Reduction of tetrazolium salts by sulfate-reducing bacteria

Abstract The reduction of tetrazolium salts by the sulfate-reducing bacteria, Desulfovibrio desulfuricans and Desulfotomaculum orientis , was examined. D. desulfuricans and D. orientis reduced triphenyltetrazolium chloride (TTC) and 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyltetrazolium chloride (INT) forming intracellular formazan deposits. The reduction rate of INT was higher than that of TTC. INT reduction was not inhibited by the addition of sulfate or molybdate, and sulfate uptake was inhibited by the addition of both INT and molybdate. The ratio of intracellular formazan forming cells to acridine orange direct counts in both strains decreased with culture age and starvation time.     

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.     

An investigation of staining methods to determine total cell numbers and the number of respiring micro-organisms in samples obtained from the field and the laboratory

Abstract Dyes were evaluated in combination with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to enable total cell numbers and the numbers of respiring cells to be determined on the same preparation. Malachite green and 4',6-diamidino-2-phenylindole (DAPI) were unsuitable counter-stains. Cells which contained INT formazan crystals could be stained with ethidium bromide or auramine. At high concentrations of INT formazan, auramine fluorescence was reduced, although this effect was partially rectified by prior fixation with glutaraldehyde. Staining with ethidium bromide produced a strong fluorescence in cells containing crystals of INT formazan. This observation was developed into a procedure which allowed total cells to be determined and provided a useful estimate of the number of respiring cells in samples obtained from the laboratory and the field.     

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.     

The role of glycerol in the nutrition of halophilic archaeal communities: a study of respiratory electron transport

Abstract Respiratory electron transport activity in the Dead Sea and saltern crystallizer ponds, hypersaline environments inhabited by dense communities of halophilic archaea and unicellular green algae of the genus Dunaliella , was assayed by measuring reduction of 2-( p -iodophenyl)-3( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan. Typical rates obtained were in the order of 5.5–17.7 nmol INT reduced h ⁻¹ per 10⁶ cells at 35 ° C. In Dead Sea water samples, respiratory activity was stimulated more than two-fold by addition of glycerol, but not by any of the other carbon compounds tested, including sugars, organic acids, and amino acids, or by addition of inorganic nutrients. Stimulation by glycerol had a half-saturation constant of 0.75 μM. A similar respiratory activity was also found when Dead Sea water samples were diluted with distilled water and incubated in the light. As Dunaliella cells did not reduce INT, it is suggested that photosynthetically produced glycerol leaking from the algae is the preferred carbon and energy source for the development of halophilic archaea in hypersaline environments. In samples from saltern crystallizer pond stimulation of INT reduction by glycerol was much less pronounced, probably because the community was less severely carbon-limited.     

A simplified dehydrogenase enzyme assay in contaminated sediment using 2-(p-Iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride

2-(p-Iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) accepts electrons from dehydrogenase enzymes and is reduced to a red-colored formazan (INTF), which can be quantified by colorimetric analysis. Use of previously published methods for this technique was unsuccessful due to background chemical reactions from high levels of polycyclic aromatic hydrocarbons (PAHs) and metals in the sediments. A modified method using acetonitrile extraction of the INTF was efficient and did not chemically reduce INT. This activity method is simple, quick, inexpensive and precise.     

Estimation of Frankia growth using Bradford protein and INT reduction activity estimations: application to inoculum standardization

Abstract The growth of Frankia spp. strain ORS 020607 in BAP medium was studied by using two methods simultaneously: determination of Bradford protein content and INT (2-( p -iodophenyl-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride ) reduction activity (IRA). With the latter test, red formazan crystals formed intracellularly were extracted with methanol. Colouration intensity was estimated by absorbance spectrophotometry at 490 nm. The protein content and IRA of the culture were monitored for 96 days. IRA appeared to reflect the 'metabolically active' biomass of Frankia more accurately than the Bradford protein estimations.     

A note on the physiological state of epiphytic bacteria compared with planktonic bacteria in two organically-enriched watercourses

Epiphytic bacteria were compared with planktonic bacteria in two watercourses enriched with sewage works effluent. Colony-forming units as a percentage of acridine orange direct counts were higher for epiphytic bacteria in one watercourse. The percentage of cells capable of intracellular reduction of 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan was higher for epiphytic bacteria in both watercourses. The greater viability of epiphytic over planktonic bacteria was less evident, however, than in previously studied non-enriched gravel pit ponds. This suggests that the greater availability of dissolved organic nutrients in the enriched watercourses reduced stress on planktonic bacteria.     

Tetrazolium reduction in Saccharomyces cerevisiae

Summary The tetrazolium salt, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) was used to determine viable respiring cells in batch cultures of Saccharomyces cerevisiae. Respiring cells reduce INT to water insoluble iodonitrotetrazolium formazan (INT-formazan) which is deposited within the respiring cell. The INT-formazan granules can be observed by brightfield microscopy. This allows a rapid quantitative determination of the percentage of respiring cells and total cells within the same microscopic field.In actively growing batch cultures of S. cerevisiae, the respiring cell count was equal to the total cell count for the first 72 h of the growth cycle. After 144 h of incubation only 22.7% of the total cell numbers were actively respiring.     

