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Food freshness monitoring, which can reflect the quality of the product at the time of use, remains a great challenge for consumers and the food industry. Herein, we report the development of a cost-effective enzyme-based paper biosensor, which can monitor fish freshness and predict spoilage. The biosensor measures the release of hypoxanthine (HX), an indicator of meat and fish degradation, using the enzymatic conversion of HX by xanthine oxidase (XOD). We demonstrate that the entrapment of XOD and an organic dye, nitro blue tetrazolium chloride (NBT), within a sol-gel biohybrid enables their stabilization on paper and promotes the enzymatic reaction with further retention of the reaction products within the cellulosic network . Linearity in the micromolar concentration range with a detection limit of 3.7 μM for HX is obtained. The biosensor has high selectivity toward HX and is manufactured in few steps from inexpensive widely available materials. The applicability of the biosensor is demonstrated by following fish degradation over time and measuring HX concentrations ranging from 117 (±9) to 198 (±5) μM within 24 h of degradation, at levels that are comparable with those measured by a commercial enzymatic kit for HX detection. As compared to the commercial kit, our biosensors are more cost-effective, do not require addition of exogenous reagents and are portable, having all of the reagents needed for analysis embedded within the sensing platform. This proof-of-concept work demonstrates that the paper-based HX biosensor has potential as a robust reagentless device for real-time monitoring of food freshness and for other applications in which HX plays an important role.

This work reports on the surface-modified woven fabrics for use as UV radiation sensors. The cotton and polyamide fabrics were printed with radiochromic hydrogels using a screen-printing method. The hydrogels used as a printing paste were composed of water, poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Pluronic F-127) as a gel matrix and nitro blue tetrazolium chloride as a radiation-sensitive compound. The development of the hydrogels’ colour occurs after exposure to UV radiation and its intensity increases with increasing absorbed dose. The features of the NBT-Pluronic F-127 radiochromic hydrogels and the fabrics printed with the hydrogels were examined using UV-Vis and reflectance spectrophotometry as well as scanning electron microscopy (SEM). The effects of NBT concentration and UV radiation type (UVA, UVB, UVC) on dose responses of the hydrogels and printed fabrics were also examined. The results obtained reveal that the fabrics printed with NBT-Pluronic F-127 hydrogels can be potentially useful as UV radiation sensors.

In this study, we describe a standard whole mount in situ hybridization method which is used to determine the spatial-temporal expression pattern of genes from Globodera spp. Unlike more invasive radioactive labeling approaches, this technique is based on a safe, highly specific enzyme-linked immunoassay where a Digoxigenin (DIG)-tagged anti-sense probe hybridized to a target transcript is detected by anti-DIG antibodies conjugated with alkaline phosphatase enzyme (AP) (anti-DIG-AP). The hybrid molecules are visualized through an AP-catalyzed color reaction using as the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium chloride (NBT). This method can be applied to both free-living pre-parasitic juveniles and early endoparasitic stages of cyst nematodes.

Oxidative damage is one of the main causes of cryopreservation injury compromising the use of cryopreserved biospecimens. The aim of this study was to evaluate the use of Fourier transform infrared spectroscopy (FTIR) as a non-invasive method to assess changes in biomolecular composition and structure, associated with oxidative stress in isolated biomolecules, acellular heart valve tissues, and ovarian cortex tissues. FTIR spectra of these specimens subjected to various treatments (H2O2- and Fenton-treatment or elevated temperatures) were vector normalized and selected spectral regions were analyzed by principal component analysis (PCA). Control and damaged biomolecules can easily be separated using PCA score plots. Acellular heart valve tissues that were subjected to different levels of oxidative damage formed separate cluster in PCA score plots. In hydrated ovarian tissue, large variation of the principal components was observed. Drying the ovarian tissues samples resulted in improved cluster separation of treatment groups. However, early signs of oxidative damage under mild stress conditions could not be detected by PCA of FTIR spectra. For the ovarian tissue samples, the standardly used nitro blue tetrazolium chloride (NBT) assay was used to monitor the amount of formazan production, reflecting reactive oxygen species (ROS) production at various temperatures. At 37 °C, formazan staining rapidly increased during the first 30 min, and then slowly reached a saturation level, but also at lower temperatures (i.e. 4 °C) formazan production was observed. In summary, we conclude that ATR-FTIR combined with PCA can be used to study oxidative damage in biomolecules as well as in tissues. In tissues, however, sample heterogeneity makes it difficult to detect early signs of oxidative damage.

