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.     

Application of a Tetrazolium Salt with a Water-Soluble Formazan as an Indicator of Viability in Respiring Bacteria

The tetrazolium salt sodium 3′-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis (4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT) was examined for use as a colorimetric indicator of viability in respiring bacteria. XTT was reduced to an orange, water-soluble formazan product by Methylosinus trichosporium OB3b, Pseudomonas putida, Escherichia coli, and Bacillus subtilis. Formazan production was proportional to live cell biomass, and XTT was reduced by all cultures in the absence of added electron-coupling agents. XTT reduction by M. trichosporium OB3b was linear over several hours and was stimulated by the presence of an exogenous substrate (methanol). Addition of cyanide to cultures incubated under oxic conditions gave an initial 10-fold increase in XTT reduction. Viability of bacteria incubated in the absence of exogenous carbon substrates was measured as XTT reduction and compared with viability estimates from plate counts. Results obtained with the two methods were generally comparable, but the XTT assay was superior when cell recovery on plates was low. Incubation of E. coli for 7 days in the absence of exogenous carbon substrates decreased viability by 90%, whereas the corresponding decreases for cultures of M. trichosporium OB3b, P. putida, and B. subtilis were less than 40%.     

检测人成纤维细胞增殖的XTT比色法

利用3种不同来源的成纤维细胞(大鼠肾上皮细胞,人胎肺成纤维细胞,人成纤维细胞)的活细胞线粒体脱氢酶在电子偶联剂硫酸酚嗪甲酯（phenazine methosulfate, PMS）的协同作用下,还原四氮唑复合物(XTT)形成可溶性的棕黄色甲簪（formazan）产物,测定细胞甲簪的生成量来反映细胞的生长与活性状态,并与传统的四甲基偶氮唑盐(MTT)方法作比较.结果表明,XTT方法直接测定水溶性的甲簪产物,敏感度高于MTT法,具有操作简单、快速、灵敏度高、结果准确的优点,为成纤维细胞的研究建立了新的检测方法.     

Hydrosoluble formazan XTT: its application to natural products drug discovery for Leishmania

A colorimetric method for measuring the viability of Leishmania promastigotes is described that is based on the reduction of the tetrazolium salt, XTT, to a water-soluble formazan. Values obtained by the XTT method correlated well with parasite number (r=0.965) and with methods that rely upon the reduction of MTT or MTS (r=0.96 and 0.97, respectively). The IC(50) values obtained by XTT method with amphotericin-B, miltefosine and ketoconazole were similar to those previously reported by other methods. The XTT method proved to be a reliable and convenient method for the screening of methanolic extracts from 1059 plants and was used for the bioassay-guided fractionation of the alkaloid aegeline from Sarcorhachis naranjoana.     

Proposal of protocols using D-glutamine to optimize the 2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay for indirect estimation of microbial loads in biofilms of medical importance

Due to technical problems, biofilm biomasses are difficult to be precisely determined. One reliable strategy is based on the colorimetry of formazan compounds derived from tetrazolium salt reduction. XTT presents some desirable properties that make the biofilm measurements easier. However, cells entrapped within the extracellular matrixes normally do not metabolize the tetrazolium equally, leading to underestimation of cell contents. This study evaluated the effectiveness of d-glutamine, a plerotic substrate of tricarboxilic acid cycle (TAC), as inducer of XTT reduction. The metabolic activities of aerobic and anaerobic 48 h-old monospecific biofilms of Pseudomonas aeruginosa ATCC®27853™, Klebsiella pneumoniae ATCC®13883™, Staphylococcus epidermidis ATCC®12228™, Streptococcus mutans ATCC®25175™, and Candida albicans SC5314 were evaluated. Results showed that d-glutamine 50 mM (for P. aeruginosa, K. pneumoniae, and S. epidermidis) and 25 mM (for S. mutans and C. albicans) may enhance the detection of soluble formazan in a significant manner, what becomes the XTT reduction assay more robust.     

