Production and viability of coccoid forms of Campylobacter jejuni

Studies were conducted into the formation and physiological state of coccoid cells of a strain of the human and animal pathogen Campylobacter jejuni. It was found that growth phase and the presence of chloramphenicol did not affect the rate of shape transformation from spiral to coccoid, while nutrient limitation, aeration of the medium and the presence of free-radical scavengers had profound effects. Coccoid cells were found to reduce the tetrazolium salts INT (2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) to their respective formazans and this was linked to cellular respiration. However, respiring coccoid cells could not sustain their existence in prolonged adverse conditions, and it was concluded that they represent a degenerative stage rather than a dormant state of the organism.     

Calibration of a flow cytometric assay of glucose-6-phosphate dehydrogenase activity.

The reduction of tetrazolium salts to colored formazans is a reaction which has been exploited both in histo- and cytochemistry. Tetrazolium salts forming fluorescent formazans prove suitable for measuring defined cellular dehydrogenase activities in automated processes. This study considers an important aspect of formazan measurement in flow cytometry, namely, calibration. Calibration is performed by correlating the number (and fluorescence intensity) of formazan-bearing cells measured by flow cytometry with simultaneously performed biochemical analyses of the same material. The method is demonstrated by an example of glucose-6-phosphate dehydrogenase. Using the data of a typical experiment, the enzyme activity is expressed in femtomol of hydrogen transferred per cell during incubation time. Furthermore, through spatially resolved double excitation of formazan and nuclear DAPI fluorescence, an independent analysis of cell cycle and cellular enzymatic activity is established.     

Detection of alkaline phosphatase activity after conventional isoelectric focusing by an indoxyl-tetrazolium salt technique

Alkaline phosphatase was solubilized from human and rat tissues using papain in the presence of TRITON X-100 and subjected to isoelectric focusing (IEF) in polyacrylamide or agarose gels. Up till now, usually 1- and 2-naphthylphosphates have been used as substrates in order to specifically stain molecular forms of this enzyme by the azo-dye technique. In this paper, the use of another histochemical substrate, 5-bromo-4-chloro-3-indoxyl phosphate, in combination with tetrazolium salts [McGadey, J. (1970) Histochemie 23, 180-184] is presented. After hydrolysis, the released indoxyl moieties reduce tetrazolium salts to insoluble formazans at the zones of alkaline phosphatase activity. Zymogrammes showing molecular forms of alkaline phosphatase from 20 rat organs and the application of this staining technique for the detection of alkaline phosphatase activity in non-dialyzed human plasma after IEF are presented.     

New tetrazolium method for the histochemical demonstration of gamma-glutamyl transpeptidase.

A new tetrazolium method for the histochemical demonstration of gamma-glutamyl transpeptidase is proposed. The method is based on a newly synthesized substrate-gamma-L-glutamic acid-1-hydroxy-4-naphthylamide, which upon the enzyme hydrolysis liberates 1,4-aminonaphthol--a powerful reducing agent that reduces tetrazolium salts quickly and quantitatively to deeply colored, water-insoluble formazans, precipitating on the sites of the enzyme activity and marking them accurately. The redox reaction is quick enough and does not need any auxiliary electron-acceptor. The method is very fast and convenient for the histochemical visualization of the enzyme.     

The role of plasma membrane in bioreduction of two tetrazolium salts, MTT, and CTC

Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. We investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. We postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc.     

New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV.

New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV (DPP IV), based on a newly synthesized substrate Gly-L-Pro-1-hydroxy-4-naphthylamide is proposed. Upon the enzyme hydrolysis of the substrate a strong reducing agent, i.e. 4-amino-1-naphthol is released, which reduces tetrazolium salts to water-insoluble, deeply colored formazans, that precipitate on the sites of enzyme activity, marking them accurately. No auxiliary electron acceptor is needed for the redox reaction. The incubation is performed at the optimal pH of the enzyme. Precise enzyme localization is achieved in all organs studied. Thus, the new method avoids most of the disadvantages of the methods in use and might open new possibilities in peptidases histochemistry.     