A liquid-liquid extraction-chromogenic system containing Fe(III), 4-(2-thiazolylazo)resorcinol (TAR), [3,3’-(3,3’-dimetoxy-4,4’-biphenylene)bis[2,5-di(4-nitrophenyl)-2H-tetrazolium] chloride (tetranitro-tetrazolium blue chloride, TNBT), water, and chloroform was studied and compared with similar systems containing such ditetrazolium salts (DTS), as neotetrazolium chloride (NTC), blue tetrazolium chloride, and nitro blue tetrazolium chloride. The results show that the complex formed in the Fe(III)–TAR–TNBT system is of different composition (1 : 3 : 2 vs. 1 : 2 : 1) and has better extraction-spectrophotometric characteristics (fraction extracted is 98.6 % and molar absorptivity, 7.8 × 104 dm3 mol–1 cm–1 at λ = 495 nm). The proposed formula of the complex is (TNBT+)[FeIII(TAR2–)2]·{(TNBT+)(HTAR–)}, where TNBT is in the monocationic form. The geometry optimization for TNBT and NTC–the DTS, which does not contain nitro- and methoxy groups, was performed by the Restricted Hartree–Fock (RHF) method with the 3-21G* basis set. The obtained results (ground-state structures and total atomic charges) for TNBT, TNBT+, TNBT2+, NTC, and NT2+ were compared and discussed.

This work presents the synthesis, characterization of copper(II) complexes (C1-C6) and the potential of these compounds to mimic the catalytic activity of the enzyme superoxide dismutase (SOD). The copper(II)complexes were obtained by reaction between the aldol condensation between substituted aromatic hydrazides and aromatic aldehydes (salicylic aldehyde and pyridoxal hydrochloride), forming two new ligands (L1 to L6), resulting in new dimeric dicopper (II) complexes (C1 and C2), new three monomeric CuII derivatives (C3, C4 and C6) and a polymeric complex (C5). The CuII complexes were fully characterized by X-ray diffraction, spectroscopic and electrochemical analysis. Subsequently, CuII derivatives were evaluated for their antioxidant activities, using the NBT (Nitro blue tetrazolium chloride) photoreduction methodology. After evaluating the antioxidant activity in vitro, it was observed that the best inhibition rates of the superoxide ion are associated to the C4 and C5 complexes. Computational analysis via molecular docking and quantum chemical calculation (Fukui map) offered a molecular level explanation on the biological activity of CuII complexes. Additionally, cytotoxicity of C1-C6 was tested in the first time in vivo in nematodes Caenorhabditis elegans, corroborating with the results identified for C4 and C5.

This work is a follow-up study for a recently-proposed 3D radiochromic gel dosimeter that contains a tetrazolium salt and a physical gel matrix made of poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Pluronic F-127). Several tetrazolium salts were examined in this work, including tetrazolium violet, blue tetrazolium chloride, nitro blue tetrazolium chloride (NBT), tetranitro blue tetrazolium chloride (tNBT) and thiazolyl blue tetrazolium bromide (TBTB). The salt-containing gel dosimeters were compared with the first Pluronic gel composition that contained 2,3,5-triphenyltetrazolium chloride (TTC) as the radiation-sensitive component (dose sensitivity of 0.0023 (Gy cm)−1). The Pluronic gels with NBT and tNBT outperformed the other gels, including the TTC-containing gel, with respect to their dose sensitivity and low dose-response. The NBT gels were found to have better stability over time than tNBT gels. Sensitization of the gels to ionizing radiation was examined by addition of tert-butyl alcohol and sodium formate. The best composition was 0.0818% NBT (1 mM), 25% Pluronic F-127 and 0.136 × 10−2% sodium formate. This gel dosimeter was insensitive to changes in dose rate for photons of different energies. The mean dose sensitivity amounted to 0.0047 ± 0.1 × 10−4 (Gy cm)−1. A diversion in the dose-response was observed for the gel irradiated with electrons. Additional characteristics of the NBT gel were a linear-dose range and a dynamic-dose range between

The antioxidant properties of oxydiacetate complexes of cobalt(II) and nickel(II) with 1,10-phenantroline and 2,2′-bipyridine have been investigated towards the superoxide radical using the nitro blue tetrazolium chloride (NBT) test and the cyclic voltammetry (CV). Moreover, the biological activity of the complexes under study has been investigated in the Human Dermal Fibroblasts adult (HDFa) cell line. In the first step, the cytotoxic and the antiproliferative activities of the complexes were examined. Subsequently, the cytoprotective properties of the complexes have been investigated in an oxidative stress conditions induced by H2O2.

The formation and liquid-liquid extraction of ion-association complexes between Co(II)-4-(2-Pyridylazo)resorcinol (PAR) anionic chelates and cations of three ditetrazolium chlorides were studied: Blue Tetrazolium chloride (BTC), Neotetrazolium chloride (NTC) and Nitro Blue Tetrazolium chloride (NBT). The optimum conditions for the formation and solvent extraction of the ion-association comlpex chelates were determined. It has been found that in the systems of Co(II)-PAR-DTS, the reactants are reacted in molar ratios 1:2:1 and the general formula of complexes was suggested. The extraction equilibria were investigated and quantitatively characterized by the equilibrium constants and the recovery factors. The analytical characteristics of the complexes were calculated.

