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PubMed2004

Rapid epidemiologic assessment of glucose-6-phosphate dehydrogenase deficiency in malaria-endemic areas in Southeast Asia using a novel diagnostic kit.

We recently reported a new rapid screening method for glucose-6-phosphate dehydrogenase (G6PD) deficiency. This method incorporates a new formazan substrate (WST-8) and is capable of detecting heterozygous females both qualitatively and quantitatively. Here, we report its evaluation during field surveys at three malaria centres and in malaria-endemic villages of Myanmar and Indonesia, either alone or in combination with a rapid on-site diagnosis of malaria. A total of 57 severe (45 males and 12 females) and 34 mild (five males and 29 females) cases of G6PD deficiency were detected among 855 subjects in Myanmar whilst 30 severe (25 males and five females) and 23 mild (six males and 17 females) cases were found among 1286 subjects in Indonesia. In all cases, severe deficiency was confirmed with another formazan method but due to limitations in its detection threshold, mild cases were misdiagnosed as G6PD-normal by this latter method. Our results indicate that the novel method can qualitatively detect both severely deficient subjects as well as heterozygous females in the field. The antimalarial drug, primaquine, was safely prescribed to Plasmodium vivax-infected patients in Myanmar. Our new, rapid screening method may be essential for the diagnosis of G6PD deficiency particularly in rural areas without electricity, and can be recommended for use in malaria control programmes.

Adolescent↗
PubMed1978

Nitroblue tetrazolium (NBT) reduction by bacteria employed for rapid determination of antibiotic concentrations in serum.

The ability of bacteria to reduce nitroblue tetrazolium (NBT) to formazan, and the inhibition of this process by antibiotics, have been utilized in a method to determine antibiotic concentrations in serum. Escherichia coli, growing in the presence of increasing but subinhibitory concentrations of antibiotics, produces diminished amounts of formazan. This is photometrically quantified and the optical density obtained with the sample is compared with those obtained with know concentrations. Levels of aminoglycosides below 1 microgram/ml can be measured in the presence of other antibiotics also, and the assay can be carried out in three hours. Good correlation was obtained when 49 serum samples containing varying concentrations of sisomicin were tested simultaneously by the NBT assay and a disc diffusion method.

Aminoglycosides↗
PubMed1990

A new sensitive bioassay for precise quantification of interferon activity as measured via the mitochondrial dehydrogenase function in cells (MTT-method).

A biological method for precise quantification of interferons has been developed. The method is based upon the dehydrogenase system of the intact target cells, which will normally convert an artificial substrate, MTT, into formazan (blue), which, in turn, can be measured spectrophotometrically. This conversion is greatly reduced by cytocidal viruses in a dose-dependent manner. The protection of target cells by interferon against challenge virus is reflected in a diminished reduction in the production of formazan, thus giving a very precise method for quantification of interferon. The lowest level of detection is around 0.10 international units. The intra- and inter-assay variability appear to be below 10%. The assay, which makes no use of expensive ingredients, is performed in 96-well micro-trays and read in an inexpensive ELISA-scanner.

Biological Assay↗
PubMed1988

Combined phagocytosis-nitroblue tetrazolium reduction test in bovine neutrophils.

An assay was developed for the simultaneous evaluation of phagocytosis and oxidative metabolism of bovine blood neutrophils. Phagocytosis was evaluated by using opsonized zymosan, and oxidative metabolism was evaluated by nitroblue tetrazolium (NBT) reduction. Normal bovine neutrophils exhibited moderate variation in ability to phagocytize zymosan, but little variation in ability to reduce NBT. The subcellular location of NBT reduction to formazan was determined by electron microscopy. Electron dense formazan precipitate was observed along the inner membrane of phagosomes enclosing zymosan particles and radiating from the membrane toward the center of the phagosome.

Journal Article↗
PubMed1986

Activity of an Attached and Free-Living Vibrio sp. as Measured by Thymidine Incorporation, p-Iodonitrotetrazolium Reduction, and ATP/DNA Ratios.

