The Reduction Of 2,3,5-triphenyltetrazolium Chloride Research Articles

Iron deficiency induces changes in riboflavin secretion and the mitochondrial electron transport chain in hairy roots of Hyoscyamus albus

Hyoscyamus albus hairy roots secrete riboflavin under Fe-deficient conditions. To determine whether this secretion was linked to an enhancement of respiration, both riboflavin secretion and the reduction of 2,3,5-triphenyltetrazolium chloride (TTC), as a measure of respiration activity, were determined in hairy roots cultured under Fe-deficient and Fe-replete conditions, with or without aeration. Appreciable TTC-reducing activity was detected at the root tips, at the bases of lateral roots and in internal tissues, notably the vascular system. TTC-reducing activity increased under Fe deficiency and this increase occurred in concert with riboflavin secretion and was more apparent under aeration. Riboflavin secretion was not apparent under Fe-replete conditions. In order to examine which elements of the mitochondrial electron transport chain might be involved, the effects of the respiratory inhibitors, barbiturate, dicoumarol, malonic acid, antimycin, KCN and salicylhydroxamic acid (SHAM) were investigated. Under Fe-deficient conditions, malonic acid affected neither root growth, TTC-reducing activity nor riboflavin secretion, whereas barbiturate and SHAM inhibited only root growth and TTC-reducing activity, respectively, and the other compounds variously inhibited growth and TTC-reducing activity. Riboflavin secretion was decreased, in concert with TTC-reducing activity, by dicoumarol, antimycin and KCN, but not by SHAM. In Fe-replete roots, all inhibitors which reduced riboflavin secretion in Fe-deficient roots showed somewhat different effects: notably, antimycin and KCN did not significantly inhibit TTC-reducing activity and the inhibition by dicoumarol was much weaker in Fe-replete roots. Combined treatment with KCN and SHAM also revealed that Fe-deficient and Fe-replete roots reduced TTC in different ways. A decrease in the Fe content of mitochondria in Fe-deficient roots was confirmed. Overall, the results suggest that, under conditions of Fe deficiency in H. albus hairy roots, the alternative NAD(P)H dehydrogenases, complex III and complex IV, but not the alternative oxidase, are actively involved both in respiration and in riboflavin secretion.

Development of a naturally miniaturised testing method based on the mitochondrial activity of fern spores: A new higher plant bioassay

One of the main concerns of current environmental toxicology is the low number of taxa used for standard bioassays. Ferns, with more than 10 000 living species, are the second largest group of vascular plants and are important components of numerous plant communities. Fern spores and gametophytes have long been recognized as useful models for plant research since they constitute a naturally miniaturised and economic higher plant model. Mitochondria are the main energy source in eukaryotic cells and any toxic damage will affect the whole organism. The reduction of tetrazolium salts to water-insoluble coloured formazan salts by the respiratory chain has been used for more than 50 years as a measure of cell mitochondrial activity and viability in eukaryotic organisms. Here, the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) by mitochondria is adapted and optimized to measure fern spore or gametophyte viability. Procedures selected as optimum in the model species Dryopteris guanchica are as follows: bleach sterilization, incubation without shaking at 20 °C in the dark for 1–4 h with 0.05–1.5% TTC in Dyer medium supplemented with 0.001–0.005% Tween 20 at pH 8. We conclude that this method constitutes a promising low cost bioassay for higher plant toxicity during development.

Isolation and identification of lactic acid bacteria from sediments of a coastal marsh using a differential selective medium

To identify lactic acid bacteria (LAB) colonies isolated from sediments of a coastal marsh by the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) in MRS medium. Single colonies isolated from sediments of a coastal marsh by enrichment in MRS broth were selected from MRS-TTC plates and classified according to colony phenotype based on TTC reduction. A total of 37 colonies grouped in seven different phenotypes were identified by analysis of its 16S ribosomal gene sequence. Most isolates belonged to the Firmicutes phylum, mainly to orders Bacillales and Lactobacillales. LAB were represented by 20 isolates, 15 of which belong to the genus Weissella. Enrichment in MRS was highly selective for the isolation of bacteria belonging to phylum Firmicutes. Several different phenotypes were developed by LAB and must be considered during LAB isolation based on TTC reduction. To our knowledge, this is the first study aimed at determining a relationship between colony phenotype from TTC reduction and a partial identification of isolates based on 16S ribosomal gene sequence similarities. Besides, this is the first report of isolation of W. cibaria from environmental samples.

