This study was aimed to determine the viability rate of rosella seed, to obtain the best result of seed testing for enhancing rosella seed viability, to find the best seed invigoration method for enhancing rosella seed viability, to obtain staining pattern through tetrazolium test of rosella seed, and to determine viability and vigor of rosella seed to be further used as estimation indicator for rosella plant growth in the field. The study was conducted in the Seed Laboratory, Indonesian Sweetener and Fiber Crops Research Institute (ISFCRI), Malang, East Java during July - August 2018. The material used included accessions of rosella seed (Hibiscus sabdariffa L.) ACC. 1148 from the year 2015 and collection of ISFCRI, 100 ml of Tetrazolium solution (40 ml KH2PO4, 60 ml Na2HPO4 and 1 gr of Tetrazolium powder). This research applied Completely Randomized Design (CRD) consisted of seed treatments of control (no immersion/0 hour), immersion for 5 hours, immersion for 10 hours, and scarification, each with 4 replications. Result of this study showed that the use of tetrazolium salt was found to be better in enhancing the viability of rosella seeds. Viable seeds was found to have bright red embryonic axis and bright red cotyledon. Testing using paper media on several seed invigoration treatments resulted in significantly different effect on parameters of vigor index, germination capacity, and dry weight of normal seedling. The best parameter of germination capacity and dry weight of normal seedling was obtained by treatment immersed in water at temperature of 27°C for 10 hours

Anti-Plasmodium activity of imidazolium and triazolium salts

Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Tooth slice organ culture and established cell line culture models for cytotoxicity assessment of dental materials

A flow-injection analysis (FIA) for superoxide dismutase (SOD) activity was developed based on the use of tetrazolium salt, WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) and an enzyme reactor packed with Sepharose 4B on which xanthine oxidase (XO) and catalase were co-immobilized. WST-1 is highly water-soluble, and no adhesion of the reduced form to the FIA line was observed during continuous operation for 3 months. As optimized conditions, a sample (9 vol) was mixed with a reagent solution (1 vol) containing 3 mM hypoxanthine and 2 mM WST-1, and the mixed solution (20 μl) was injected into a carrier stream of 50 mM carbonate buffer (pH 10.2) at a flow rate of 0.4 ml/min. Under the conditions, the concentration of the SOD preparation giving 50% inhibition (IC50) was 2.7 μg/ml and the sampling frequency was 30 samples/h. When the SOD activity in erythrocytes of rats was determined by the present FIA method, the values were linearly related to those obtained by the conventional nitroblue tetrazolium (NBT) assay (r=0.975; n=18). The enzyme reactor was stable for at least 200 repetitive injections.

A method is described utilizing the tetrazolium salts neotetrazolium chloride (NTC), triphenyltetrazolium chloride (TTC), C,N-diphenyl-N'-4,5-dimethylthiazol-2-yltetrazolium bromide (MTT) and various substrates to elucidate damage to the mitochondrial electron transport chain of intact cells following in vitro photodynamic therapy (PDT). Using this methodology, a portion of the dark toxicity manifested by Photofrin II (PII) was found to occur prior to entry of electrons into the transport chain through Complex I, as evidenced by the fact that the inhibition of MTT reduction was reversible by the addition of malic acid to the culture media. A second site of dark toxicity was found to be Complex IV (cytochrome oxidase). After photoirradiation of the cells, Complex I was found to be affected since malic acid could no longer reverse the inhibition of MTT reduction but it could be reversed by the addition of succinic acid, whose electrons enter the transport chain at Complex II. A second and more sensitive site of photoirradiation damage was found to be Complex IV. A region near cytochrome C was also affected by photoirradiation but appreciably less so than noted for Complexes I and IV. A kinetic analysis of MTT and TTC reduction following photoirradiation indicated that MTT reduction was sustained at a normal rate for 1 h after which it slowed down and eventually plateaued. In contrast, TTC reduction was found to be inhibited almost immediately indicating Complex IV is extremely susceptible to photoirradiation damage. Compared to other assays of mitochondrial function requiring subcellular fractionation, the use of tetrazolium salts is simpler to perform and can be done using physiologically relevant conditions.