Epiphytic bacteria: development of a method for determining respiring bacteria on leaves

A method was developed for studying the total numbers and the proportion of active bacteria on leaf surfaces. It involves staining gelatin impressions of leaves treated with an electron transport system indicator, 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT). The method is rapid, inexpensive and allows simultaneous observation of numbers, ecology and respiratory activity of epiphytic bacteria. The total numbers of epiphytic bacteria for four species of aquatic plants varied between 0.6 to 10.2 times 10⁶/cm². The proportion of active bacteria on leaves of aquatic plants ranged from 2.2 to 42.9%. The method was also applied to a comparison of surface fouling of glass slides and aquatic leaf surfaces, indicating significant differences in numbers of bacteria but little difference in the proportion active on the two surfaces.     

Measurement of Electron Transport System (ETS) activity in soil

Electron transport system (ETS) activity was measured in amended and nonamended soil by measuring the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium formazan (INT-formazan), which can be easily extracted with methanol without interference from other compounds found in soil. A high correlation between ETS activity and oxygen consumption was observed. This technique allows rapid quantitative measurements of microbial ETS activity in soil.     

A modified method for studies of electron transport system activity in freshwater sediments

A method for measuring respiratory electron transport system activity (ETSA) in freshwater sediments is presented. It is a modification of the methods used for marine sediments by Christensen & Packard (1977) and Olànczuk-Neyman & Vosjan (1977). The assay is based on the reduction of the electron acceptor 2-(p iodophenyl) 3 (p nitrophenyl) 5 phenyl tetrazolium chloride (INT) by cell-free homogenates of sediment samples. This study shows that the method is sensitive, has good accuracy and can be used for freshwater sediments. The accuracy decreases at high turbidity and as yet has only been tested for sediments of the gyttja type. Untreated samples can be stored at low temperature for several weeks without any loss of activity. ETSA decreases with the depth of sediment and with increased amount of allochthonous material in the sediment.     

Physiological state of epiphytic bacteria on submerged stems of the reed Phragmites australis compared with planktonic bacteria in gravel-pit ponds

Suspensions of epiphytic bacteria from submerged stems of Phragmites australis , collected from a gravel-pit pond, were prepared by treatment in a stomacher and by brushing. The two procedures were equally successful in dislodging bacteria. These epiphytic bacteria were compared with planktonic bacteria in water samples from within the reed bed. Colony-forming units (cfu) as a percentage of acridine-orange direct counts (AODCs), percentage of cells capable of intracellular reduction of 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, percentage of cells able to form microcolonies, and cell length were all greater for epiphytic bacteria. Epiphytic bacteria of P. australis from six further gravel-pit ponds were also compared with bacterioplankton; cfu as a percentage of AODCs, and percentage of cells capable of INT reduction were again greater for epiphytic bacteria. Because the epiphytic bacteria in these non-organically-enriched, gravel-pit ponds were physiologically different from the planktonic bacteria it is suggested that there was not continual casual exchange, by largely identical bacterial cells, between the epiphytic and planktonic mode. Instead, epiphytic and planktonic populations were independent of each other and/or if there was exchange then bacteria were more successful while in the attached mode, perhaps because of greater organic-nutrient availability at the stem surface.     

Tetrazolium staining by optical scanning overestimates colony size and number of colonies counted

We measured the effect that staining with 2-(P-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) had on the number and size distribution of tumor colonies counted using an optical image analyzer (FAS II). Staining increased the number of tumor colonies counted. By using opaque tumor cells or pigmented melanoma cells and measuring colony growth kinetics, we demonstrated that the use of INT staining to assist in counting tumor colonies artificially increased the size of viable tumor cell aggregates by adding a red precipitate to the outside surface of the cells. Laboratories that are using the INT method for drug screening are probably measuring colonies down to and below 42 microns in diameter. These small colonies could result from as few as one or two divisions. Thus, potentially useful drugs may be missed in the screen because of the presence of abortive colonies: i.e., lethally damaged cells completing only one or two divisions.     

An NADH-tetrazolium-coupled sensitive assay for malate dehydrogenase in mitochondria and crude tissue homogenates

A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 microg protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application.     

Two short, daily activity bouts vs. one long bout: are health and fitness improvements similar over twelve and twenty-four weeks?