Osmotic-treatment-induced cell death and osmotic processing kinetics of apples with characterised raw material properties

Three apple varieties cultivated in southern Sweden namely Jonagold, Kim and Mutsu were subjected to osmotic treatment at 5, 20 and 40 °C with a 50% sucrose solution. The evaluation of cell viability after osmotic processing, was carried out in Granny Smith apples from Argentina. The processing conditions were 50% sucrose solution at 20 °C and the viability assay used was based on the reduction of 2,3,5-triphenyltetrazolium chloride (TTC). The experimental samples were separated into inner (close to the apple core) and outer (close to the skin) specimens, due to the existence of pronounced structural differences between them. The kinetics revealed that for each apple variety and at each process temperature, samples of the outer structure exhibited higher water loss and lower solids gain than those of the inner structure. Overall, the Jonagold and Kim apples exhibited similarly high water loss, while Mutsu showed a lower loss. Jonagold absorbed the lowest amount of solids with Kim rating second and Mutsu apples showing the highest solids gain. The cell viability assay on the experimental samples revealed the first layer of cell in a depth 1–2 mm from the surface to die as a result of the severe osmotic shock.

A comparison of two methods of inducing water stress in wheat (Triticum aestivum L.)

The aim of this study was to compare the withholding of water and the use of polyethylene glycol (PEG) as methods of inducing water stress in spring wheat cultivars (Triticum aestivum L.) grown in the Western Cape. Water stress was induced in two cultivars that had previously shown differences in drought tolerance. Proline accumulation in plants and the reduction of 2,3,5-triphenyltetrazoliumchloride (TTC) were used as indicators of water stress in the test plants. Proline accumulation proved to be a more sensitive indicator of water stress compared to TTC, showing significant increases with both the withholding of water and PEG as methods to induce water stress. Although both methods of stress induction proved to be effective, the withholding of water may be more appropriate due to the possibility of root injuries with PEG. Good control of climatic conditions and measurements of plant water potential will, however, be essential if the withholding of water is used to induce stress.

Ethylene production by leaves of rice ( Oryza sativa L.) in relation to salinity tolerance and exogenous putrescine application

The effects of salt stress on the production of ethylene in leaves of five rice cultivars differing in salinity tolerance were investigated after 15 and 30 days of stress exposure in plants sprayed every 2 days with deionised water or putrescine 100 μM. NaCl induced an increase in ethylene synthesis which was higher in salt-tolerant than in salt-sensitive cultivars (cvs.). Salt-stress also induced an increase in 1-aminocyclopropane-1-carboxylic acid (ACC) content which was larger in salt-tolerant cvs. whereas a decrease in ACC conversion to ethylene suggested a reduction in ACC oxidase activity. Putrescine increased the growth and the leaf tissue viability (as indicated by the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to formazan) of salt-treated plants in all cvs. This positive effect was associated with an increase in ethylene biosynthesis through an increase in ACC content and a suppression of NaCl-induced inhibition of ACC conversion to ethylene. These data suggest that putrescine could be involved in salinity tolerance in rice.

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 √Valt plot, and was established to be 89 %.

EVALUATION OF A DEHYDROGENASE ASSAY BASED ON TETRAZOLIUM REDUCTION FOR RAPID IN VITRO ESTIMATION OF FERMENTATION ACTIVITY IN RUMEN CONTENTS