Three independent techniques, [H]thymidine incorporation, the reduction rate of p-iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA, were adapted to measure the activity of attached and unattached estuarine bacteria. In experiments employing the estuarine isolate Vibrio proteolytica, nutrient concentrations were manipulated by varying the concentration of peptone-yeast extract. In the presence of exogenous nutrients, the activity of free-living cells was greater than that of attached cells as measured by [H]thymidine incorporation and ATP/DNA ratios. In the absence of peptone-yeast extract, however, the activity of attached cells surpassed that of free-living cells as determined by [H]thymidine incorporation and INT formazan normalized to DNA. Of the three techniques, [H]thymidine incorporation was deemed most sensitive for detecting changes in activity resulting from slight differences in nutrient concentration. By this technique, attached cells were much less sensitive to changing nutrient concentrations than were free-living cells. Below a threshold concentration, attached cell activity remained constant, while the activity of unattached cells decreased as a function of decreasing nutrient concentration. The results suggest that loss of cell surface area available for substrate uptake due to attachment may be an important factor in determining the relative activities of attached and free-living cells.

Journal Article↗
PubMed1986

Activity measurements of planktonic microbial and microfouling communities in a eutrophic estuary.

[H]thymidine incorporation, the rate of reduction of iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA were adapted to measure the activity of attached and unattached microbial assemblages of Bayboro Harbor, Fla. Activity measurements by [H]thymidine incorporation were made of cells attached to polystyrene culture dishes, in unfiltered water samples, and in the <1-mum-filtered fraction. In most cases, the activity of attached cells was greater than that of unattached cells either in unfiltered water samples or in the <1-mum fraction. The calculated thymidine incorporation rates for cells in the >1-mum fraction were higher than those for cells either in unfiltered water or in the <1-mum-filtered fraction. By the rate of reduction of INT to INT formazan normalized to DNA and by ATP-to-DNA ratios, attached cells were also more active than cells in unfiltered water samples. These results indicate that the microenvironment afforded by attachment is a more beneficial habitat for microbial growth. Reasons for greater activity by natural populations of attached bacteria are discussed.

Journal Article↗
PubMed1991

Metabolic status of bacteria and fungi in the rhizosphere of ponderosa pine seedlings.

We determined the quantity and metabolic status of bacteria and fungi in rhizosphere and nonrhizosphere soil from microcosms containing ponderosa pine seedlings. Rhizosphere soil was sampled adjacent to coarse, fine, or young roots. The biovolume and metabolic status of bacterial and fungal cells was determined microscopically and converted to total and active biomass values. Cells were considered active if they possessed the ability to reduce the artificial electron acceptor 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) to visible intracellular deposits of INT formazan. A colorimetric assay of INT formazan production was also used to assess dehydrogenase activity. INT-active microorganisms made up 44 to 55% of the microbial biomass in the soils studied. The proportion of fungal biomass that exhibited INT-reducing activity (40 to 50%) was higher than previous estimates of the active proportion of soil fungi determined by using fluorescein diacetate. Comparison between soils from different root zones revealed that the highest total and INT-active fungal biomass was adjacent to fine mycorrhizal roots, whereas the highest total and active bacterial biomass was adjacent to the young growing root tips. These observations suggest that fungi are enhanced adjacent to the fine roots compared with the nonrhizosphere soil, whereas bacteria are more responsive than fungi to labile carbon inputs in the young root zone. Colorimetric dehydrogenase assays detected gross differences between bulk and rhizosphere soil activity but were unable to detect more subtle differences due to root types. Determination of total and INT-active biomass has increased our understanding of the role of spatial compartmentalization of bacteria and fungi in rhizosphere carbon flow.

Journal Article↗
PubMed1992

Use of a fluorescent redox probe for direct visualization of actively respiring bacteria.

The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was employed for direct epifluorescent microscopic enumeration of respiring bacteria in environmental samples. Oxidized CTC is nearly colorless and is nonfluorescent; however, the compound is readily reduced via electron transport activity to fluorescent, insoluble CTC-formazan, which accumulates intracellularly. Bacteria containing CTC-formazan were visualized by epifluorescence microscopy in wet-mount preparations, on polycarbonate membrane filter surfaces, or in biofilms associated with optically opaque surfaces. Counterstaining of CTC-treated samples with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole allowed enumeration of active and total bacterial subpopulations within the same preparation. Municipal wastewater, groundwater, and seawater samples supplied with exogenous nutrients yielded CTC counts that were generally lower than total 4',6-diamidino-2-phenylindole counts but typically equal to or greater than standard heterotrophic (aerobic) plate counts. In unsupplemented water samples, CTC counts were typically lower than those obtained with the heterotrophic plate count method. Reduction of CTC by planktonic or biofilm-associated bacteria was suppressed by formaldehyde, presumably because of inhibition of electron transport activity and other metabolic processes. Because of their bright red fluorescence (emission maximum, 602 nm), actively respiring bacteria were readily distinguishable from abiotic particles and other background substances, which typically fluoresced at shorter wavelengths. The use of CTC greatly facilitated microscopic detection and enumeration of metabolically active (i.e., respiring) bacteria in environmental samples.