Zur Substantivität von Tetrazoliumsalzen in der Histochemie

Zusammenfassung Die in der Literatur beschriebenen Beziehungen zwischen Substantivität und chemischem Bau der in der Histochemie verwendeten Tetrazoliumsalze wurden an weiteren Tetrazoliumsalzen überprüft. Die Untersuchungen ergaben, daß die Anzahl und die Stellung der Nitrogruppen im Molekül an Bedeutung für die Substantivität des Tetrazoliumsalzes hinter dem Einfluß einer linearen, aromatischen Substitution am N(₃) des Tetrazolringes bzw. einer Molekülverdoppelung zurücktreten. Es ist zwischen einer Substantivität der Tetrazoliumsalze und einer Substantivität der Formazane zu unterscheiden.

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On substantivity of tetrazolium salts in histochemistry

Summary The known correlations between substantivity and chemical constitution of tetrazolium salts used in histochemistry are further examined on several tetrazolium salts. The investigations are demonstrating that number and position of the nitro groups in the molecule are less important for substantivity of the salt than the influence of a linear, aromatic substitution on N(₃) of the tetrazol ring or of doubling the molecule. It is to differentiate between substantivity of tetrazolium salts and the substantivity of the formazans.

     

Histochemical Analysis of Microdissected Nephrons: Enzyme Stains

Enzyme stains were done on 120-160 μ, sections of fresh-frozen human kidney obtained at autopsy or biopsy. These sections were then either fixed in a 3:7 mixture of ethanol and glacial acetic acid and macerated in collagenase, or fixed in 10% formalin and macerated in concentrated HCl. Following maceration by either method, the sections were washed in water and microdissected. With collagenase maceration, good results were obtained for dehydrogenase methods using tetrazolium salts. HCl maceration caused loss of formazans, but did not destroy the colored products of staining procedures for alkaline and acid phosphatase, aliesterase, and cytochrome oxidase.     

Use of physiological substrates for zymograms of disaccharidases after separation in immobilized pH gradients

A new technique is described for in situ visualization of the activity of intestinal disaccharidases after isoelectric focusing in immobilized pH gradients using their physiological substrates. The reaction principle is based on the oxidation of D-glucose, liberated by the disaccharidases, into D-gluconolactone and the production of NADH by glucose dehydrogenase. At the sites of enzymatic activity, tetrazolium salts present in the reaction mixture are reduced to relatively water-insoluble formazans by NADH. The rate of formazan production is increased by the presence of phenazine methosulfate. An additional modification of the technique involves the use of polyvinyl alcohol in the substrate solution. Due to the increase in the viscosity of the substrate solution, leakage of the enzyme from the IPG gels is minimized. Incubation times can thus be prolonged without loss of resolution and band-blurring.     

Serum albumin leads to false-positive results in the XTT and the MTT assay

Tetrazolium salts like 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) or sodium 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) that form formazans after reduction are widely used to investigate cell viability. Besides cellular enzymes, some constituents of cell media and other substances reduce tetrazolium salts, thereby interfering with these assays. We describe here that different preparations of serum albumin from bovine or human origin can lead to a concentration-dependent increase in the signals of the XTT assay; therefore leading to an overestimation of cell numbers and to an underestimation of potential cytotoxic effects of compounds to be tested. The same effect was seen in the MTT assay with human serum albumin (HSA). We demonstrate that this reductive activity cannot be inactivated by proteolytic digestion, but that it is due to the free cysteine residue in albumin, and is also observed when cysteine or glutathione (GSH) are used. Binding of N-ethylmaleimide (NEM) to the free cysteine residue leads to a decrease of the albumin interference in the XTT assay.     

Fluorescent redox dyes

Summary The reduction of a new series of tetrazolium salts to red fluorescent formazans by Ehrlich ascites tumor cells is described. The qualitative effect on this reaction of two cell surface-active compounds and of six exogenous electron carriers was investigated by varying the incubation conditions. After incubation of Ehrlich ascites cells with the new colourless, watersoluble 5-cyan-2.3-ditolyltetrazolium salts, bright red water-insoluble formazan crystals on the cell surface can be observed under fluorescence microscopy. The production of formazan is enhanced by 12-0-tetradecanoylphorbol-13-acetate (TPA) or digitonin (DIG), two potent stimulators of oxygen consumption or by the electron carriers phenzazine methosulphate (PMS), 1-methoxy-phenazine methosulphate (MPMS), meldola blue (MB), methylen blue (MTB), and 2.6-dichlorindophenol (DCIP). These results provide further evidence for the existence of redox enzymes bound to the plasma membrane of intact ascites cells and for a free radical mechanism of tetrazolium salt reduction. The fluorescence property of the new redox dyes offers the advantage of high sensitivity. Moreover, their greater homogeneity relative to the commonly used di-tetrazolium salts lowers the chances of misinterpretations due to impurities. The possible application of these new mono-tetrazolium salts to cytochemical investigations of oxidative metabolic reactions is discussed.     

