INT Reduction Research Articles

Polycrystalline inclusion-free growth of thick, lattice-matched SiGeC with high substitutional Carbon concentrations up to 2%

Lattice-matched SiGeC with ~20% of Ge was grown at 550°C, 10 Torr on a 300 mm Si substrate in an industrial standard Reduced Pressure Chemical Vapor Deposition reactor using commercially available Si2H6/GeH4/SiH3CH3 precursors. Thicknesses exceeding 250 nm resulted in cluster formation in SiGeC with [C]Sub~2.0% and [C]Int~0.4%, with a surface density up to 6.13x106 µm-2. Room temperature photoluminescence measurements showed that the clusters were non-radiative defects and Transmission Electron Microscopy, using the ASTAR system, proved they were polycrystalline with random orientation. Cluster-free growth was achieved for 1 µm thick SiGeC with [C]Sub~1.0% and no interstitial carbon atoms, however, without lattice-matched growth on a Si substrate. Injecting HCl during a co-flow process resulted in a smooth surface with a RMS roughness of 0.23 nm and a reduction of [C]Intfrom ~0.7% down to ~0.1%, close to the detection limit of X-ray Photoelectron Spectroscopy at 0.1%. This led to a significant cluster surface density reduction down to 1.15x104 µm-2. The injection of HCl caused an increase in the Ge concentration from 19.7% up to 28.9% and a non-lattice-matched growth. A Cyclic Deposition Etch (CDE) process resulted in no [C]Int reduction, but significantly reduced the cluster surface density down to 4.60x104 µm-2. CDE-grown SiGeC was lattice-matched to the Si substrate and yielded a smooth surface with a RMS roughness of 0.22 nm. This enabled the growth of high crystalline quality, thick, lattice-matched layers on a 300 mm Si substrate with material properties beyond pure Si.

Potential oxygen consumption and community composition of sediment bacteria in a seasonally hypoxic enclosed bay.

The dynamics of potential oxygen consumption at the sediment surface in a seasonally hypoxic bay were monitored monthly by applying a tetrazolium dye (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride [INT]) reduction assay to intact sediment core samples for two consecutive years (2012–2013). Based on the empirically determined correlation between INT reduction (INT-formazan formation) and actual oxygen consumption of sediment samples, we inferred the relative contribution of biological and non-biological (chemical) processes to the potential whole oxygen consumption in the collected sediment samples. It was demonstrated that both potentials consistently increased and reached a maximum during summer hypoxia in each year. For samples collected in 2012, amplicon sequence variants (ASVs) of the bacterial 16S rRNA genes derived from the sediment surface revealed a sharp increase in the relative abundance of sulfate reducing bacteria toward hypoxia. In addition, a notable shift in other bacterial compositions was observed before and after the INT assay incubation. It was Arcobacter (Arcobacteraceae, Campylobacteria), a putative sulfur-oxidizing bacterial genus, that increased markedly during the assay period in the summer samples. These findings have implications not only for members of Delta- and Gammaproteobacteria that are consistently responsible for the consumption of dissolved oxygen (DO) year-round in the sediment, but also for those that might grow rapidly in response to episodic DO supply on the sediment surface during midst of seasonal hypoxia.

INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structure.