Bacteria↗
PubMed1994

Viability of indigenous soil bacteria assayed by respiratory activity and growth.

The bacterial population in barley field soil was estimated by determining the numbers of (i) cells reducing the artificial electron acceptor 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to CTC-formazan (respiratory activity), (ii) cells dividing a limited number of times (microcolony formation) on nutrient-poor media, (iii) cells dividing many times (colony formation) on nutrient-poor agar media, and (iv) cells stained with acridine orange (total counts). The CTC reduction assay was used for the first time for populations of indigenous soil bacteria and was further developed for use in this environment. The number of viable cells was highest when estimated by the number of microcolonies developing during 2 months of incubation on filters placed on the surface of nutrient-poor media. The number of bacteria reducing CTC to formazan was slightly lower than the number of bacteria forming microcolonies. Traditional plate counts of CFU (culturable cells) yielded the lowest estimate of viable cell numbers. The microcolony assay gave an estimate of both (i) cells forming true microcolonies (in which growth ceases after a few cell divisions) representing viable but nonculturable cells and (ii) cells forming larger microcolonies (in which growth continues) representing viable, culturable cells. The microcolony assay, allowing single-cell observations, thus seemed to be best suited for estimation of viable cell numbers in soil. The effect on viable and culturable cell numbers of a temperature increase from 4 to 17 degrees C for 5 days was investigated in combination with drying or wetting of the soil. Drying or wetting prior to the temperature increase, rather than the temperature increase per se, affected both the viable and culturable numbers of bacteria; both numbers were reduced in predried soil, while they increased slightly in the prewetted soil.

Journal Article↗
PubMed2002

Detection of microbial growth on polycyclic aromatic hydrocarbons in microtiter plates by using the respiration indicator WST-1.

We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 [4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate], in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.

Bacteria↗
PubMed1963

INHIBITORY ACTION OF PHAGE K ON STAPHYLOCOCCAL DEHYDROGENASES. II. ITS POSSIBLE RELATIONSHIP TO SENSITIZATION AND CELL LYSIS.

Ralston, D. J. (University of California, Berkeley) and M. D. Perry. Inhibitory action of phage K on staphylococcal dehydrogenases. II. Its possible relationship to sensitization and cell lysis. J. Bacteriol. 86:740-748. 1963.-By measuring the reduction of the dye triphenyl tetrazolium chloride to the insoluble red formazan, an analysis was made of the inhibition by phage K of the dehydrogenase capacity of populations of Staphylococcus aureus K(1), to determine to what extent this might be associated with the ability of phage K to sensitize the cells-a reaction characterized by the conversion of the cell wall to susceptibility to lysis by the staphylococcal enzyme, virolysin. Increasing multiplicities of phage progressively increased the fractions of sensitized cells and also caused increasing inhibition of the dehydrogenase activity of the populations. The dehydrogenase activities of the sensitized fractions were compared with those of the nonsensitized fractions. Over a wide range of phage-bacterium ratios, the dehydrogenase activities of the sensitized fractions were found to be lower than those of the nonsensitized fractions. Microscopically, this was reflected by the appearance of large numbers of cells with a reduced amount of visible formazan granules. When lysin was added to the phage-treated cells, lysis occurred mainly from cell fractions which possessed little or no tetrazolium-reducing capacity. The data indicated that sensitization by phage was accompanied by a marked decrease in cellular dehydrogenase activity but was not associated with a complete inhibition of these enzymes. A comparison was made between the dehydrogenases of phage-sensitized cells and cells found to be "spontaneously" sensitive to virolysin, i.e., lysed by the enzyme without any prior exposure to phage. Like phage-sensitized cells, the spontaneously sensitized staphylococci possessed low dehydrogenase activity and lacked the capacity to support phage synthesis. In tests of a given cell preparation, the dehydrogenase levels of the phage-sensitized fractions were found to be close to, or even lower than, the level of the spontaneously sensitized fraction, suggesting that in S. aureus K(1) the sensitized state is associated in some manner with a reduction of the dehydrogenase activity to a critical level. There is as yet no evidence for any direct causal relationship between sensitization and dehydrogenase inhibition.