Abstract A method is described utilizing the tetrazolium salts neotetrazolium chloride (NTC), triphenyltetrazolium chloride (TTC), C,N‐diphenyl‐N'‐4,5‐dimethylthiazol‐2‐yltetrazolium bromide (MTT) and various substrates to elucidate damage to the mitochondrial electron transport chain of intact cells following in vitro photodynamic therapy (PDT). Using this methodology, a portion of the dark toxicity manifested by Photofrin II (PII) was found to occur prior to entry of electrons into the transport chain through Complex I, as evidenced by the fact that the inhibition of MTT reduction was reversible by the addition of malic acid to the culture media. A second site of dark toxicity was found to be Complex IV (cytochrome oxidase). After photoirradiation of the cells, Complex I was found to be affected since malic acid could no longer reverse the inhibition of MTT reduction but it could be reversed by the addition of succinic acid, whose electrons enter the transport chain at Complex II. A second and more sensitive site of photoirradiation damage was found to be Complex IV. A region near cytochrome C was also affected by photoirradiation but appreciably less so than noted for Complexes I and IV. A kinetic analysis of MTT and TTC reduction following photoirradiation indicated that MTT reduction was sustained at a normal rate for 1 h after which it slowed down and eventually plateaued. In contrast, TTC reduction was found to be inhibited almost immediately indicating Complex IV is extremely susceptible to photoirradiation damage. Compared to other assays of mitochondrial function requiring subcellular fractionation, the use of tetrazolium salts is simpler to perform and can be done using physiologically relevant conditions.

The reduction of four tetrazolium cations (TCs), nitroblue tetrazolium (NBT), neotetrazolium (NT), methylthiazolyldiphenyltetrazolium (MTT) and iodonitrophenyltetrazolium (INT), by viable micro-organisms, immobilized on glass cover slips, was examined by light microscopy with a view to determining a systematic basis for applying these reagents as cytochemical indicators of microbial viability and activity. The potential value of histochemical information about TC reactions for developing their microbiological applications was also assessed. INT and MTT detected viable cells more readily than NBT and NT. In order to obtain cell-localized formazan, MTT required cobalt ions in the reaction mixture and INT reactions had to be assessed soon after mounting. In general, formazan deposition could be accelerated by the addition of glucose and an intermediate electron carrier (IEC) to the reaction mixture, although inhibitory effects of IECs were also detected. Cultures in exponential phase, in stationary phase and inhibited by chloramphenicol could be differentiated with MTT but not with INT. For some organisms, notably Candida albicans, Pseudomonas aeruginosa and Enterococcus faecalis. TC reactions proved to be a relatively insensitive means of demonstrating viability. Two parameters used in selecting TCs for histochemical reactions, lipophilicity and reducibility appeared to be predictive for the relative sensitivity of these reagents as indicators of cell viability. The concepts of substantivity, a measure of non-specific interactions between reagents and staining substrates, and TC oxygen sensitivity, the effect of competition between oxygen and TCs for electrons, were found to be relevant to formazan deposition in live microbes. These findings support the use of TCs as cytochemical probes of microbial activity in defined settings and the use of histochemical knowledge to support further development of these techniques.

In an attempt to understand the significance of microbiol activity measurements in anaerobic sediments by the tetrazolium reduction assay I examined the effect of addition of tetrazolium salts on a variety of anaerobic bacteria of different metabolic types. Triphenyletrazolium chloride was found to be reduced by a variety of obligately anaerobic bacteria that lack cytochromes, and evolve hydrogen during fermentation; thus the assay not only measures respiratory electron transport as was often assumed. Hydrogen evolution is inhibited in hydrogen-producing fermentative bacteria upon addition of tetrazolium salts, thereby depriving, e.g., the surfate-reducing bacteria of an important electron donor. Moreover, a dissimilatory sulfate-reducing bacterium tested did not reduce tetrazolium salts at significant rates. It is thus suggested that the tetrazolium reduction assay applied to anaerobic ecosystems gives a measure for activity of hydrogen-evolving fermentative bacteria, rather than for overall bacterial metabolism or of anaerobic respiration.