Rumen contents obtained from fistulated steers fed brome–alfalfa hay was used to evaluate a dehydrogenase assay based on the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to triphenylformazan (TPF) for rapid in vitro estimation of fermentation activity. Maximum absorbance of TPF occurred at 485 nm over the pH range 3–10. TPF formed from TTC was stable to atmospheric O2. Absorbance decreased if TPF was exposed to light. In the presence of excess TTC, production of TPF in strained rumen fluid (SRF) at 39 °C was linear with elapsed incubation times up to at least 5 min. Dehydrogenase activity was abolished in the presence of air. It was significantly increased if the CO2 gas phase above SRF was replaced with N2 or H2, owing, at least in part, to an increase in the pH of the fermentation. The dehydrogenase activity of SRF was maximal at pH 7.8–8.0. Fractionation of SRF by differential centrifugation showed that about 50% of the dehydrogenase activity was associated with a protozoal fraction sedimenting at 188 × g, and 42% with a bacterial fraction sedimenting at 1,475 × g. Neither the cell-free supernatant obtained by centrifuging SRF at 21,500 × g, nor boiled SRF, nor the hay fed to the animals showed dehydrogenase activity. Whole rumen contents (WRC) showed a mean dehydrogenase activity that was 58% higher than that of SRF. The dehydrogenase activity of SRF was increased in the presence of substrates representing major plant carbohydrates, the greatest stimulatory effect being shown by ground brome–alfalfa hay. When rumen contents were sampled at the same time by stomach tube and through the fistula, the mean dehydrogenase activity of SRF prepared from the stomach tube samples was 137% higher than that of SRF prepared from the fistula samples. In an animal fed at 12-h intervals, the dehydrogenase activity of SRF rose within 2 h after feeding, declined for the next 6 h, and showed a second peak at 9 h. Activity then declined to the pre-feeding level. The dehydrogenase activity of WRC showed a similar pattern but the peaks of activity were less pronounced. It was concluded that dehydrogenase activity based on TTC reduction provides a simple and rapid means of estimating the fermentation activity of rumen contents which may reflect the availability of energy substrates to the microbiota.

Studies on the reduction of 2,3,5-triphenyltetrazolium chloride as a viability assay for plant tissue cultures

Optimum conditions for the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to a water-insoluble red formazan have been determined for suspension tissue cultures of Acer saccharum and Haplopappus gracilus. The standard incubation mixture is 0.8% TTC dissolved in a 2:1 solution of 0.05 M sodium phosphate buffer:Acer growth medium, pH 7.5. After the cells are incubated for 18–20 h in the dark at room temperature, the red formazan is extracted with 95% ethanol, and the absorbance is read at 485 nm. The use of the TTC procedure as a viability assay is supported by studies with Haplopappus tissue cultures, in which viability can be assayed by both plating in agar and the TTC procedure. These studies show that there is a quantitative correspondence between the decrease in the plating assay and in the TTC assay when these cells are subjected to freezing stress and plasmolysis stress.

Mechanism of the reduction of triphenyltetrazolium chloride by succinoxidase

1. 1. A low concentration of antimycin A inhibits the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) in the presence of succinate and rat liver and heart mitochondria under aerobic conditions. 2. 2. A greater concentration of antimycin A is needed for the inhibition of the reduction of TTC with rat heart mitochondria than with rat liver mitochondria in the presence of succinate.

Release of Mitochondria from Yeast Cells by the Action of Metal-Chelating Agents

The reduction of 2,3,5-triphenyltetrazolium chloride (TTC) by resting cells of a yeast, Candida albicans, appears to be localized in particulate structures within the cells. The reduced tetrazolium (triphenylformazan, TPF) can readily be seen in structures similar to those identified as mitochondria by Mudd, et al.1, 2 in bakers' yeast and in bacteria. In the course of our studies3 on the reduction of TTC by resting cells of Candida albicans, it was observed that certain activators of the reduction, particularly potassium cyanide and the disodium salt of ethylenediaminetetraacetic acid (Na2EDTA), caused a dispersion of the waterinsoluble formazan into the medium surrounding the cells. Light microscopic studies revealed that the coloration was due only in small part to the crystallization or colloidization of TPF, and resulted mainly from the release of TPF-stained particles from the yeast cells. The particles that were released were identical with particles observed inside the cell. Indeed, particles could often be observed on the exterior of the cell wall (Fig. 1). In some instances, partly lysed cells appeared to have released large clusters of particulate material. It was possible in the following manner to demonstrate the identity of the TPF-stained bodies inside and outside of the cells. Washed resting cells which had been in contact with 0.005 per cent TTC for 5-10 min. were mounted on slides and observed under the light microscope. Illumination from the microscope light hastened the reduction of TTC inside the cells.4 When numerous TPF