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Microencapsulation with various materials has been used as an efficient method to improve the viability of probiotic bacteria in multiple food products and the human gastrointestinal tract. Although plate count agar is the most commonly used method for evaluating the viability of encapsulated bacteria, it is still far from providing reliable information about the intermediate state between viable and dead bacteria. This study optimized a tetrazolium salt-based colorimetric method for the detection of viable but non-culturable state within encapsulated Lactobacillus rhamnosus and Lactobacillus plantarum probiotic strains. The viability of encapsulated bacteria was assessed after different spray-drying conditions and also during two months of storage at room temperature. The ability to reduce tetrazolium salts of two lactic acid bacteria was verified and calibrated according to the experimental conditions (strains, incubation time, and microencapsulation material). The loss of bacterial cultivability was species-specific and more problematic throughout the processing than during the storage period. An outlet temperature of 73-75 °C yielded a higher viable but non-culturable state level than at 68-69 °C, especially in maltodextrin and trehalose powders. Whey protein was statistically the best carrier in preserving viable and culturable encapsulated bacteria after spray-drying and during storage, as compared to sugar-based carriers. The tetrazolium-optimized method was more sensitive and accurate for the evaluation of viable bacteria in microcapsules as compared to the conventional plate count methods available. It showed the high variability of CFU counts on Man–Rogosa–Sharpe (MRS) agar. This colorimetric technique could be considered a real-time, simple, cost-effective, and reliable alternative to culture-based methods in evaluating probiotic microencapsulation efficiency.

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Tamarind (Tamarindus indica) is a fruit tree of African origin cultivated in more than 50 countries, including Colombia, mainly in the Colombian Caribbean departments, where its harvest generates significant income in rural communities. Its establishment presents a difficulty because the sexual seed presents physical dormancy and poor germination. The objective of this study was to evaluate Tamarind seed viability by using the tetrazolium test and to reduce the time needed for the determination of its physiological quality. Ripe and healthy fruits were collected from patio trees, from which their seeds were extracted, disinfected with 1% sodium hypochlorite, and washed with plenty of water before use. Subsequently, they were scarified with No. 100 sandpaper by the edges, except in the area of the micropyle. The completely random design was used in a 3x3 bifactorial arrangement, with four repetitions. The first factor, tetrazolium concentration (%), was tested at levels of 0.50, 0.75 and 1.00%, and the second factor, immersion time, at 2, 4, and 6 hours. The analysis of variance did not show significance for the tetrazolium concentration, while it did for the immersion time (p≤0.01), and the tetrazolium-time interaction (p≤0.05). Therefore, the concentration of 0.50% tetrazolium for six hours of immersion is a reliable alternative to determine the physiological quality of tamarind seeds compared to conventional germination testing due to its low cost and execution time. Likewise, the percentages of germination and germination speed index were higher when the seed was scarified with sandpaper than the conventional method.

To ensure clean drinking water, viable pathogens in water must be rapidly and efficiently screened. The traditional culture or spread-plate process—the conventional standard for bacterial detection—is laborious, time-consuming, and unsuitable for rapid detection. Therefore, we developed a colorimetric assay for rapid microorganism detection using a metabolism-based approach. The reaction between a viable microorganism and the combination of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-8) and 1-methoxy-5-methylphenazinium methyl sulfate (mPMS) results in a color change. In combination with a microplate reader, WST-8-mPMS reactivity was leveraged to develop a colorimetric assay for the rapid detection of various bacteria. The detection limit of the WST-8-mPMS assay for both gram-negative and gram-positive bacteria was evaluated. This WST-8-mPMS assay can be used to perform colorimetrical semi-quantitative detection of various bacterial strains in buffers or culture media within 1 h without incubation before the reaction. The easy-to-use, robust, rapid, and sensitive nature of this novel assay demonstrates its potential for practical and medical use for microorganism detection. [ABSTRACT FROM AUTHOR]