A model for NADH-tetrazolium reductase

Summary In the presence of light, reduced nicotinamide adenine dinucleotide (NADH) and riboflavin formed a complex which was able to reduce certain tetrazolium salts. Neither NADH (10^–3 M) nor riboflavin (10^–4 M) alone was able to induce tetrazolium reduction in the presence of oxygen, but in a nitrogen atmosphere photoreduction of riboflavin induced reduction of tetrazolium salts. Only electrophilic nitro and thiazolyl substituted tetrazolium salts with more positive redox potentials were reduced by the NADH-riboflavin complex, and only monoformazans were produced from the ditetrazolium salts. The reduction kinetics of these tetrazolium salts are given, and the spectral area capable for induction of electron transfer in the NADH-riboflavin complex is screened. It is concluded that the electron transfer in flavin nucleotide dependent dehydrogenase systems will probably proceed without direct interference with the apoenzyme. This may have practical implications for the histochemistry of tetrazolium reductases especially as regards fixation. The catalytic action of light on tetrazolium reduction should also be taken into consideration when tetrazolium salts are used as electron acceptors in a histochemical reaction.     

Effects of tetrazolium salts on oxidative phosphorylation in rat-liver mitochondria

1. The effects of five different tetrazolium salts on oxidative phosphorylation in rat-liver mitochondria have been investigated. 2. In all cases the mitochondria were uncoupled by very low concentrations of the tetrazolium salts. Further, the transition from a system just exhibiting respiratory control to one in which the mitochondria were totally uncoupled has been shown to occur over very small concentration ranges of the tetrazolium salts. 3. The effectiveness of the five tetrazolium salts as uncoupling agents is discussed in the light of their standard electrode potentials and effectiveness as electron acceptors in dehydrogenase-linked reactions.     

Histochemical demonstration of a circadian rhythm of succinate dehydrogenase in rat pineal gland. Influence of coenzyme Q10 addition.

Succinate dehydrogenase activity was investigated histochemically in the rat pineal gland. The influence of fixation on the activity pattern, the possible diffusion of enzyme, the nothing dehydrogenase reaction, and the substantivity of the tetrazolium salts and formazans were investigated in control experiments. In rats maintained on a 17/7 h light/dark schedule a distinct circadian rhythm of the succinate dehydrogenase was demonstrated in the pineal gland. Activity was lowest during the day and highest during the night. The dorsocaudal part of the gland showed the highest activity and within the same part of the gland the activity varied between individual pinealocytes. A relative lack of endogenous coenzyme Q, as well as a circadian rhythm of this coenzyme, highly influenced the activity of succinate dehydrogenase. It is concluded that succinate dehydrogenase activity in the pineal gland of the rat is regulated by changing the concentration of the active enzyme itself as well as the level of the endogenous coenzyme Q. Whether this is caused by a circadian rhythm in the synthesis or in the catabolism of the enzyme and the coenzyme was not revealed by the present study .     

Histochemical demonstration of a circadian rhythm of succinate dehydrogenase in rat pineal gland. Influence of coenzyme Q10 addition

Summary Succinate dehydrogenase activity was investigated histochemically in the rat pineal gland. The influence of fixation on the activity pattern, the possible diffusion of enzyme, the nothing dehydrogenase reaction, and the substantivity of the tetrazolium salts and formazans were investigated in control experiments.In rats maintained on a 17/7 h light/dark schedule a distinct circadian rhythm of the succinate dehydrogenase was demonstrated in the pineal gland. Activity was lowest during the day and highest during the night. The dorsocaudal part of the gland showed the highest activity and within the same part of the gland the activity varied between individual pinealocytes. A relative lack of endogenous coenzyme Q, as well as a circadian rhythm of this coenzyme, highly influenced the activity of succinate dehydrogenase. It is concluded that succinate dehydrogenase activity in the pineal gland of the rat is regulated by changing the concentration of the active enzyme itself as well as the level of the endogenous coenzyme Q. Whether this is caused by a circadian rhythm in the synthesis or in the catabolism of the enzyme and the coenzyme was not revealed by the present study.     