The reduction of 2-para (iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) is increasingly being used as an indirect method to measure plankton respiration. Its greater sensitivity and shorter incubation time compared to the standard method of measuring the decrease in dissolved oxygen concentration, allows the determination of total and size-fractionated plankton respiration with higher precision and temporal resolution. However, there are still concerns as to the method's applicability due to the toxicity of INT and the potential differential effect of plankton cell wall composition on the diffusion of INT into the cell, and therefore on the rate of INT reduction. Working with cultures of 5 marine plankton (Thalassiosira pseudonana CCMP1080/5, Emiliania huxleyi RCC1217, Pleurochrysis carterae PLY-406, Scrippsiella sp. RCC1720 and Oxyrrhis marina CCMP1133/5) which have different cell wall compositions (silica frustule, presence/absence of calcite and cellulose plates), we demonstrate that INT does not have a toxic effect on oxygen consumption at short incubation times. There was no difference in the oxygen consumption of a culture to which INT had been added and that of a replicate culture without INT, for periods of time ranging from 1 to 7 hours. For four of the cultures (T. pseudonana CCMP1080/5, P. carterae PLY-406, E. huxleyi RCC1217, and O. marina CCMP1133/5) the log of the rates of dissolved oxygen consumption were linearly related to the log of the rates of INT reduction, and there was no significant difference between the regression lines for each culture (ANCOVA test, F = 1.696, df = 3, p = 0.18). Thus, INT reduction is not affected by the structure of the plankton cell wall and a single INT reduction to oxygen consumption conversion equation is appropriate for this range of eukaryotic plankton. These results further support the use of the INT technique as a valid proxy for marine plankton respiration.

The Chemical Transformation of the Cellular Toxin INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(Phenyl) Tetrazolium Chloride) as an Indicator of Prior Respiratory Activity in Aquatic Bacteria

In the ocean, the prokaryote respiration rates dominate the oxidation of organics, but the measurements may be biased due to pre-incubation size filtration and long incubation times. To overcome these difficulties, proxies for microbial respiration rates have been proposed, such as the in vitro and in vivo estimation of electron transport system rates (ETS) based on the reduction of tetrazolium salts. INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(Phenyl) Tetrazolium Chloride) is the most commonly applied tetrazolium salt, although it is toxic on time scales of less than 1 h for prokaryotes. This toxicity invalidates the interpretation of the rate of in vivo INT reduction to formazan as a proxy for oxygen consumption rates. We found that with aquatic bacteria, the amount of reduced INT (F; µmol/L formazan) showed excellent relation with the respiration rates prior to INT addition (R; O2 µmol/L/hr), using samples of natural marine microbial communities and cultures of bacteria (V. harveyi) in batch and continuous cultures. We are here relating a physiological rate with the reductive potential of the poisoned cell with units of concentration. The respiration rate in cultures is well related to the cellular potential of microbial cells to reduce INT, despite the state of intoxication.

ВЫЯВЛЕНИЕ ЛИМИТИРУЮЩЕГО ФАКТОРА В ВОССТАНОВЛЕНИИ ЙОДНИТРОТЕТРАЗОЛИЯ ХЛОРИДА СУСПЕНЗИЕЙ КЛЕТОК БАКТЕРИЙ В ФИЗИОЛОГИЧЕСКОМ РАСТВОРЕ

In this work, we set out to investigate the role of diffusion in the microbial reduction of iodonitrotetraolium chloride (INT) using the methods of chemical kinetics. In environmental studies, re-searchers use the intensity of tetrazolium salt reduction to evaluate an overall viability of microorganisms and their communities. In particular, tetrazolium salts could be used as an indicator of the corrosive activity of bacteria. However, it is known that the reduction of tetrazolium salts can only register the response of those microbial community parts that are capable of actively reducing these substances. Therefore, research into the causes of a wide-range reduction ability of microorganisms towards tetrazolium salts can improve the objectivity of environmental and other studies, including the study of the corrosion activity of organotrophic bacteria. The kinetic parameters of INT reduction by Clostridium spp., Proteus vulgaris and Escherichia coli suspensions have been determined. On the basis of the values of the pre-exponential factor in the Arrhenius equation, an assumption is made that the reaction is characterized by a diffusion-controlled character.