On the use of tetrazolium salts for the measurement of microbial activity in sediments

On the use of tetrazolium salts for the measurement of microbial activity in sediments : Oren, Aharon, 1987. FEMS Microbiol. Ecol., 45(3):127–133

An effective and reliable method for the quantitative estimation of creatine kinase-MB, creatine kinase-MM variants and mitochondrial forms of creatine kinase in serum is presented. The high resolving power of isoelectric focusing allows the use of tetrazolium salts and meldola blue for the quantitative measurement without interfering non-specific reduction. The addition of thiol compounds to the agarose medium increases the sensitivity of the method, due to the inhibition of sulfhydryl group oxidation, and prevents enzyme degradation, which is a possible cause of an artificial heterogeneity. Depending upon the type of muscle and the degree of cell damage, we found 3-4 creatine kinase-MM sub-bands in sera with activities below 80 U/l. At elevated creatine kinase activities 3-11 creatine kinase-MM sub-bands were found. The appearance of creatine kinase-MB in serum indicates that damage has occurred to certain organs, especially the cardiac muscle. An organ with moderate or massive cell damage could release, in addition to the sarcoplasmatic creatine kinase variants, other forms with more alkaline isoelectric points (mitochondrial creatine kinase). The presence of such bands in serum of patients correlates with poor prognosis. Besides the separation of creatine kinase-MM sub-bands, creatine kinase-MB, creatine kinase-BB and of macroforms 1 and 2, the advantage of this method is the detection of mitochondrial creatine kinase forms, which in cellulose acetate electrophoresis migrate with creatine kinase-MM.

Tetrazolium salts have been suggested as analytical reagents for metals and non-metals. The elements TI(III), Cr(VI), Fe(III) (HASHMI et al. 1965, ALEXANDROV et al. 1976), Sn (NUTESCU & TODOR 1979), Fe(II) (HASHMI et al. 1972), Co(II) (MEHRA & LEBLANC 1979), Au(III), Ce(IV), Pt(II) (HASHMI & RASHID 1966), and Zn(II) (ALEXANDROV & KAMBUROWA 1976) have been determined in aqueous systems in the microgram range through the use of tetrazolium salts. Non-metallic species such as BrO 3and IO3(HASHMI et al. 1970) and the insecticide "Gardona" (VASSILEVA & SHISHMANOV 1977) have also been determined through their colored products which were generated with tetrazolium compounds.

We describe a procedure for assay of diaphorase activity in commercial purified preparations and in clinical chemical reagents by use of iodonitrotetrazolium chloride or other tetrazolium salts. The method is based on measurement of the formazan produced by enzymic reduction of tetrazolium salts in the presence of NADH. The assay procedure has been optimized for linear kinetics, simplicity of operation, nondetectable blank rates, and extended activity/enzyme concentration proportionality. The proposed method has several advantages over the older assay by use of dichlorophenolindophenol.

Close correlation is known to exist between the form of deposition of products of enzyme histochemical reactions involving the use of tetrazolium salts, ultrastructural changes in the mitochondria, and the functional capacity of the muscle cells of the isolated heart [i]. On this basis types of deposition of the enzyme histochemical reaction product reflecting the degree of degenerative and necrobiotic changes in a preserved organ have been defined. A diffuse-granular type of reaction product corresponds to the normal average physiological state of the cell. Intensification of the diffuse component in this type of reaction is a histochemical sign of functional (relating to energy) stress on the cell. The granular and dispersed types of diformazan precipitation are connected with profound but reversible disturbances in the energy metabolism of the mitochondria. Finally, the secondary diffuse type of deposition of enzyme histochemical reaction products corresponds to profound degenerative and autolytic changes in the parenchymatous cells of the preserved organ. These criteria were used by the writers to compare the morphological and functional state of the kidneys preserved by a nonperfusion method in various solutions.