Bacteriophages↗
PubMed2001

Colorimetric assay for antifungal susceptibility testing of Aspergillus species.

A colorimetric assay for antifungal susceptibility testing of Aspergillus species (Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, and Aspergillus ustus) is described based on the reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) in the presence of menadione as an electron-coupling agent. The combination of 200 microg of XTT/ml with 25 microM menadione resulted in a high production of formazan within 2 h of exposure, allowing the detection of hyphae formed by low inocula of 10(2) CFU/ml after 24 h of incubation. Under these settings, the formazan production correlated linearly with the fungal biomass and less-variable concentration effect curves for amphotericin B and itraconazole were obtained.

Antifungal Agents↗
PubMed1972

Nitro blue tetrazolium test in children with kwashiorkor with a comment on the use of latex particles in the test.

The nitro blue tetrazolium test was applied on neutrophils from children with kwashiorkor and from control children. The percentage of formazan cells formed in the kwashiorkor patients was significantly less than in the control group in spite of the presence of associated infections in the former. A direct correlation exists between the percentage of formazan cell formation and total serum proteins, albumin, and haemoglobin. These findings may reflect a decreased bactericidal activity of neutrophils in kwashiorkor. The use of latex particles is an unnecessary technical refinement of the test.

Blood Bactericidal Activity↗
PubMed1993

Effect of quartz and alumina dust on generation of superoxide radicals and hydrogen peroxide by alveolar macrophages, granulocytes, and monocytes.

Phagocytosis of quartz particles by rabbit alveolar macrophages and monocytes and human granulocytes and monocytes was accompanied by stimulation of substrate free reduction of nitroblue tetrazolium to formazan. This reflects activation of an oxygen dependent bactericidal system of phagocytes and total (exogenic and endogenic) generation of active oxygen species. Low fibrogenic and cytotoxic alumina dust tended to increase formazan production by comparison with quartz dust. During phagocytosis of quartz dust by alveolar macrophages and monocytes there was no exogenic generation of superoxide radicals and hydrogen peroxide by these cells. By contrast, incubation of human granulocytes with quartz dust caused a significant increase in exogenic generation of superoxide radicals and hydrogen peroxide. Under such conditions, low fibrogenic alumina dust had no effect on hydrogen peroxide generation and substantially decreased the level of superoxide radical generation by human granulocytes. During incubation of rabbit granulocytes with quartz dust, an increase in the level of superoxide radical generation was also detected. It is considered that the differences between alveolar macrophages and granulocytes in their response to quartz dust are important from a physiological point of view. Alveolar macrophages are permanently present in pulmonary alveolae in large quantities; therefore their uncontrolled generation of superoxide radicals and hydrogen peroxide might immediately cause damage to pulmonary parenchyma. At the same time, destruction products from alveolar macrophages that died during phagocytosis of quartz particles contain a factor attracting granulocytes. Presence of a significant number of granulocytes in bronchopulmonary lavage fluid in cases of silicosis indicates development of a pathological process. This agrees well with the data obtained on exogenic generation of superoxide radicals and hydrogen peroxide by granulocytes, and on stimulation of this process due to phagocytosis of the quartz dust.

Aluminum Oxide↗
PubMed1994

Matrix protein regulation of PMN oxidative metabolism during ischemia.

Matrix proteins upregulate polymorphonuclear neutrophil (PMN) oxidative metabolism in a normoxic environment. We sought to investigate the relationship between matrix proteins and adherent PMN oxidative metabolism during acute ischemia. PMN adherent to buffer, fibronectin, Arg-Gly-Asp-Ser (RGDS), or laminin were placed in either normoxic or ischemic media. PMN adherence, superoxide anion production, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formazan production, and surface receptor expression (CD64, CD32w, CD16, CD35, and CD11b/CD18) using monoclonal antibodies directed against these receptors were assayed. Ischemia increased PMN adherence unless the PMN were adhered to fibronectin or RGDS. Ischemia reduced PMN superoxide anion, MTT formazan, and H2O2 production unless the PMN were adhered to fibronectin or RGDS. Fibronectin and RGDS prevented ischemic-induced suppression of FcR expression. Immunofluorescent studies demonstrated capping and clustering of PMN Fc and complement receptors during ischemia while adhered on matrix proteins. These results demonstrate that 1) ischemia suppresses matrix protein upregulation of PMN oxidative metabolism, which is restored by fibronectin; 2) fibronectin-mediated restoration of PMN oxidative metabolism involves the binding epitope of fibronectin; and 3) fibronectin maintains PMN oxidative metabolism during ischemia in part by maintaining PMN FcR on the cell surface and by recruiting a new population of PMN capable of undergoing oxidative metabolism.