Potential of a soluble tetrazolium/formazan assay for the evaluation of filarial viability

Using female Acanthocheilonema viteae we have investigated the bioreduction of the tetrazolium reagent XTT (2,3-bis(2-methoxy-4-nitro-sulphonyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide). Unlike the formazan formed by other tetrazolium salts, that derived from XTT readily diffuses out of A. viteae in vitro. Formazan formation can therefore be quantified by direct absorbance reading of the incubation medium, eliminating the need for a DMSO solubilization step. Optimum assay conditions involved a 4 h incubation, in the presence of the electron coupling agent phenazine methosulphate (PMS). Repeat 4 h incubations with XTT-PMS were well tolerated by worms for 5 consecutive days. This confirmed the low toxicity of XTT formazan and its usefulness in the semi-continuous assessment of filarial viability. In comparison to our previously reported MTT (3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)-reduction assay XTT-PMS reduction showed comparable drug sensitivity and accuracy, however XTT-PMS appears to be at least 10-15 times less efficiently reduced by A. viteae females. A possible application of the XTT assay using female Onchocerca volvulus is discussed.     

An Evaluation of Three New-Generation Tetrazolium Salts for the Measurement of Respiratory Activity in Activated Sludge Microorganisms

XTT (3′-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), and WST–1 (4-(3-4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzenedisulfonate) are tetrazolium salts that have become commercially available only in relatively recent years; they differ from earlier such compounds in that their reduction gives rise to a formazan product that is water soluble. We have established the sites in the prokaryotic respiratory chain at which each of the dyes is reduced to its corresponding formazan and have evaluated the suitability of each for the colorimetric estimation of electron transport system activity in populations of activated sludge microorganisms. Reduction of all three tetrazolium salts was shown to be proportional to cell biomass and oxygen uptake and to be susceptible to low levels of the reference toxicant 3,5-dichlorophenol. XTT, which was not inhibitory at concentrations of up to 2 mM and was reduced by 91% of isolates from a sample of culturable activated sludge bacteria, was chosen for further assay development. XTT-formazan production was found to be stimulated by the availability of an exogenous carbon and energy source, and by the presence of the electron-coupling agent phenazine methosulfate. Less than 3% of XTT reduction by an activated sludge sample was abiotic. An assay based on this compound could be a valuable and simple tool for the routine monitoring of the performance of wastewater treatment systems.     

Comparison of three assays for the quantification of Candida biomass in suspension and CDC reactor grown biofilms

A common assay to measure yeast metabolic activity in biofilms is based on the reduction of the tetrazolium salt XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} to a colored formazan. However, a recent report, also confirmed by our own findings about the shortcomings of the chromogenic XTT assay, has prompted us to investigate alternative methods for yeast biomass quantification. To this end, two fluorogenic assays using fluorescein diacetate (FDA) and SYTO 9 as well as the XTT assay were comparatively evaluated with regard to the linear range of Candida albicans and Candida parapsilosis cell number-response curves, precision and intra- and interspecies variability. Reading of fluorescence and absorbance was carried out in a multilabel microtiter plate reader. All three assays were adequate for the determination of planktonic yeast biomass, but the FDA and SYTO 9 assays present practical advantages. When applied to the quantification of yeast biofilm biomass obtained in the CDC biofilm reactor, the FDA assay proved superior.     