C-source metabolic profilings of foodborne Shiga-toxin producing E. coli match serogroup differentiations and highlight functional adaptations

The tropism of pathogenic STEC for foodstuffs and cattle reservoir is related to functional specializations. An investigation of C-source utilization patterns among and between STEC serogroups was performed using omnilog phenotypic microarrays (OM). OM functional groupings were compared with STEC phylogroups, seropathotypes, EFSA's molecular risk assessment groups and serogroups. OM INT reduction activities of 37 STEC strains growing on 190 C-substrates were compared. Each strain had its own specific C-utilization profile but 23% of the substrates was used by all strains, 47% by none, and 30% was variably metabolized. Galactose, mannose, N-acetyl-glucosamine (GlcNAc), and N-acetyl neuraminic acid (Neu5Ac) found in the mucus layer of the bovine small intestine were metabolized by all strains. The 56 most informative substrates divided the C-utilization patterns (CP) into three clusters with: (A) harboring all O157 and O145 strains; (B) all O26 strains, and (C) strains of the other serogroups. Significant correlations between INT reduction values of pair of strains per CP group supported these differentiations. CP of group A and B strains were respectively defective in the use of galactonic acid-γ-lactone and rhamnose. Most CP group C strains grew with l-lyxose. Adjusted Wallace coefficients analyses of the datasets indicated high probabilities for the prediction of the use of glycolic acid, β-hydroxybutyric acid, l-lyxose and d-galactonic acid-γ-lactone and 5-keto-d-gluconic acid by a serogroup. The use of a C-substrate could be predicted from the classification of a strain into a phylogroup or seropathotype. Significantly lower numbers of C-substrates were used by seropathotype A strains like O157 ones. Improvements of STEC identification keys were proposed using the most discriminant C-substrates found in this study.

Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on microbial activity and polycyclic aromatic hydrocarbons (PAH) degradation in contaminated river sediments

Polycyclic aromatic hydrocarbons (PAHs) are of continued interest because of their carcinogenic nature and persistence in the environment. Among volatilization, sorption, and chemical oxidation, microbial degradation is the main path of PAHs disappearance. The majority of degradation studies have used pure cultures, were spiked with added PAHs, or have been performed using single pollutants. Because contaminated aquatic ecosystems retain mixtures of xenobiotics, experiments using environmental samples that incorporate the role of microbes provide more realistic conditions to study degradation. We tested the inhibitory effects of [carbonyl cyanide m-chlorophenylhydrazone (CCCP)] on microbial respiration, which in turn altered PAH degradation. In this study, 3.5 mM of CCCP (two orders of magnitude higher to what was needed to inhibit growth of E. coli in pure culture) was shown to inhibit respiration of indigenous microbes in complex sediment samples by 79 % at 25 °C and 64 % at 37 °C. After 15 days of incubation sediment slurries without CCCP showed higher PAH degradation rates (between 60 and 90 %) compared to sediment slurries with CCCP, which showed much lower degradation rates (<40 %). This study also reported the highest recorded rates of INT reduction (29 nkat/g) due to both short incubation times and the potential to stimulate high microbial activity in the sediments. These data indicated the effectiveness of CCCP as a microbial respiratory inhibitor in sediments, and that indigenous microbes in long-term PAH contaminated anaerobic sediments can be stimulated to degrade PAHs present in their environment.

Differences in tolerance to anthropogenic stress between invasive and native bivalves

Tolerance towards environmental stress has been frequently considered as one of the key determinants of invasion success. However, empirical evidence supporting the assumption that invasive species can better endure unfavorable conditions compared with native species is limited and has yielded opposing results. In this study, we examined the tolerance to different stress conditions (thermal stress and trace metal zinc pollution stress) in two phylogenetically related and functionally similar freshwater bivalve species, the native Anodonta anatina and the invasive Sinanodonta woodiana. We assessed potential differences in response to stress conditions using several cellular response assays: efficiency of the multixenobiotic resistance mechanism, respiration estimate (INT reduction capacity), and enzymatic biomarkers. Our results demonstrated that the invasive species overall coped much better with unfavorable conditions. The higher tolerance of S. woodiana was evident from (i) significantly decreased Rhodamine B accumulation indicating more efficient multixenobiotic resistance mechanism; (ii) significantly higher INT reduction capacity and (iii) less pronounced alterations in the activity of stress-related enzymes (glutathione-S-transferase, catalase) and of a neurotoxicity biomarker (cholinesterase) in the majority of treatment conditions in both stress trials. Higher tolerance to thermal extremes may provide physiological benefit for further invasion success of S. woodiana in European freshwaters, especially in the context of climate change.

INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(Phenyl) Tetrazolium Chloride) Is Toxic to Prokaryote Cells Precluding Its Use with Whole Cells as a Proxy for In Vivo Respiration.

Prokaryote respiration is expected to be responsible for more than half of the community respiration in the ocean, but the lack of a practical method to measure the rate of prokaryote respiration in the open ocean resulted in very few published data leaving the role of organotrophic prokaryotes open to debate. Oxygen consumption rates of oceanic prokaryotes measured with current methods may be biased due to pre-incubation size filtration and long incubation times both of which can change the physiological and taxonomic profile of the sample during the incubation period. In vivo INT reduction has been used in terrestrial samples to estimate respiration rates, and recently, the method was introduced and applied in aquatic ecology. We measured oxygen consumption rates and in vivo INT reduction to formazan in cultures of marine bacterioplankton communities, Vibrio harveyi and the eukaryote Isochrysis galbana. For prokaryotes, we observed a decrease in oxygen consumption rates with increasing INT concentrations between 0.05 and 1 mM. Time series after 0.5 mM INT addition to prokaryote samples showed a burst of in vivo INT reduction to formazan and a rapid decline of oxygen consumption rates to zero within less than an hour. Our data for non-axenic eukaryote cultures suggest poisoning of the eukaryote. Prokaryotes are clearly poisoned by INT on time scales of less than 1 h, invalidating the interpretation of in vivo INT reduction to formazan as a proxy for oxygen consumption rates.

Plankton community and bacterial metabolism in Arctic sea ice leads during summer 2010

Microbial plankton metabolism was examined during summer 2010 in sea ice-influenced waters of the Fram Strait, eastern Arctic Ocean. Rates of gross primary production and community respiration were tightly coupled over a wide range of values (33±3–143±6 and 20±3–126±6mmol O2m−2−1, respectively) leading to a prevalence of positive net community production. The high variability in community respiration, similar to that of gross primary production, suggests that heterotrophic metabolism may exhibit a significant response to environmental change. Bacterial respiration was assessed at similar time scales to bacterial production measurements, by determining the in vivo INT reduction capacity without pre-filtering the community. Bacteria seem to play a major role in total community respiration, contributing between 5% and 61% of total community respiration, indicating that a high fraction of the organic carbon in Arctic planktonic food webs could flow through these microbes.

Plankton metabolism and bacterial growth efficiency in offshore waters along a latitudinal transect between the UK and Svalbard

Euphotic zone gross primary production, community respiration and net community production were determined from in vitro changes of dissolved oxygen, and from in vivo INT reduction capacity fractionated into two size classes, in offshore waters along a latitudinal transect crossing the North, Norwegian and Greenland Seas between the UK and Svalbard. Rates of gross primary production were higher and more variable than community respiration, so net autotrophy prevailed in the euphotic zone with an average net community production of 164±64mmol O2m−2d−1. Respiration seemed to be mainly attributed to large eukaryotic cells (>0.8µm) with smaller cells, mainly bacteria, accounting for a mean of 25% (range 5–48%) of community respiration. Estimates of bacterial growth efficiency were very variable (range 7–69%) due to uncoupling between bacterial respiration and production. Larger cells tended to contribute more towards total respiration in communities with high gross primary production and low community respiration, while bacteria contributed more towards total respiration in communities with lower gross primary production, typical of microbial-dominated systems. This suggests that community respiration is related to the size structure of the plankton community.