A simple method to identify plasma amine oxidase activity band on polyacrylamide gel

The distribution of succinic semialdehyde dehydrogenase in the brain and retina of the tiger salamander ( Ambystoma tigrinum)

1. Various dehydrogenases can be demonstrated in tissue sections by the use of tetrazolium salts as hydrogen acceptors. These substances become reduced to insoluble formazans which are deposited in the section. 2. A solvent system for the quantitative elution of nitro-blue formazan is described. 3. The solvent is alkaline dimethylformamide (DMF); the alkalinity is achieved by the addition of 10% (v/v) of a buffer, pH about 12, to the DMF. 4. The piece of glass containing the section is cut out of the slide, placed into a test tube, and the DMF is added, followed by the buffer. The usual volumes are 0.9 ml DMF +0.1 ml buffer, but these can be altered as necessary. The formazan first turns green, and is then eluted into the DMF. Elution is usually complete within a few minutes, but may be assisted by placing the tubes into a water-bath at 40–50° C. 5. The colour is measured in a spectrophotometer at 715 mμ. It is stable for about 40 minutes. Serial dilutions are linear up to an optical density of at least 0.850. 6. The method records a linear production of formazan with respect to the amount of tissue (enzyme) present, and also with respect to the time of incubation. 7. With each test, it is necessary to incubate a control section in medium lacking both substrate and co-enzyme. This permits a correction to be made for the effects of any endogenous substrates, and also for the possibility that additional formazan is produced during the elution due to binding of the tetrazole to the tissue. 8. An optical density at 715 mμ of 0.100 in 1.0 ml solvent corresponds to 0.940 μg formazan in the sample. 9. When tested with nitro-blue tetrazolium, the optimal activity of both pentose shunt dehydrogenases occurred at very similar pH values to those found with neotetrazolium, although about three times the actual activity (in terms of hydrogen) was recorded with the former than with the latter tetrazole.

Succinic dehydrogenase activity was determined in fresh or cryostat sections of tissues from Allium cepa, Vicia faba, Pisum sativum and Helianthus tuberosus using different tetrazolium salts as electron accepters. In 10 μ fresh or frozen sections a reaction was obtained with TNBT, NBT, MTT, and INT but not with NT, BT or TTC. In contrast a reaction was obtained with each of the tetrazolium salts in 50–150 μ fresh or frozen sections. The observed differences in the abilities of the tetrazolium salts to demonstrate succinic dehydrogenase activity are discussed.

SUMMARY— Triphenyltetrazolium chloride, (TTC), selectively precipitates carrageenan and other sulfated hydrocolloids in the presence of neutral, carboxylic and phosphorylated polysaccharides. Agar is not precipitated. Tetrazolium blue (TB) precipitates both the sulfated and carboxylic polysaccharides but does not precipitate phosphorylated and neutral polysaccharides.Except for “weak” reactions with mucin, gum karaya, sodium alginate, and polysaccharides B‐1459 (NRRL); tetrazolium red, neotetrazolium chloride and tetrazolium violet precipitate only sulfated polysaccharides. Although in aqueous dispersion the proteins were not precipitated by any of the tetrazolium salts, when these salts are added to milk (containing no added carrageenan), copious precipitation occurred.Carrageenan was isolated from milk, milk products and other complex milieu by precipitation with 2, 3, 5, ‐triphenyl‐tetrazolium chloride. The proteins were precipitated simultaneously. The mixed carrageenan‐protein precipitate was then analyzed for carrageenan by hydrolyzing it with hydrochloric acid and assaying for sulfate by the barium chloranilate method.Mixtures of sulfated, carboxylic and neutral polysaccharides were resolved by precipitating the sulfated polysaccharide with 2, 3, Atriphenyltetrazolium chloride. After centrifugation and/or filtration to remove the precipitate, the filtrate was treated with tetrazolium blue to precipitate the carboxylic polysaccharide. The neutral polysaccharide remained in the supernatant. Solubility studies have shown that the carrageenan‐TT precipitate is highly insoluble in sodium chloride, sodium sulfate, magnesium chloride and other salt solutions.The method developed allows the use of tetrazolium salts to differentiate between and to separate the main groups (sulfated, carboxylic, and neutral) polysaccharides. After separation, the individual polysaccharides may be determined quantitatively by suitable analytical methods.