A quantitative colorimetric method to evaluate the functional state of human polymorphonuclear leukocytes

The colorimetric assay previously described by Mosmann for the measurement of cell viability and proliferation has been modified for the assessment of the functional state of human polymorphnuclear cells (PMNs). The ability of PMNs to reduce the tetrazolium salt MTT to formazan reflects directly the degree of stimulation induced by various agents. The underlying mechanism of MTT-reduction to formazan seems to be similar to that of nitroblue tetrazolium (NBT)-reduction. In contrast to the NBT-reduction assay, the formazan produced from MTT can easily be measured by an ELISA reader. Parallel experiments revealed a qualitative correlation between the concentration of formazan produced from MTT and the concentration of cytochrome C reduced by PMNs. Although oxidative burst may not be the actual lytic mechanism in cellular cytotoxicity of PMN, we also observed an association between MTT-reduction capacity and the cytotoxic activity of PMNs from normal donors in antibody dependent cellular cytotoxicity. Our results indicate that the MTT-reduction assay can be employed to estimate the functional state of polymorphnuclear granulocytes.     

Biological responses of Bacillus stratosphericus to Floating Electrode‐Dielectric Barrier Discharge Plasma Treatment

Aims: Dielectric barrier discharge (DBD) plasma is used for sterilization of contaminated inanimate surfaces but seldomly optimized and depends upon the type of organisms and the plasma treatment duration, (net energy deposited) this efficacy varies. The proposed study was designed to see biological responses of one of the robust organism, Bacillus stratosphericus. Methods and Results: DBD plasma was applied over various durations to B. stratosphericus either surface‐dried or suspension in de‐ionized water, and viability, culturability, and viable but nonculturability (VBNC) were assayed using standard techniques. Depending upon the exposure of B. stratosphericus to DBD plasma resulted in three viability states, viable and culturable at low plasma doses and VBNC or disintegrated bacteria at higher plasma doses. Although organism’s respiration levels at relatively low levels, immediately after plasma treatment, over the course of 24‐ h respiratory activity was increased c. eight times (and found still nonculturable during colony assays). Conclusions: The loss of culturability is hypothesized to be induced as one of the responses to oxidative stress and it remains to be unclear if the response is temporary or indefinite. Appropriate plasma powers should be used to avoid VBNC‐like status. 2,3‐Bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium‐5‐carboxanilide (XTT) assay is a good alternative method to detect VBNC state. Significance and Impact of the Study: Bacillus stratosphericus has the potential to turn into VBNC upon plasma application, and XTT assay can be an alternative method to detect VBNC state.     

Colorimetric cell proliferation assay for microorganisms in microtiter plate using water-soluble tetrazolium salts

A colorimetric method to assay cell proliferation of microorganisms in 96-well microtiter plates using water-soluble tetrazolium salts and electron mediators was developed. Combinations of 6 kinds of water-soluble tetrazolium salts and 27 kinds of electron mediators that considered the metabolic efficiency of microorganisms and the influence with medium components were investigated. 2-Methyl-1,4-naphthoquinone (NQ) was reduced most effectively by various species of microorganisms, and a combination of WST-8 as a water-soluble tetrazolium salt with 2-methyl-1,4-NQ repressed the increase in background due to medium components. In the presence of 2-methyl-1,4-NQ, WST-8 was reduced by microbial cells to formazan, which exhibited maximum absorbance at 460 nm. The proposed tetrazolium method could be applied to measure proliferations of various microbial cells including 3 kinds of yeast, 9 kinds of Gram-positive bacteria, and 10 kinds of Gram-negative bacteria. Linear relationships between the absorbance and viable microbial cell density were obtained in all microorganisms, suggesting that the absorbance change reflected the microbial cell proliferation.     

A colorimetric assay for determination of cell viability in algal cultures

In this work, we propose the determination of cell viability in algal cultures by using a colorimetric assay widely used for estimation of cell proliferation in animal cell cultures. The method is based on in vivo reduction by metabolically active cells of a tetrazolium compound (MTS=3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt) to a colored formazan, with maximal absorbance at 490 nm, that is released to the culture medium. For this purpose, we have tested two microalgae with high commercial value (Dunaliella and Spirulina) and two seaweeds with different morphology (Ulva and Gracilaria). Color development in this assay is directly proportional to the number of viable cells, to the incubation time in the presence of the assay solution, and to the incubation temperature. A direct significant correlation was found between algal photosynthesis rate and color development in all species used through this work. Moreover, the intensity of absorbance at 490 nm was significantly lower in stressed cells (e.g. in nutrient-limited cultures, in the presence of toxic substances, and in osmotically-stressed cultures). We conclude that cell viability of algal cultures can be rapidly and easily estimated through colorimetric determination of the reduction of MTS to formazan.     