Antibodies, Monoclonal↗
PubMed1991

Human recombinant superoxide dismutase protects primary cultured neurons against hypoxic injury.

Induction of free radicals and succeeding cascade reactions of lipid peroxidation are known to cause neuronal damage in cases of cerebral ischemia. We studied whether hypoxic stress in vitro induces the formation of these cytotoxic materials, and whether superoxide dismutase (SOD) protects cultured neurons from hypoxic stress. We used human recombinant SOD (rSOD), which is structurally homologous to native human SOD, the only exception being that the N terminus is not acetylated. First, we detected free radicals by means of nitroblue tetrazolium (NBT) and assured that hypoxic stress to the cultured neurons induced the formation of peroxidation materials. Immediately after the hypoxic stress, the neurons showed no reduction from NBT to formazan. Striking NBT coloration of the somata, however, started to appear 30 min after reoxygenation, and the percentage of formazan-positive neurons increased during up to 32 h of the experimental period. About 50% of the neurons stained positively 24 h after hypoxia. This shows that rSOD is needed immediately after reoxygenation. Secondly, we examined whether rSOD exerts a cytoprotective activity against hypoxia. rSOD, with the application of proper treatment which incorporates rSOD into the target neuron, prevents the neuronal damage induced by oxide anions, which plays an important role in acute neuronal death accompanied by hypoxia. It can be concluded that, when intracellularly present in sufficient amounts, rSOD is useful for the treatment of hypoxic damage.

Animals↗
PubMed1975

The ultracytochemical demonstration of a formaldehyde-resistant dehydrogenase of leuco nitroxyl analogues.

A dehydrogenase which is relatively stable in formaldehyde fixative is demonstrated ultracytochemically by the reduction of various leuco nitroxyl analogues in rat hepatic, renal, myocardial, skeletal muscle and prostatic tubuloalveolar glandular tissues. The nonosmiophilic tetrazolium salt, t-(2'-benzothiazolyl)-5-styryl-3-(4'-phtalhydrazidyl) tetrazolium chloride, is subsequently reduced to an insoluble osmiophilic formazan by the hydrogen ions resulting from the dehydrogenase activity. Exposure of the formazan to osmium tetroxide results in electron density enabling visualization of the reaction product in the electron microscope. Known inhibitors of various dehydrogenases were utilized in an attempt to determine the existence and/or extent of any specific characteristics of the dehydrogenase(s) involved.

Animals↗
PubMed1991

A novel tetrazolium method for peroxidase histochemistry and immunohistochemistry.

We developed a new method for the histochemical demonstration of peroxidase. This method, which has a novel reaction mechanism, is based on the oxidation of phenol by peroxidase and coupling of this reaction to the reduction of a tetrazolium salt, with the deposition of an insoluble formazan at sites of enzyme activity. This new method was compared with an established diaminobenzidine (DAB) technique for peroxidase histochemistry and immunohistochemistry. Although both methods identified peroxidase activity in myeloid cells of bone marrow biopsy specimens, there was no interference from red cell pseudoperoxidase activity with the phenol-tetrazolium method, in contrast to the diaminobenzidine method. The detection of cytokeratin using an indirect immunoperoxidase technique was compared with both methods for demonstrating peroxidase activity. The phenol-tetrazolium method gave results similar to that obtained with DAB and appeared to be at least as sensitive as DAB in detecting low amounts of antigen. In addition, the production of a formazan as the final reaction product means that the phenol-tetrazolium method is ideally suited for quantitative peroxidase histochemistry. Therefore, the phenol-tetrazolium method represents a useful alternative method to DAB and for certain applications offers significant advantages over DAB.

Bone Marrow↗