Background and Aims: Survival of periodontal ligament (PDL) cells after avulsion is an important factor in treatment prognosis. Grapefruit Seed Extract (GSE) can be a proper environment for preserving periodontal ligament cells. The objective of this study was to analyze the effect of different concentrations of GSE on the proliferation of fibroblast PDL cells. Materials and Methods: In this study, the undifferentiated PDL fibroblasts were obtained from two human premolars teeth and cultured in Dulbecco’s modified Eagle’s medium (DMEM). The cultured cells were exposed to different concentrations of GSE. The positive and negative control groups were cultured in fetal bovine serum (FBS) 10% and in a medium without FBS 10%, respectively. The plates were incubated for 1, 6, 12, 24, and 48 hrs. The PDL cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Statistical analysis of data was accomplished using repeated measure ANOVA with Post HOC Tukey, P0.05). Furthermore, all the samples were similar to the positive control group in three of the five timeperiods (P>0.05). Conclusion: GSE was more effective in fewer concentration and longer periods and it had no toxic effect on PDL cells. Therefore, GSE can be considred as a promoting medium in PDL regeneration of avulsed permanent teeth in the future.

The ethanol extracts of 155 different foodstuffs containing medicinal plants were investigated for their biofilm eradication activities against pathogenic bacteria. A combined method of a colorimetric microbial viability assay based on reduction of a tetrazolium salt (WST-8) and a biofilm formation technique on the 96-pins of a microtiter plate lid was used to screen the biofilm eradication activities of foodstuffs. The ethanol extracts of licorice (Glycyrrhiza glabra) showed potent biofilm eradication activities against Streptococcus mutans, Staphylococcus aureus, and Porphyromonas gingivalis. Among the antimicrobial constituents in licorice, glabridin had the most potent eradication activities against microbial biofilms. The minimum biofilm eradication concentration of glabridin was 25–50 μg/ml. Furthermore, the combination of glabridin with ɛ-poly-l-lysine, a food additive, could result in broad biofilm eradication activities towards a wide variety of bacteria associated with infection, including Escherichia coli and Pseudomonas aeruginosa. [ABSTRACT FROM AUTHOR]

In this work, a method for the measurement of one-dimensional (1D) UV radiation dose is described. It comprises a new tablet dosimeter that measures the dose using reflectance spectrophotometry. The tablet dosimeter elaborated is a solid structure with a cylindrical form and has been manufactured with polycaprolactone (PCL) doped with a representative of tetrazolium salts: 2,3,5−triphenyltetrazolium chloride (TTC). The PCL used makes the dosimeter biodegradable and therefore proecological. The TTC dopant is distributed uniformly in the whole PCL tablet, and the whole tablet changes color to red under UVC irradiation. The intensity of this color increases if the PCL–TTC tablet absorbs higher doses. The color of the tablet is stable for at least 30 days after irradiation. It is proposed that the PCL-TTC tablet be used for measurement with reflectance spectrophotometry in order to determine the reflectance of light versus the absorbed dose in a fast and easy manner. On this basis, the PCL-TTC tablet could be characterized by providing information on its dose range, which amounted to 0–2 J/cm2. Moreover, other parameters were derived, such as dose sensitivity, quasilinear dose range, and dose threshold. The morphology of the tablets studied using scanning electron microscopy revealed their high porosity, which however did not influence the reflectance measurements with the aid of the chosen instrument. UVC irradiation at a dose (15 J/cm2) much above the PCL-TTC tablets’ dose range did not alter the morphology of the tablets. The PCL-TTC tablet read with reflectance spectrophotometry is shown to be a promising and fast method for 1D UV dose measurements.