In vivo electron transport system activity: a method to estimate respiration in natural marine microbial planktonic communities

Oxygen consumption during in vitro dark bottle incubations is the most common method to estimate planktonic respiration. This method is time consuming and labor instensive and, consequently, the database of planktonic respiration rates is scarce. Electron Transport System (ETS) activity measurement has gained acceptance as a routine technique to estimate respiration due to its high sensitivity. However, the in vitro ETS assay commonly used yields potential rates. Hence, the empirically determined ratio between in situ respiration and potential ETS activity is not constant, varying over two orders of magnitude, highly limiting the routine application of this technique to estimate microbial respiration. We hypothesized that in vivo ETS activity should be a reliable estimator of in situ respiration in marine plankton communities. The aim of this study was to develop a methodology to estimate in vivo ETS activity rates by using tetrazolium salt 2‐para (iodo‐phenyl)‐3(nitrophenyl)‐5(phenyl)tetrazolium chloride (INT) as electron acceptor. We established a procedure to apply the in vivo INT reduction method to natural marine microplankton samples. Optimum incubation times were found to be lower than 2–6 h, even for oligotrophic waters. The method was tested in natural waters, covering a very wide range of respiration rates and trophic conditions. A significant linear relationship was found between in situ respiration and in vivo INT reduction. Moreover, the ratio between oxygen consumption and INT reduction was constant across contrasting environments, which reveals the INT reduction method as a simple, quick, sensitive, and robust technique suitable to substantially improve the microbial plankton respiration database.

INT reduction as a basis for determining bakers' yeast viability

Summary Two rapid and simple methods for determining the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) to INT-formazan by bakers' yeast were compared. The first method involves counting with a light microscope the number of yeast cells containing INT-formazan crystals. In the second method, INT-formazan is extracted from cells retained on membrane filters, using dimethyl sulphoxide. The INT-formazan is then quantitated by absorption spectrophotometry. Several brands of bakers' yeast of various ages were tested. Results obtained with the two methods were closely correlated. The extraction method is the more rapid and convenient of the two assays investigated and may be used to follow change in activity with time, compare various yeast brands or investigate the effect of various environmental conditions on yeast INT-dehydrogenase activity.

Ectoadenylate Kinase and Plasma Membrane ATP Synthase Activities of Human Vascular Endothelial Cells

Formation of ATP from ADP on the external surface of vascular endothelial cells has been attributed to plasma membrane ATP synthase, ectoadenylate kinase (ecto-AK), and/or ectonucleoside diphosphokinase. These enzymes or their catalytic products have been causatively linked to the elaboration of vascular networks and the regulation of capillary function. The amount of ATP generated extracellularly is small, requiring sensitive analytical methods for quantification. Human umbilical vein endothelial cells were used to revisit extracellular ATP synthesis using a reliable tetrazolium reduction assay and multiwell plate cultures. Test conditions compatible with AK stability were established. Extracellular AK activity was found to be <1% of the total (intracellular and extracellular), raising the possibility that the external enzyme could have leaked from living cells and/or a few dying cells. To determine whether AK inadvertently leaked from the cells, the activity of another cytoplasmic enzyme, glucose-6-phosphate dehydrogenase (G6PD), was also measured. G6PD is present in the cytoplasm in similar abundance to AK. The activity ratio of G6PD (extracellular/total) was found to be similar to that of AK. Because G6PD in the medium was probably due to leakage, other cytoplasmic macromolecules, including AK, should be released proportionately from the cells. The role of plasma membrane ATP synthase in extracellular ATP formation was examined using Hanks' balanced salt solution with and without selective inhibitors of AK and ATP synthase activities. With P(1),P(5)-di(adenosine 5')-pentaphosphate (inhibitor of AK activity), no extracellular ATP synthesis was detected, whereas with oligomycin, piceatannol, and aurovertin (inhibitors of F(1)F(0)-ATP synthase and F(1)-ATPase activities), no inhibition of extracellular ATP synthesis was observed. AK activity alone could account for the observed extracellular ATP synthesis. The possible impact of ADP impurity in the assays is discussed.

