very young, presumable active roots (0.8% of total active root length) failed to stain red with TTC. The colour analysis of inactive and active roots was based on four colour classes for boiled roots and seven classes for fresh roots, respectively, as defined upon visual examination of images. Pixel colours falling outside the defined classes were allocated to the nearest defined class – an option that increased objectivity and stability and reduced the required number of colour classes. For the fresh white clover roots, 75–86% of the total root length was determined as active, while 3–7% of the boiled roots fell into the same category. The percentage of total root length measured by WinRHIZO that was identified as metabolically active was linearly correlated with the percentage of fresh roots in mixtures of fresh and boiled roots (R²=0.99). Colour classes chosen à priori from one experiment could be used to distinguish fairly satisfactorily between active and inactive roots of another white clover cultivar grown under other conditions, but failed to classify activity in ryegrass (Lolium multiflorum Lam.) root samples. In the latter case, colour classes needed to be re-defined in order to produce reliable data. Our work shows that WinRHIZOs colour identification sub-module provides a new promising tool to classify root activity as identified after staining with TTC, but colour classes must be carefully evaluated on every new occasion.     

Presumptive identification of an emerging yeast pathogen: Candida dubliniensis (sp. nov.) reduces 2,3,5-triphenyltetrazolium chloride

Developments in medical intervention and the increasing population of patients with immunodeficiencies and transient or long-term immunosuppression have increased the list of yeast species that can cause disease. Candida dubliniensis is a novel species with close genetic relatedness to C. albicans. The two species share many common physiological and biochemical properties thus making their distinction cumbrous. A rapid and inexpensive way to presumptively differentiate between the two species, having previously performed a germ tube test, is the ability of C. dubliniensis to reduce the tetrazolium salt and it is reported for the first time. Microbiological information about new and emerging yeast pathogens, including rapid means for their identification, equips medical microbiologists with the means to identify and physicians to treat effectively infections attributed to unusual yeasts.     

ByDioscorea deltoidea free and immobilized plant cells

Summary 2-(4-methoxybenzyl)-1-cyclohexanone (1) was converted to its glucoside (4,5) via corresponding alcohols (3,4) usingDioscorea deltoidea Wall. free and immobilized plant cells.     

Bioconversion of gitoxigenin by immobilized plant cells in a column bioreactor

Summary Daucus carota cells immobilized in a Ca-alginate performed the bioconversion of gitoxigenin to 5-hydroxygitoxigenin in a column bioreactor. The smooth spherical shape of the alginate beads was preserved for more than three weeks. The bioreactor was functional for more than thirty days as detected by the bioconversion activity. The rate of bioconversion was influenced by means of aeration.Issued as National Research Council of Canada Publication Number 19556     

Development of bioreactors for the culture of surface immobilized plant cells

The scaleup of the technique of plant cell surface immobilization was performed successfully in specifically designed laboratory size bioreactors. The immobilizing matrix was formed into a vertically wound spiral providing for a high immobilizing area-to-volume ratio (0.8-1.2 cm(-1)). A modified airlift and a mechanically stirred vessel delivered a best bioreactor performance characterized by low biomass frothing and highly efficient plant cell attachment and retention (>/=96%). The growth of Catharanthus roseus cells investigated in these bioreactors was found not to be mass transfer limited. It required mild mixing and aeration levels (k(L)a approximately 10-15 h(-1)). The biomass formation pattern of surface immobilized plant cells generally exhibited a linear growth phase followed by a stationary phase characterized by the presence of residual carbohydrates in the medium, contrary to suspension cultures. This behavior was found to depend on the plant cell type and/or line cultured, as well as on the inoculum age. The space restriction and unidirectional growth of the SIPC biofilm combined with the limited availability of essential intracellular nutrients rapidly accumulated from the medium by the stationary phase inoculated plant cells all likely contributed to the culture behavior.     

A colorimetric assay for immobilized chloroperoxidase

A rapid and sensitive colorimetric assay was developed for the estimation of chloroperoxidase activity. N,N,N',N'-Tetramethyl-p-phenylenediamine was chosen from four potential chromogenic substrates because the blue product resulting from chloroperoxidase conversion gave the highest molar absorption. This product exhibited two absorbance maxima, at 563 and 610 nm. Activity was monitored at 563 nm, and the product absorbance was stable for at least 1 h at 10 degrees C after treatment with an equal volume of a mixture (40:1) of methanol and phosphoric acid (85% w/v), pH 2. The linear range of the assay with respect to enzyme amount was determined. The assay was developed using soluble chloroperoxidase but worked well with the enzyme immobilized on glass beads.     