Aim. To explore the functional activity of neutrophils in healthy individuals and patients with acute intestinal infections and the effect of selimakcid on it in vitro. Methods. 48 patients with acute intestinal infections aged 20 to 60 years were examined. The control group consisted of 10 healthy people of the same age. Functional and metabolic activity of neutrophils was studied by a nitro blue tetrazolium chloride test and by «BURSTTEST» (Neutrophil Oxidative Burst Test). Results. Examination of the functional and metabolic activity of neutrophils in patients with acute intestinal infections revealed increased results of spontaneous nitro blue tetrazolium chloride test in acute period of the disease. The same changes were discovered while studying the Serratia marcescens stimulated cell activity in patients with dysentery. However, in patients with salmonellosis neutrophils activity in induced nitro blue tetrazolium chloride test did not differ from that of healthy controls. The number of neutrophils synthesizing reactive oxidants recorded by «BURSTTEST» in patients corresponded to the indicators of the induced nitro blue tetrazolium chloride test and did not differ in patients with different nosologies. Studying the effect of selimakcid on neutrophils functional activity in healthy controls and patients with acute intestinal infections by nitro blue tetrazolium chloride test and «BURSTTEST» showed that pre-incubation of neutrophils with selimakcid intensified the response to Serratia marcescens. The ability of the drug to modulate the neutrophils activity in patients with acute intestinal infections was higher compared to healthy individuals. Conclusion. Increased functional activity of neutrophils associated with reduced reserve capacity was revealed in patients with salmonellosis and shigellosis of moderate severity. Selimakcid showed immunomodulatory effect, increasing the functional and metabolic potential of neutrophils in patients with acute intestinal infections in vitro.

The Escherichia coli LacZ gene is a widely used reporter for gene regulation studies in transgenic mice. It encodes bacterial β-galactosidase (Bact β-Gal), which causes insoluble precipitates when exposed to chromogenic homologues of galactose. We and others have recently reported that Bact β-Gal detection with Salmon-Gal (S-Gal) in combination with nitro blue tetrazolium chloride (NBT) is very sensitive and not prone to interference by acidic endogenous β-galactosidases. Unfortunately, as we show here, the method appears to be inadequate for evaluation of Bact β-Gal expression in keratinized epithelial appendages but not in other keratinized epithelia. NBT in the reaction mixture, just as other tetrazolium salts, inevitably causes unwanted staining artifacts in lingual filiform papillae, penile spines, and hair fibers by interacting with keratin sulfhydryl-rich regions. The methodological limitation can be overcome in part by pretreating the tissues before the S-Gal/NBT staining with an iodine–potassium iodide solution. Alternatively, the use of iodonitrotetrazolium chloride instead of NBT in the S-Gal reaction mixture provides enough color resolution to distinguish the specific Bact β-Gal staining in orange from the artifact staining in dark red. In summary, we provide evidence that S-Gal/NBT histochemistry has limitations, when staining keratinized epithelial appendages.

The immune-related enzymes in marine animals are very sensitive to divalent metal ions. To investigate the roles divalent metal ions play in the influence on the immunity of sea cucumber Apostichopus japonicus, one of the most important commercial species in Asian countries, the effects of eight divalent metal ions at concentrations of 2.5, 5, 10, 15, 20, 25 and 30 mmol L−1 on the activities of superoxide dismutase (SOD), phenoloxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP) and myeloperoxidase (MPO) in coelomic fluid were determined with the nitro blue tetrazolium chloride (NBT) method, dopachrome formation method, p-nitrophenyl phosphate (pNPP) method and 3,3′,5,5′-tetramethyl benzidine (TMB) method. The results indicated that Mg2+ enhanced the activities of SOD, PO, ACP and AKP significantly and showed no obvious effect on MPO activity; Zn2+ increased the activities of SOD, ACP and AKP, and showed no obvious effect on the activities of PO and MPO; Cu2+ enhanced the activities of ACP, AKP and MPO and activated SOD and PO at a certain concentration range; Ca2+ and Mn2+ inhibited the activities of ACP and AKP; Fe2+ had strong inhibitory effect on SOD activity; Pb2+ showed inhibitions on the activities of SOD, PO, ACP and AKP; and Cd2+ inhibited MPO activity greatly. The data obtained in this study collectively suggest that Mg2+, Zn2+ and Cu2+ have potential in promotion of A. japonicus immunity, while Ca2+, Fe2+, Pb2+, Cd2+ and Mn2+ might be limiting factors to the immune response of A. japonicus.