A quantitative real-time RT-PCR assay for mature C. albicans biofilms

Background  

Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high C. albicans cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR) that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells.     

Microplate assay for detection of bacterial contamination in tissue culture media

In the present study, a rapid and simple colorimetric technique has been described to determine the presence of bacteria in tissue culture medium used in animal cell culture. The microplate assay is based on utilization of MTT [3(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] by bacteria resulting in formation of formazan crystals which can be measured colorimetrically. Contaminated medium, a standard gram-negative and gram-positive bacteria produce formazan from MTT which is related to the bacterial load. The assay has utility in screening tissue culture reagents to detect the presence of bacteria.     

Use of the tetrazolium salt MTT to measure cell viability effects of the bacterial antagonist Lysobacter enzymogenes on the filamentous fungus Cryphonectria parasitica

Despite substantial interest investigating bacterial mechanisms of fungal growth inhibition, there are few methods available that quantify fungal cell death during direct interactions with bacteria. Here we describe an in vitro cell suspension assay using the tetrazolium salt MTT as a viability stain to assess direct effects of the bacterial antagonist Lysobacter enzymogenes on hyphal cells of the filamentous fungus Cryphonectria parasitica. The effects of bacterial cell density, fungal age and the physiological state of fungal mycelia on fungal cell viability were evaluated. As expected, increased bacterial cell density correlated with reduced fungal cell viability over time. Bacterial effects on fungal cell viability were influenced by both age and physiological state of the fungal mycelium. Cells obtained from 1-week-old mycelia lost viability faster compared with those from 2-week-old mycelia. Likewise, hyphal cells obtained from the lower layer of the mycelial pellicle lost viability more quickly compared with cells from the upper layer of the mycelial pellicle. Fungal cell viability was compared between interactions with L. enzymogenes wildtype strain C3 and a mutant strain, DCA, which was previously demonstrated to lack in vitro antifungal activity. Addition of antibiotics eliminated contributions to MTT-formazan production by bacterial cells, but not by fungal cells, demonstrating that mutant strain DCA had lost complete capacity to reduce fungal cell viability. These results indicate this cell suspension assay can be used to quantify bacterial effects on fungal cells, thus providing a reliable method to differentiate strains during bacterial/fungal interactions.     

Eradication of Pseudomonas aeruginosa Biofilms by Atmospheric Pressure Non-Thermal Plasma

Bacteria exist, in most environments, as complex, organised communities of sessile cells embedded within a matrix of self-produced, hydrated extracellular polymeric substances known as biofilms. Bacterial biofilms represent a ubiquitous and predominant cause of both chronic infections and infections associated with the use of indwelling medical devices such as catheters and prostheses. Such infections typically exhibit significantly enhanced tolerance to antimicrobial, biocidal and immunological challenge. This renders them difficult, sometimes impossible, to treat using conventional chemotherapeutic agents. Effective alternative approaches for prevention and eradication of biofilm associated chronic and device-associated infections are therefore urgently required. Atmospheric pressure non-thermal plasmas are gaining increasing attention as a potential approach for the eradication and control of bacterial infection and contamination. To date, however, the majority of studies have been conducted with reference to planktonic bacteria and rather less attention has been directed towards bacteria in the biofilm mode of growth. In this study, the activity of a kilohertz-driven atmospheric pressure non-thermal plasma jet, operated in a helium oxygen mixture, against Pseudomonas aeruginosa in vitro biofilms was evaluated. Pseudomonas aeruginosa biofilms exhibit marked susceptibility to exposure of the plasma jet effluent, following even relatively short (～10's s) exposure times. Manipulation of plasma operating conditions, for example, plasma operating frequency, had a significant effect on the bacterial inactivation rate. Survival curves exhibit a rapid decline in the number of surviving cells in the first 60 seconds followed by slower rate of cell number reduction. Excellent anti-biofilm activity of the plasma jet was also demonstrated by both confocal scanning laser microscopy and metabolism of the tetrazolium salt, XTT, a measure of bactericidal activity.     