Hypericum perforatum plant cells reduce Agrobacterium viability during co-cultivation

Plant recalcitrance is the major barrier in developing Agrobacterium-mediated transformation protocols for several important plant species. Despite the substantial knowledge of T-DNA transfer process, very little is known about the factors leading to the plant recalcitrance. Here, we analyzed the basis of Hypericum perforatum L. (HP) recalcitrance to Agrobacterium-mediated transformation using cell suspension culture. When challenged with Agrobacterium, HP cells swiftly produced an intense oxidative burst, a typical reaction of plant defense. Agrobacterium viability started to decline and reached 99% mortality within 12 h, while the plant cells did not suffer apoptotic process. This is the first evidence showing that the reduction of Agrobacterium viability during co-cultivation with recalcitrant plant cells can affect transformation.     

Continuous production of steroid glycoalkaloids by immobilized plant cells

Summary Non-permeablilized cells ofSolanum aviculare were immobilized by their adsorption and covalent linkage on activated polymeric adsorbent. The retention of extracellular production of steroid glycoalkaloids was studied in a packed-column recycle reactor.     

On-line low-volume transesterification-based assay for immobilized lipases

A method has been developed for fast evaluation of transesterification activity of immobilized lipases using microlitre and submicrolitre volumes of substrate solutions. The model reaction (acylation of isopropanol with vinyl acetate) is catalyzed by microbial lipases immobilized on ceramic particles, diatoms or acrylic resin, packed into a Teflon tube connected to fused-silica capillary tubing. The substrate solution is pumped through the microreactor and the product of transesterification, acetaldehyde, quantified on capillary by UV absorbance at 280 nm. Using this system in the continuous-flow mode, comparisons were made of transesterification catalysed by PS-C II lipase in two different solvent mixtures. Acetonitrile was found to be a compatible solvent that can be used as a solubilizer without suppressing enzymatic activity. The method has been used to compare conversions at a fixed flow rate using a single pass of substrate through packed beds containing approximately 0.2 mg of supported enzyme. Spatial distributions of the product have been visualized using a complementary metal oxide semiconductor (CMOS) imaging detector in conjunction with the microreactor system operated in the stopped-flow mode.     

Suspension-cultured plant cells as a platform for obtaining recombinant proteins

Production of recombinant proteins in suspension cultures of genetically modified plant cells is a promising and rapidly developing area of plant biotechnology. In the present review article, advantages related to using plant systems for expression of recombinant proteins are considered. Here, the main focus is covering the literature on optimization of cultivation conditions of suspension-cultured plant cells to obtain a maximal yield of target proteins. In particular, certain examples of successful use of such cells to produce pharmaceuticals were described.     

Permeabilization of plant cells for release of intracellularly stored products: viability studies

Summary The effects of various chemical substances on the permeability of plasma membranes and tonoplasts of three suspension cultures (Catharanthus roseus, Thalictrum rugosum and Chenopodium rubrum) have been studied. The permeability of the plasma membrane is monitored by measuring the activity of the cytosolic enzyme isocitrate dehydrogenase and the permeability of the tonoplast is measured by determining the release of substances stored in the vacuoles (inorganic phosphate, berberine and betanin for the three cell lines, respectively). The minimum concentration required for quantitative release of vacuolar products have been established for five different permeabilization agents. Cell viability is lost upon permeabilization except for treatment of Catharanthus roseus with DMSO and Triton X-100.Abbreviations DMSO dimethylsulfoxide - PEA phenethylalcohol - HDTMAB hexadecyltrimethylammonium bromide - ICDH isocitrate dehydrogenase     

Real-time viability assay based on 51Cr retention in adherent cells

The chromium (51Cr) release assay has been widely used for viability measurements, even though it has major disadvantages such as high manual workload and poor time resolution. By the use of LigandTracer 51Cr release viability measurements on adherent cells can be significantly simplified and improved. LigandTracer enables a time-resolved detection of 5SCr in target cells, with the result that the effect of toxic material is updated continuously throughout the experiment. Here we explain the principle behind this novel real-time viability assay and show viability curves for known toxic compounds on A431 and U343MGaCl2:6 cell lines.