In this study, an ultrasensitive surface-enhanced Raman scattering (SERS) detection of alkaline phosphatase (ALP) has been developed, in which nile blue A (NBA) was chosen to replace nitro blue tetrazolium chloride (NBT) in a reactive system of 5-bromo-4-chloro-3-indolyl phosphate (BCIP), NBT, and ALP. In the reactive process, NBA was converted to a low SERS-active molecule, and its SERS intensity at 592 cm−1 decreased when NBA was reduced by 5-bromo-4-chloro-3-indolyl, which converted from BCIP by ALP. The SERS signal of NBA was inversely proportional to the amount of ALP in the reactive system. Based on convenient SERS materials of gold nanoshells absorbed on acupuncture needles, the detectable concentration range of ALP was 1–104 mU L−1 in a reactive system of BCIP, NBA, and ALP.

The present study is the first to describe the detection of Reactive Oxygen Species (ROS) in spearmint (Mentha spicata L.) grown under nutrient stress conditions through 3, 30-diaminobenzidine (DAB) and nitro blue tetrazolium chloride (NBT) histochemical methods. Spearmint cuttings grown under manganese and salt conditions showed toxicity and stunted growth after 10 and 20 days of the stress treatments (> 2.5 mM for the manganese and >100 mM for the saline stress). These abiotic stresses induced the production of ROS in the leaf tissue of the spearmint. Accumulation of hydrogen peroxide was characterised as brown spots by DAB polymerisation which was noticed on leaf tissues from the plants grown under 2.5 and 5 mM concentrations of manganese as well as 300 mM concentration of salt. Furthermore, accumulation of superoxide anion was also characterised as blue pigments based upon the ability of cells to reduce NBT. Level of the secondary metabolite menthol was also significantly increased in response to the ROS production. Our study confirmed the validity of use of DAB and NBT stains in elucidating ROS accumulation in cells of spearmint, the important medical plant under nutrient stress conditions.

Flat woven polyamide textiles were chosen for modification with nitro blue tetrazolium chloride (NBT) or 2,3,5-triphenyltetrazolium chloride (TTC). Such samples change colour from white to blue (NBT) or red (TTC) if exposed to ionizing radiation or UV light. When inhomogenously irradiated, a clear pattern of the absorbed dose distribution is visible to the naked eye. Performance of quantitative 2D analysis with the aid of a flat-bed document scanner was proposed. Most importantly, the application of a scanner is an easy method for the assessment of irradiated samples. Therefore, scanning parameters such as resolution, sharpness, scanning reproducibility and sample preparation were assessed in this work; and optimal parameters were chosen. The cause of uncertainty in the measurements is discussed.

Complex formation and liquid-liquid extraction were studied in systems containing indium(III), 4-(2-pyridylazo)resorcinol (PAR), phenylmethoxybis(tetrazolium) salt (MBT), water and chloroform. The following MBTs, which differ only by the number of -NO2 groups in their cationic parts, were used: 3,3′-(3,3′-dimetoxy-4,4′-biphenylene)bis(2,5-diphenyl-2H-tetrazolium chloride) (Blue Tetrazolium chloride, BT), 3,3′-(3,3′-dimetoxy-4,4′-biphenylene)bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (Nitro Blue Tetrazolium chloride, NBT) and 3,3′-(3,3′-dimetoxy-4,4′-biphenylene)bis[2,5-di(4-nitrophenyl)-2H-tetrazolium chloride] (Tetranitro Blue Tetrazolium chloride, TNBT). The composition of the formed ternary complexes was determined, In:PAR:MBT=1:2:2, and the optimum conditions for their extraction found: pH, shaking time, concentration of the reagents and the sequence of their addition. Some key constants were estimated: constants of extraction (Kex), constants of association (β) and constants of distribution (KD). BT appears to be the best MBT for extraction of the In(III)-PAR species, [In3+(OH)3(PAR)2]4−, (Log Kex=10.9, Log β=9.8, Log KD=1.12, R%=92.7%). Several additional characteristics concerning its application as extraction-spectrophotometric reagent were calculated: limit of detection (LOD = 0.12 µg cm−3), limit of quantification (LOD = 0.40 µg cm−3) and Sandell’s sensitivity (SS =1.58 ng cm−2); Beer’s law is obeyed for In(III) concentrations up to 3.2 µg mL−1 with a molar absorptivity coefficient of 7.3×104 L mol−1 cm−1 at λmax=515 nm.

The development of flat woven fabric-based ionizing radiation 2D dosimeters is reported in this work. Polyamide fabric was surface modified with radiation-sensitive 2,3,5-triphenyltetrazolium chloride (TTC) or nitro blue tetrazolium chloride (NBT). These samples responded to gamma radiation of 60 Co through a colour change: red and blue for TTC and NBT, respectively. The tinge intensity was observed to depend on the absorbed dose, which allowed for the reflectance of light ( R ) measurements and calculation of the calibration parameters: dose range, quasi-linear range, threshold dose and sensitivity. Oxygen was shown to be an important factor determining the dose response of the samples. For this reason, a range of additional modifications to the TTC- and NBT-polyamide fabric was proposed which lead to a decrease in the threshold dose and increase in the sensitivity to irradiation of the samples. For instance, a dosimeter made of polyamide fabric modified with 10 g/dm 3 TTC, 0.5 mol/dm 3 tert-butyl alcohol, 7.5% gelatine hydrogel at pH 10 (vacuum packed) showed the lowest dose threshold (50 Gy), dose range up to 2.8 kGy and the highest sensitivity to irradiation (−0.0396%/Gy) among the compositions studied. In consequence, this dosimeter was examined in terms of response to inhomogeneous irradiation from a 192 Ir brachytherapy radiation source. The relative dose distribution profiles across the source's longitudinal axis were calculated. This showed potential of the textile dosimeters for 2D dose distribution measurements; however, further modifications towards improvement of the dosimeter's low dose response can be considered.