Is reduction of the sulfonated tetrazolium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide a reliable measure of intracellular superoxide production?

The sulfonated tetrazolium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide (XTT) is advantageous in that it yields a water-soluble formazan, unlike most other available tetrazoliums. XTT is reducible by superoxide, as are other tetrazoliums, but is not directly reduced by xanthine oxidase plus xanthine or by glucose oxidase plus glucose. This led to the suggestion that XTT reduction might serve as a reliable index of intracellular O(2)(-) production. We now show that soluble extracts of Escherichia coli contain two NADPH:XTT reductases that act aerobically or anaerobically. That being the case, XTT reduction is not a reliable measure of intracellular O(2)(-).     

Evaluation of a tetrazolium salt test to determine absence of live mycobacteria in tuberculin purified protein derivative.

Current methodology to determine absence of live mycobacteria in tuberculin purified protein derivative (PPD) takes up to 8 weeks to perform and may also involve testing on animals. In this paper we describe an in vitro test utilising the tetrazolium salt, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) to monitor the absence of live Mycobacterium tuberculosis (Mtb) in PPD. In the presence of live cells XTT is converted to a coloured formazan product that can be measured spectrophotometrically. Live mycobacteria present in spiked PPD were detected by a marked change in optical density above background levels. This test is easy to perform and is complete in just 48 hr.     

The use of the tetrazolium salt XTT for the estimation of biological activity of activated sludge cultivated under steady-state and transient regimes

The tetrazolium salt 3′-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis (4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) was used as a tool for estimating the activity of the electron transport system (ETS) in activated sludge cultivated under steady-state and transient regimes in chemostat culture. Production of formazan by reduction of XTT depended on the initial concentration of the XTT following a saturation law and was proportional to live cell biomass. Addition of cyanide (KCN) to activated sludge gave an initial 1.5-fold increase in XTT reduction, while addition of 3,5-dichlorophenol (3,5-DCP) reduced this value drastically. At steady-state and transient regimes of an activated sludge chemostat, oxygen uptake rate (OUR) and XTT reduction rate were highly correlated and indicated significant variations depending on the growth conditions.     

Colorimetric quantitation of filarial viability

A simple three-step colorimetric assay based on the tetrazolium salt MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) has been developed for quantifying filarial viability. Living (but not dead) filariae take up MTT and rapidly reduce it to formazan, so staining themselves dark blue. This colour change which is easily seen provides a rapid qualitative test for filarial viability. Quantitative data can be obtained by solubilizing formazan out of the worm with DMSO and measuring the absorbance of the resulting solution at 510 nm. To date the technique has been demonstrated in several species of filariae including Onchocerca volvulus. MTT reduction is thought to be selective for NADH-dependent dehydrogenase activity in viable worms. The reaction occurs readily in all developmental stages of Dipetalonema viteae including fragments of filarial tissue. Enzyme activity in viable intact D. viteae appears to be primarily associated with the hypodermis/muscle cells, with minimal formazan formation in the gut and reproductive tracts. The application of this MTT assay as a parameter for quantifying in vitro drugs effects is described. Assay procedures have been developed and optimized with D. viteae and Brugia pahangi for the assessment of effects of macrofilariae and microfilarial release, and the activity of a range of antifilarial standards reported. Several potential applications of the technique to studies on filarial biology are discussed.