Continuous operation of foamed emulsion bioreactors treating toluene vapors

Continuous operation of a new bioreactor for air pollution control called the foamed emulsion bioreactor (FEBR) has been investigated. The effect of several liquid feeding strategies was explored. The FEBR exhibited high and steady toluene removal performance (removal efficiency of 89%-94%, elimination capacity of 214-226 g/m3h at toluene inlet concentration of 1 g/m3) for up to 360 h, when 20% of the culture was replaced every 24 h by a nutrient solution containing 4 g/L of potassium nitrate as a nitrogen source. This feeding mode supported a high cell activity measured as INT reduction potential and active cell growth without being subject to nitrogen limitation. In comparison, operating the FEBR with the liquid in a closed loop (i.e., batch) resulted in a significant decrease of both the removal efficiency of toluene and INT reduction activity. Operation with feeding active cells resulted in stable and effective treatment, but would require a significant effort for mass culture preparation. Therefore, the continuous process with periodically feeding nutrients was found to be the most practical and effective operating mode. It also allows for stable operation, as was shown during removal of low concentration of toluene or after pollutant starvation. Throughout the study, INT reduction measurements provided insight into the process. INT reduction activity data proved that under normal operating conditions, the FEBR performance was limited by both the kinetics and by mass transfer. Overall, the results illustrate that engineered gas-phase bioreactors can potentially be more effective than conventional biofilters and biotrickling filters for the treatment of air pollutants such as toluene.

Indicators of biofilm development and activity in constructed wetlands microcosms

Methods to measure protein, exopolysaccharide, viable cell number and INT reduction activity were tested on biofilm growing in a wastewater batch reactor. They were shown to be meaningful indicators of biofilm growth and correlated well with each other. Protein, exopolysaccharide, viable cells and INT reduction rates increased linearly over time. Viable cell number exhibited strong linear correlations with protein ( R 2=0.98) and exopolysaccharide ( R 2=0.99) while INT reduction rate was somewhat less well correlated ( R 2=0.90). Our results indicate production rates of 0.91×10 −7 μg EPS per viable cell and 1.0×10 −7 μg protein per viable cell. Protein and polysaccharide specific INT reduction rates decreased by approximately 50%, whereas viable cell specific INT reduction rates decreased by 65% and the protein to polysaccharide ratio stayed relatively constant at between 1.1 and 1.2 as the biofilm developed. Measurement of protein, polysaccharide, viable cells and INT reduction rate at depth within the bioreactor showed that they were concentrated in the top 1 cm of the influent end of the reactor and each decreased to a base level within 4.5 cm of the inlet. Protein to polysaccharide ratios increased with depth in the reactor and the specific INT reduction rates were maximal at 4.5 cm depth. The results indicate that the biomass can take upwards of 100 days to stabilize during batch (fill and draw) operation of subsurface wetlands and that the relative ratios of biomass components remain relatively constant during biofilm growth. Also, it appears that filtration of suspended solids results in biomass concentration at the inlet to the wetland.

The Antioxidant Properties of Thyme (Thymus zygis L.) Essential Oil: an Inhibitor of Lipid Peroxidation and a Free Radical Scavenger

Antioxidants minimize the oxidation of lipid components in cell membranes by scavenging free radicals. However, imbalance between free radical production and removal tends to increase with age causing progressive damage. For the food industry it is of considerable interest to delay the autoxidation of food lipids, which cause the reduction in food quality, affecting color, taste, nutritive value, and functionality. A general orientation toward the use of natural compounds has stimulated research into the potential use of aromatic and medicinal plants as possible antioxidant replacements. This study characterized the antioxidant and pro-oxidant properties of thyme oil and a number of its components. The major components identified in thyme oil were found to inhibit ferric-ion-stimulated lipid peroxidation of rat brain homogenates, although none was as effective as the whole oil The order of antioxidant activity was; thyme oil > thymol > carvacrol > γ-terpinene > myrcene > linalool > p-cymene > limonene > 1,8-cineole > α-pinene. Both thyme oil and thymol were also found to inhibit tert-butyl-hydroperoxide-stimulated peroxidation and INT reduction by superoxide radicals generated by the xanthine-xanthine oxidase system. Of these compounds tested only p-cymene, 1,8-cineole and myrcene were found to exhibit pro-oxidant activity, albeit to a very small extent. Overall, the data suggest that thyme oil possesses useful antioxidant properties that may be utilized in the food industry and as a dietary supplement.