This paper reports on the preparation and features of a UV light dosimeter composed of nitro blue tetrazolium chloride (NBT) and polyamide woven fabric. This textile dosimeter is based on the conversion reaction of NBT into formazan, which was initially examined in aerated aqueous solutions through steady state UV irradiation. Irradiated solutions change their colour as a consequence of the formation of polydisperse NBT formazan particles. This was analysed in relation to the absorbed dose of UV light through UV–VIS spectrophotometry and dynamic laser light scattering measurements. When NBT substrate molecules are embedded in polyamide textile, UV irradiation leads to similar effects as in solution. However, the tinge intensity changes at much lower absorbed doses. The dependence of the tinge intensity on the absorbed dose was followed by measurements of the remission of light from the NBT-polyamide samples. Consequently, the calibration parameters were calculated such as the dose sensitivity, dose range, and quasi-linear dose range. An improvement of the NBT-polyamide samples by application of a colour levelling agent and improvement of their resistance to humidity is presented. Finally, the samples were used for estimation of absorbed UV energy distribution showing their capability as new dosimeters for in-plane high resolution radiation dose measurements.

Two new copper(II) complexes, [Cu(BH)(bipy)](ClO4)2(H2O)2 (1) and [Cu(BH)2(NO3)2] (2) (BH = benzoylhydrazine, bipy = 2,2′-bipyridine), have been synthesized and characterized. X-ray crystallographic analysis shows the copper to be four- and six-coordinate in the complexes. The magnetic and spectroscopic data indicate a distorted square-planar geometry for 1, while 2 is assigned a distorted octahedral geometry. The electron paramagnetic resonance spectra indicate a ground state (g ‖ > g ⊥ > 2.0023). Both complexes display quasireversible redox waves due to Cu(II)/Cu(I) reduction. The superoxide dismutase-mimetic activity of the complexes was investigated by nitro blue tetrazolium chloride DMSO assay.

Complex formation and liquid-liquid extraction have been studied for ternary complexes of vanadium(IV) with 4-(2-pyridylazo)-resorcinol (PAR) and ditetrazolium chlorides (DTC) in a water-chloroform medium. The specific ditetrazolium compounds investigated were i) 3,3′-(4,4′-biphenylene)-bis(2,5-diphenyl-2H-tetrazolium) chloride (Neotetrazolium chloride, NTC); ii) 3,3′-(3,3′-dimetoxy-4,4′-biphenylene)-bis(2,5-diphenyl-2H-tetrazolium) chloride (Blue Tetrazolium chloride, BTC); and iii) 3,3′-(3,3′-dimetoxy-4,4′-biphenylene)-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium] chloride (Nitro Blue Tetrazolium chloride, NBT). Molar absorptivity coefficients and the composition of the complexes have been calculated. Association constants (β) have also been obtained for the interactions between the vanadium(IV) — PAR anionic chelates [VO(PAR)2]2− (I) and [VO(OH)2(PAR)2]4− (II), and ditetrazolium cations (DT2+). Some special features of NBT as an extraction-spectrophotometric reagent for vanadium(IV) have been discussed. Unlike NTC and BTC which form complexes with both I and II, NBT associates only with II. The pH interval for complete extraction of (NBT2+)2[VO(OH)2(PAR)2] is broader and allows work at lower pH values the other ion-associates of V(IV,V)-PAR that were studied. NBT is -therefore the appropriate reagent both for direct V(IV) determination and for V(IV)/V(V) separation. Some additional characteristics for the V(IV)-PAR-NBT-water-chloroform system have been determined: extraction constant, distribution constant, recovery factor, limit of detection and limit of quantification. Beer’s law is valid up to 1.4 μg mL−1 vanadium(IV) with molar absorptivity coefficient of 3.55×104 L mol−1 cm−1 at λmax=559 nm.