This study sought to determine whether a 12-week intermittent (INT; 2 x 15 min.d(-1)) exercise program yielded similar improvements in cardiovascular health and fitness, compared with a traditional 12-week, 30-minute continuous (CON; 1 x 30 min.d(-1)) exercise program. A second purpose was to determine the effects of switching exercise programs and continuing training for an additional 12 weeks. Twenty women and 17 men, (age 48.8 +/- 9.0 years) were divided randomly into 2 groups: INT (n = 20) and CON (n = 17). Aerobic exercise was performed 4 d.wk(-1) for 12 weeks. Subjects then crossed over to the opposite training program for an additional 12 weeks of training. Subjects exercised incrementally for weeks 1-4 and training was conducted at 70-80% heart rate reserve for weeks 5-24. Both groups showed comparable exercise adherence, completing 96.6 +/- 12.2% (CON) and 96.3% +/- 17.7% (INT) of the prescribed exercise time. The INT walked at a lower percentage of Vo(2)max, maximum heart rate, systolic blood pressure, and diastolic blood pressure (p < 0.05). Maximal oxygen consumption increased by 4.5% in CON and by 8.7% in INT. Following the second 12 weeks, Vo(2)max increased by 3.6 and 7.7% in CON and INT, respectively. Treadmill test time increased by 41 seconds in CON (p < 0.05) and 71 seconds in INT (p < 0.05) after 12 weeks of training. High-density lipoproteins significantly increased in the INT group following the first 12 weeks of training. This study suggests that an INT exercise program, which is incremental in nature, provides comparable, and in some cases greater, health and fitness benefits than those expected following traditional CON exercise training.     

Enzymatic cycling assay for D-carnitine determination.

An enzymatic method for d-carnitine determination using the enzyme d-carnitine dehydrogenase is described. The assay is based on the amplified signal produced during NAD(+) cycling in the presence of a tetrazolium salt and using phenazine methosulfate as electron carrier. Optimum assay conditions were studied with two tetrazolium salt pairs: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT)/MTT-formazan and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT)/INT-formazan. The first pair (MTT) showed higher sensitivity. The calibration curve was linear from 0.1 to 5 mM d-carnitine, with a quantification limit of 0.1 mM and a relative standard deviation of 1.51%. The procedure is simple, rapid, accurate, and easily automated. It was satisfactorily applied to following d-carnitine levels during the microbial transformation of d-carnitine into l-carnitine and to determining the d-carnitine content of pharmaceutical preparations.     

Comparative metabolism of spermatozoa from subfertile Delaware and Wyandotte roosters

Sperm metabolism was determined via reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to formazan. When the reaction mixture contained cyanide, which blocks cytochrome oxidase and thus maximizes intermediate electron transfer to INT, and calcium which stimulates fowl sperm motility, the metabolic capacity of spermatozoa from subfertile Delaware and Wyandotte roosters was 90 and 63% of that of spermatozoa from fertile Leghorn roosters. When the assay was performed at 40 degrees C without calcium or cyanide, no difference in metabolism was observed between Delaware and Leghorn spermatozoa (P greater than 0.05). However, the metabolism of Wyandotte spermatozoa was 66% of that observed with Delaware or Leghorn spermatozoa. These results provide further evidence that heritable subfertility in Delaware and Wyandotte roosters is attributable to distinct sperm defects.     

Adaptation of the tetrazolium reduction test for the measurement of the electron transport system (ETS) activity during embryonic development of medaka

The quantitative electron transport system (ETS)-assay based on tetrazolium reduction has been adapted for determining the terminal ETS activity during embryonic development of the subtropical teleost fish Oryzias latipes , medaka. Homogenization with a glass potter for 1–2 min was required for the complete extraction of the ETS. Additional sonication and centrifugation had a degradatory effect on the ETS activity. The main substrate of the ETS of fish embryos was NADH. NADPH also donated electrons for the ETS but with much less intensity. The impacts of the NADH and the NADPH on the enzyme activity was not additive. Succinate was ineffective as a substrate for the ETS. NADH (1.7 mM) and NADPH (0.25 mM) in combination with 0.8 mM of the artificial electron acceptor, 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT), ensured a V _max for the ETS if the reaction mixture contained 400 μg wet weight egg ml⁻¹ of cell-free homogenate. The pH-optimum of the ETS was between pH 8.0 and 8.6. The enzyme reaction at 24°C was linear during 40 min incubation. The ETS activity increased exponentially during embryonic development. The assay could be a useful tool for detecting the effect of pollutants on the development of the respiratory enzyme system in fish during embryogenesis.     

Purification and characterization of cold-active L-glutamate dehydrogenase independent of NAD(P) and oxygen

L-Glutamate dehydrogenase (GLDH) independent of NAD(P) and oxygen was first obtained from the psychrotrophic bacterium Aeromonas sp. L101, originally isolated from the organs of salmon (Oncorhynchus keta). GLDH was purified by a series of chromatography steps on DEAE-Sepharose, Superdex 200pg, Q-Sepharose, CM-Sepharose, and Phenyl-Sepharose. The purified protein was determined to have a molecular mass of 110 kDa and a pI of 5.7. Maximum activity was obtained at 55 degrees C and pH 8.5. The activity of GLDH at 4 and 20 degrees C was 38 and 50%, respectively, of that at 50 degrees C. GLDH was coupled to cytochrome c and several redox dyes including 1-methoxy-5-methylphenazinium methylsulfate (1-Methoxy PMS), 2, 6-dichlorophenylindophenol (DCIP), 9-dimethylaminobenzo[alpha]phenoxazin-7-ium chloride (meldola's blue), 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4, 4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H tetrazolium chloride] (nitroblue tetrazolium; NBT), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H tetrazolium (INT). The presence of NAD(P) and oxygen gave no oxidation activity to GLDH. Spectroscopic profile and ICP data indicated a b-type cytochrome containing iron.