Measurement of Superoxide Dismutase in Erythrocytes and Whole Blood Using Iodonitrotetrazolium Violet

A simple analytical method for superoxide dismutase (SOD) is described. It is based on the competitive reduction of a superoxide anion (O2 −) by piodonitrotetrazolium (2-[4-iodopheny]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride, INT). The magnitude of inhibition of INT reduction by SOD was dependent on the rate of O2 − generation by xanthine oxidase: within the range of xanthine oxidase we have examined (2.2 – 4.4 μg/mL or 1.1 – 2.2 mU), the inhibition ranged from 49 to 64% and was inversely related to the xanthine oxidase activity. With 4.4 μg/mL of xanthine oxidase, as high as 1.0 units (0.32 μg/mL) of SOD inhibited about 50% of INT reduction, and the calibration curve derived from the INT assay correlates quite well with that of the cytochrome c method (r = 0.991). Analysis of the patient samples (n = 19) for erythrocytes SOD by both the INT and the cytochrome c methods compares reasonably well (r = 0.901). Student's t-test on the results shows no significant difference between the values obtained by these methods. Considering convenience, simplicity, and cost effectiveness of the reagents for the INT method, this assay has the potential of being useful in routine analysis of the red blood cells and serum samples for SOD.

Tetrazolium reduction in acidicSphagnum-derived peat

Assessment of how carbon storage in peat and gas fluxes across the peatland/atmosphere interface may be affected by predicted climate changes requires an understanding of the controls on C mineralization in peat. Toward this end, our goal was to develop the INT[2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride] reduction procedure for use in the field as a rapid and reliable approach to estimating C mineralization (CO2 and CH4 production) in peat under near-natural conditions with considerable spatial and temporal replication.Sphagnum peat samples collected from three sites were divided and analyzed for INT reduction activity, as well as for production of CO2 and CH4, under both oxic and anoxic conditions. For peat from each site, we found no significant relationships between INT reduction and oxic or anoxic CO2 or CH4 production. Although INT is reduced in acidicSphagnum peat, INT reduction rates do not provide a usable index of C mineralization.

Mechanisms of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride), and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli K-12

The tetrazolium salts INT and CTC are frequently used as indicators of bacterial respiration. Mechanisms of tetrazolium reduction to formazan in eukaryotic cells have been proposed, however those in prokaryotes remain largely unresolved. We undertook studies to determine the sites, and degree of coupling for INT and CTC reduction in the aerobic Escherichia coli K-12 respiratory chain using both intact cells and inverted membrane vesicles. In addition, reduction in whole cells was assayed under anaerobic conditions which elicit different electron transport pathways. Results of experiments with inhibitors of specific electron transport components indicated that both CTC and INT were reduced prior to ubiquinone in the E. coli respiratory chain by the primary aerobic [succinate and NAD(P)H] dehydrogenases. INT was also reduced at ubiquinone and possibly cytochromes b 555, 556. Quantitative CTC reduction was more closely correlated with respiration in whole cells than INT, but the reverse was true in inverted membrane vesicles. Both tetrazolium salts were reduced to significant degrees under anaerobic conditions, particularly glucose fermentation. Mid-point reduction potential of CTC was determined to be −200 mV by redox titration. However, it appears that CTC forms a weakly fluorescent, poorly-localized intracellular formazan at redox potentials higher than ca. −200 mV. Inhibition of the aerobic terminal oxidases with KCN or azide strongly increased INT-, but significantly decreased CTC reduction in whole cells, indicating that these agents may not be useful for optimizing CTC-formazan or CTC (+) cell numbers in some bacteria. However, several other chemical agents stimulated CTC and INT reduction. These results suggest strategies for optimizing methods using these tetrazolium salts for assessment of bacterial respiratory activity.