The formation and extraction of ion-associate complexes between the vanadium(V)-4-(2-thiazolylazo)resorcinol (TAR) anionic chelate and the cations of some mono-and ditetrazolium salts {3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (Thiazolyl blue, MTT), 3-(2-naphtyl)-2,5-diphenyl-2H-tetrazolium chloride (Tetrazolium violet), 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (Iodonitrotetrazolium chloride), 3,3′-[3,3′-dimetoxy(1,1′-biphenyl)-4,4′-diyl]-bis[2,5-diphenyl-2H-tetrazolium] chloride (Tetrazolium blue chloride) and 3,3′-(3,3′-dimetoxy-4,4′-biphenylene)bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (Nitro blue tetrazolium chloride)} have been studied. The optimum extraction conditions have been found. The composition of the V-TAR-monotetrazolium and V-TAR-ditetrazolium complexes extracted into chloroform has been determined to be 1:2:3 and 2:4:3 respectively. The extraction, distribution and association constants, and the recovery factors have been calculated. The relationship between the molecular weight of tetrazolium cations, and the association constants of their complexes has been discussed. The special behavior of the tetrazolium cations, containing-NO2 groups has been noticed. The effects of foreign ions and reagents on the extraction of vanadium with TAR and the best tetrazolium salt-MTT have been studied. A sensitive, selective, simple and fast method for the determination of vanadium has been developed.

3Alpha-hydroxysteroid dehydrogenase (3-HSD) from Pseudomonas testosteroni and diaphorase (lipoyl dehydrogenase) from Clostridium spp. have been immobilized individually onto arylamine glass beads through diazotization. A cost-effective enzymic colorimetric method for determination of bile acid in serum and bile employing a mixture of these immobilized enzymes was developed. The method is based on measurement of reduced nicotinamide adenine dinucleotide generated from bile acid in serum/bile by immobilized 3alpha-HSD with a color reagent consisting of nitro blue tetrazolium chloride salt, oxidized nicotinamide adenine dinucleotide, and immobilized lipoyl dehydrogenase in 0.065 M sodium phosphate buffer, pH 7.0. Analytical recovery of added bile acid (50 and 200 micromol/L) was 95.57 and 85.46% in serum and 97.6 and 91.6% in bile, respectively. Within- and between-batch coefficients of variation (CV) for bile acid determination were1.2 and0.2% in serum and0.1 and0.1% in bile, respectively. Good correlations for bile acid in serum (r1=0.92) and in bile (r2=0.97) were obtained by use of a standard chemical method and the present method. The mixture of immobilized 3alpha-HSD dehydrogenase and lipoyl dehydrogenase lost 50% of its initial activity after 6 months of regular use. The cost of bile acid determination in 100 serum and bile samples by the present method has been compared with that of the Sigma kit method.

The dosimetry characteristics of a polyvinylalcohol based radiochromic dye film containing the ditetrazolium salt nitro blue tetrazolium chloride were studied with respect to the potential use of the films for routine dosimetry in radiation processing. The useful dose range for the dosimeter film for gamma and electron irradiation is 5–50 kGy depending on the concentration of the dye. The effects of irradiation temperature and humidity, as well as the stability of the response of the film before and after irradiation, were investigated and determined. Formulations for preparation of the films with different concentrations of the dye and with different pH were tested. The films were also tested in industrial gamma irradiation facilities for process control purposes by comparing their performance with transfer standard dosimeters.

The radiolytic reactions of the ditetrazolium salt nitro blue tetrazolium chloride (NBTCl2) were studied by pulse radiolysis technique in aqueous solution under reducing and oxidising conditions with the aim of potential dosimetry application. Under reducing conditions the fast formation of the tetrazolinyl radical is observed that is followed by the appearance of monoformazan (MF+), i.e. one of the tetrazolium rings is reduced to formazan. The formation of the water-insoluble diformazan, i.e. the result of the second reduction step was not observed in pulse radiolysis. Formazan formation was not found under oxidative conditions.

Background Fractional laser resurfacing treatment has been extensively investigated and is widely used. However, the mechanism underlying its effects is poorly understood because of the ethical and cosmetic problems of obtaining skin biopsies required to study the changes after laser treatment. Objective To evaluate the usefulness of human skin explants for the investigation of fractional photothermolysis. Methods Full-thickness discarded skin was treated in 4 ways: no treatment (control), fractional carbon dioxide laser, fractional Er:YAG laser, and fractional 1,550-nm erbium-doped fiber laser. Both treated and non-treated skin samples were cultured ex vivo at the air-medium interface for 7 days. Frozen tissue was sectioned and stained with hematoxylin & eosin for histologic examination and nitro blue tetrazolium chloride for viability testing. Results Skin explants cultured for up to 3 days exhibited histologic changes similar to those observed in in vivo studies, including microscopic treatment zones surrounded by a thermal coagulation zone, re-epithelialization, and formation of microscopic epidermal necrotic debris. However, the explant structure lost its original form within 7 days of culture. The viability of skin explants was maintained for 3 days of culture but was also lost within 7 days. Conclusion The skin explant model may be a useful tool for investigating the immediate or early changes following fractional photothermolysis, but further improvements are required to evaluate the long-term and dermal changes.