Some effects of tetrazolium salt on the metabolism of mitochondria isolated from Jerusalem artichoke tubers

Summary A series of tetrazolium salts were found to accept electrons more readily from succinate than malate even though the rate of oxygen uptake was similar with both substrates. This difference was explained by showing that all the tetrazolium salts tested caused a reduction in electron flow between NAD⁺ and Cyt.b. The tetrazolium salts were also found to be able to uncouple phosphorylation from electron transport. The monotetrazolium salts causing complete uncoupling around 100 moles/litre and the ditetrazolium salts causing complete uncoupling around 20 moles/litre.     

Superoxide anion radical-independent pathway for reduction of tetrazolium salts in aerobic mixtures consisting of NADH and 5-methylphenazinium methyl sulfate in the presence of aqueous micelles of nonionic and cationic detergents

The effect of Triton X-100 and certain other nonionic as well as cationic detergents on 5-methyl-phenazinium methyl sulfate (PMS)-mediated reduction of tetrazolium salts was studied under aerobic conditions using an exogenous source of reducing equivalents, such as NADH or by generating NADPH through an enzymatic reaction. In the absence of detergents, 5,10-dihydro-5-methylphenazine (MPH), formed on reduction of 5-methylphenazinium cation (MP+) of PMS by NAD[P]H, was reoxidized allowing first the univalent reduction of molecular oxygen (O2) to the superoxide anion radical (O2-.) which, in turn, reduced tetrazolium salts. In the presence of detergents, however, a significant fraction of the PMS-mediated reduction of tetrazolium salts appeared to proceed without the intervention of O2-. The reasons for this were examined experimentally and it was suggested that the reduced phenazine (i.e., MPH), which is sparingly soluble in aqueous solutions, migrates into detergent micelles where tetrazolium salts are reduced in preference to O2. By lowering the pH and thereby facilitating the H+-mediated dismutation of O2-., it was possible to obtain the reduction of tetrazolium salts, mediated selectively and directly by MPH in the micellar pseudophase. Employing the technique of saturation analysis, further evidence was obtained that lends support for preferential reduction of tetrazolium salts (e.g., nitroblue tetrazolium chloride) to that of O2 by the micelle-bound MPH.     

Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC). In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT). To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3'-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated. Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays. For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude. A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation. Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation. However, one bacterium (Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation. This strain would be considered nonviable based on traditional tetrazolium salt reduction assays. The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may be falsely considered nonviable by this assay due to poor uptake or reduction of the salts.     

Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis

The testa of higher plant seeds protects the embryo against adverse environmental conditions. Its role is assumed mainly by controlling germination through dormancy imposition and by limiting the detrimental activity of physical and biological agents during seed storage. To analyze the function of the testa in the model plant Arabidopsis, we compared mutants affected in testa pigmentation and/or structure for dormancy, germination, and storability. The seeds of most mutants exhibited reduced dormancy. Moreover, unlike wild-type testas, mutant testas were permeable to tetrazolium salts. These altered dormancy and tetrazolium uptake properties were related to defects in the pigmentation of the endothelium and its neighboring crushed parenchymatic layers, as determined by vanillin staining and microscopic observations. Structural aberrations such as missing layers or a modified epidermal layer in specific mutants also affected dormancy levels and permeability to tetrazolium. Both structural and pigmentation mutants deteriorated faster than the wild types during natural aging at room temperature, with structural mutants being the most strongly affected.     

The use of tetrazolium salts in the histochemical demonstration of succinic dehydrogenase activity in plant tissues

Summary 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 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 fresh or frozen sections. The observed differences in the abilities of the tetrazolium salts to demonstrate succinic dehydrogenase activity are discussed.The sites of acceptance of electrons from the electron transport pathway in plant cells by the tetrazolium salts has been demonstrated cytochemically, and shown to differ from those observed in animal cells in that unlike animal cells, there is no apparent acceptance of electrons by MTT and INT from cytochrome C₁-C region of the pathway.