This study evaluated the superoxide dismutase (SOD) activity, copper and zinc levels in saliva of caries-active and caries-free individuals. Eighty healthy adults were divided according to WHO criteria: caries-free (DMFT = 0) and caries-active (DMFT >10). Saliva collected was estimated for SOD activity as well as copper and zinc levels using the atomic nitro blue tetrazolium chloride reduction method and absorption spectrophotometry, respectively. The Student t test was applied. In conclusion, SOD activity as well as copper and zinc levels increased in the caries-active group and showed statistically significant results.

The main aim of this study was to compare selected nonspecific immunity parameters in 14 allergic and 12 healthy horses. Each animal was assessed according to the following parameters: in vitro functional capacity of phagocytic cells using the nitro blue tetrazolium chloride reduction test, both spontaneous (NBT) and zymozan stimulated (NBTs), and ingestion capacity of phagocytic cells using a phagocytic index test (IF) and percentage of phagocytosing neutrophils activity (%KF). Differences were demonstrated between the group of allergic horses, especially with severe allergy symptoms, and healthy horses in NBTs values, with higher values in healthy horses. The values of the phagocytic index were significantly higher in horses with allergy.

Leek, maize, and pigmented soybean roots colonized by vesicular-arbuscular mycorrhizal (VAM) fungi were assessed for succinate dehydrogenase (SDH) activity using the nitro blue tetrazolium chloride (NBT)-succinate method. NBT-succinate-reacted roots, cleared in a 55° C drying oven in 5% (w/v) KOH for 24 h or longer and observed as whole mounts, revealed signs of intraradical VAM fungus colonization more clearly than roots cleared by the standard 20% (w/v) boiling chloral hydrate method. Combined clearing of NBT-succinate-reacted roots using boiling chloral hydrate followed by clearing in 5% KOH at 55° C for prolonged periods also improved the visualization of intraradical fungal structures. Bleaching of NBT-succinate-reacted roots using the standard NH3-H2O2 method removed pigmentation from roots and did not alter the viability indicator, formazan. Pigmented, field-collected soybean roots were successfully cleared and bleached to reveal signs of viable and nonviable intraradical fungal structures. Counterstaining of NBT-succinate-reacted roots with acid fuchsin clearly revealed both viable and nonviable intraradical fungal structures. The NBT-succinate solution infiltrated all intraradical fungal structures after 24 h; formazan products were observed at similar concentrations in viable structures after 24, 36, and 48 h.

Diagnosis of CGD is made by demonstrating absent or markedly reduced oxidase activity in stimulated neutrophils. The screening test proposed is based upon the naked eye evaluation of the reduction of NBT on a solid surface. It seems to be a useful tool for rapid and inexpensive detection of CGD patients, especially for large-scale screening purposes. The test was carried out on forty-five subjects: two males affected by CGD, three female carriers and forty healthy donors. The test confirmed the results obtained with flow cytometric and NBT assays.

Recently, an automated mRNA in situ hybridization application was introduced for the Ventana Discovery instrument. The application was designed so that all necessary steps from baking through signal detection were completed within 1 day on the instrument. We applied this technology for visualizing the expression site of versican in formalin-fixed mouse skin paraffin tissue sections. Our focus of this study was to demonstrate the effects of protease digestion or heating pretreatment, termed cell conditioning, on the hybridization signal using a well characterized versican antisense riboprobe. Paraffin sections were automatically deparaffinized, fixed, and acid-treated. Then, the tissue sections were subjected to protease digestion alone (3 strengths), cell conditioning alone, or the combination of cell conditioning and protease digestion. Hybridization was performed with digoxigenin-labeled versican antisense probe (20 ng/slide) for 6 hours, and the signal was detected using a Nitro blue Tetrazolium chloride 5-Bromo-4-cloro-3-indolyl phosphate toluidine salt (NBT/BCLIP) substrate solution for 3 hours on the instrument. Cell conditioning alone did not produce any signal, whereas the highest strength of protease digestion produced noticeable background staining. However, when cell conditioning and mild protease digestion were combined, the signal for versican mRNA was clearly demonstrated in the hair papilla region. Thus, we demonstrated the effects of the cell conditioning step followed by mild protease digestion for enhancing the mRNA target staining compared with protease digestion or the cell conditioning step alone. We verified that the automated in situ hybridization process was applicable for formalin-fixed mouse skin paraffin sections and that the automated 1-day protocol is simple and reproducible. The precise control of automation allows fine tuning of temperature and enzyme dose to find the optimized assay condition for the signal to noise ratio and morphology.

2-Deoxy-D-glucose-6-phosphate ketol-isomerase (EC 2.6.1.16) forms glucosamine-6-phosphate and glutamate from fructose-6-phosphate and glutamine and plays an important role in chitin synthesis in fungi. We have established a new assay for fungal ketol-isomerase activity that is amenable to high throughput screening to identify enzyme inhibitors. Aspergillus fumigatus crude lysate was incubated with substrates and after incubation, reactions were terminated. Glutamate dehydrogenase, nitro blue tetrazolium chloride, phenazine methosulfate and beta-NAD were added and the amount of glutamate formed by ketol-isomerase activity was determined by measuring OD585nm. A feedback inhibitor, UDP-N-acetylglucosamine, of fungal ketol-isomerase was successfully detected by this assay (IC50=0.48 mM). In a pilot scale screening, an active extract from an extremophilic bacterium was found, and the extract showed antifungal activity against A. fumigatus, Candida albicans and C. glabrata.

We present a histochemical method using the redox dye nitro blue tetrazolium chloride (NBTC) for the evaluation of argon-laser-induced tissue damage. By this method viable cells are stained blue, devitalized cells not. Our findings suggest that the NBTC staining method is more appropriate for the accurate definition of the necrosis and cell viability after laser treatment than routine histology.

Techniques for the localization of mRNAs in situ were first developed in the late 1970s and are now widely used in many areas of biological research. They provide an extremely powerful tool for analysing patterns of gene expression, both temporally and spatially. Although other very sensitive methods are available for assaying transcript levels in different tissues and at different developmental or physiological stages (Chapters 19, 20 and 22), in situ hybridization is unique in its ability to reveal patterns of gene expression with cellular resolution.

Amalakayas Rasayana (AR) is a polyherbal formulation mentioned in Ayurveda to treat aging and age-associated diseases. Being an antiaging drug, AR may have antioxidants and free radical scavenging activity to minimize free radical-induced damage which is a key cause of aging. The methanolic extract of AR was evaluated in vitro for total phenolic and tannin content, free radical scavenging activity, superoxide radical scavenging activity, and reducing power. The total phenolic content was measured using Folin-ciocalteu reagent against gallic acid [relative standard deviation (R(2)) = 0.998]. Total tannin was estimated using the Stephen method and was found to be 2.82% w/w. Free radical scavenging activity was measured by 2,2-diphenyl-1-picryl hydrazyl assay and R(2) was 1. Superoxide radical scavenging activity was done by ethylene diamine tetra acetate and Nitro Blue Tetrazolium Chloride assays against ascorbic acid and R(2) was 0.976 (EC(50)= 77.5 μg/ml). Ferrous reducing power was evaluated by Oyaizu method where R(2) was 0.986. All studies showed that AR possesses antioxidant activity. The results of this study suggest that the antioxidant and free radical scavenging activity of AR may explain its rasayana effect and justify its use as a medicine for age associated diseases.

A two-step excisional treatment of a port-wine stain (PWS) on the back of a 43-yr-old female patient was performed. Immediately before the first surgical treatment, two corresponding series of argon laser impacts were performed, each on one PWS half. Different laser parameters with irradiances ranging from 95 to 382 W/cm2 and energy fluences ranging from 19 to 114,6 J/cm2 were used. Laser spots on the first part ot be excised were biopsied 10 min after laser treatment and prepared for histochemical analysis by staining with nitro blue tetrazolium chloride (NBTC). Reduction of this redox dye by nicotinamide adenine dinucleotide diaphorase (NADH-diaphorase) leads on frozen tissue sections to an intense blue precipitate. The activity of NADH-diaphorase subsides immediately upon cell damage. All vital epidermal and dermal cells presented a dense blue granular pigment in their cytoplasm, sparing the nuclei. Laser induced arc-shaped epidermal and dermal necrosis did not stain, showing a clear demarcation from surrounding vital tissue. The depth of the thermal injury ranged from 0.28 to 0.45 mm; it did not correlate with the chosen fluences. With these penetration depths, the vast majority of PWS vessels was affected. Assessment of the remaining part of the PWS 8 months later yielded blanching of all laser-treated areas. With the NBTC method, an accurate definition of laser-induced tissue damage is feasible. It could be shown that the exposure time is the most relevant parameter influencing the penetration depth.

Light-dependent reduction of p -nitro blue tetrazolium chloride (NBT) and 2,6-dichlorophenol-indophenol (DCPIP) was stimulated by nitrofen (2,4-dichlorophenyl p -nitrophenyl ether) in the presence or absence of the photosensitizer riboflavin. Enhancement of NBT reduction occurred at a concentration as low as 17 μ M nitrofen. Nitrofen had no effect on dark reduction of NBT by dithionite, ascorbate, or reduced phenazine methosulfate. NBT reduction proceeded in solution saturated with either air or nitrogen. Stimulation by nitrofen in the presence or absence of riboflavin also occurred independently of oxygen. Superoxide dismutase (superoxide:superoxide oxidoreductase; EC 1.15.1.1) inhibited light-dependent, aerobic reduction of NBT and DCPIP with and without riboflavin. The nitrogen-stimulated component was also eliminated. Thus, it appears nitrofen can effect reduction of NBT (and DCPIP) anaerobically by transferring electrons directly to NBT (and DCPIP) through an oxygen-independent mechanism or aerobically via superoxide radicals.