To ensure clean drinking water, viable pathogens in water must be rapidly and efficiently screened. The traditional culture or spread-plate process—the conventional standard for bacterial detection—is laborious, time-consuming, and unsuitable for rapid detection. Therefore, we developed a colorimetric assay for rapid microorganism detection using a metabolism-based approach. The reaction between a viable microorganism and the combination of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-8) and 1-methoxy-5-methylphenazinium methyl sulfate (mPMS) results in a color change. In combination with a microplate reader, WST-8-mPMS reactivity was leveraged to develop a colorimetric assay for the rapid detection of various bacteria. The detection limit of the WST-8-mPMS assay for both gram-negative and gram-positive bacteria was evaluated. This WST-8-mPMS assay can be used to perform colorimetrical semi-quantitative detection of various bacterial strains in buffers or culture media within 1 h without incubation before the reaction. The easy-to-use, robust, rapid, and sensitive nature of this novel assay demonstrates its potential for practical and medical use for microorganism detection.

Background and Aims: Survival of periodontal ligament (PDL) cells after avulsion is an important factor in treatment prognosis. Grapefruit Seed Extract (GSE) can be a proper environment for preserving periodontal ligament cells. The objective of this study was to analyze the effect of different concentrations of GSE on the proliferation of fibroblast PDL cells. Materials and Methods: In this study, the undifferentiated PDL fibroblasts were obtained from two human premolars teeth and cultured in Dulbecco’s modified Eagle’s medium (DMEM). The cultured cells were exposed to different concentrations of GSE. The positive and negative control groups were cultured in fetal bovine serum (FBS) 10% and in a medium without FBS 10%, respectively. The plates were incubated for 1, 6, 12, 24, and 48 hrs. The PDL cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Statistical analysis of data was accomplished using repeated measure ANOVA with Post HOC Tukey, P<0.05 was considered statistically significant. Results: We found out that among different concentrations of GSE, 1:128 had the most impact on undifferentiated PDL fibroblasts. Although, the cell vitality was higher in the twelfth hour, 1:128 GSE and in the forty-eighth hour, 1:1024 GSE than the positive control group but they were not statistically significant (P>0.05). Furthermore, all the samples were similar to the positive control group in three of the five timeperiods (P>0.05). Conclusion: GSE was more effective in fewer concentration and longer periods and it had no toxic effect on PDL cells. Therefore, GSE can be considred as a promoting medium in PDL regeneration of avulsed permanent teeth in the future. [ABSTRACT FROM AUTHOR]

Salvia hispanica L. is an annual herbaceous plant, belonging to the Lamiaceae family, it stands out as the natural resource of plant origin with the highest content of fatty acids known so far. To obtain success in the production of seeds it is necessary to use lots of high quality, which can be evaluated, through the vigor of the same, at present, one of the main requirements for the evaluation of vigor refers to the obtaining of reliable results in a relatively short period of time. The tetrazolium test stands out for being fast and reliable. However, the methodology for the genus Salvia is not referenced within the standards of the International Seed Testing Association (ISTA), considering these facts, it becomes important to carry out the experiment for the development of a protocol that allows this analysis. The research was carried out in the Seed Laboratory of the Federal University of Pelotas, six batches of black chia seeds were used and four concentrations of tetrazolium salt (0.075%, 0.1%, 0.5% and 1%) were tested to evaluate the seed viability. The experimental delineation was in randomized blocks, submitted to the analysis of variance through the F test and subsequently the means compared to each other by the Tukey test, at 5% probability, for comparison of the means. The tetrazolium test conducted in the concentration of 0.075% is efficient to evaluate the viability of the seeds of S. hispanica L., as well as to differentiate lots with different physiological quality. [ABSTRACT FROM AUTHOR]

• 2,3,5-triphenyl tetrazolium chloride (TTC) is a promising tetrazolium salt to be used as a colorimetric indicator. • TTC assay could identify antimicrobial resistance in nontuberculous mycobacteria. • TTC assay allows an easier reading of results. This study aimed to propose and evaluate a drug susceptibility testing (DST) using the 2,3,5-triphenyl tetrazolium chloride (TTC) as a colorimetric indicator against Mycobacterium abscessus complex (MABC), M. avium complex (MAC), and M. kansasii strains, main nontuberculous mycobacteria (NTM) of clinical relevance. Our results demonstrated that the assay using TTC and the broth microdilution method recommended by the Clinical and Laboratory Standards Institute had essential agreement above 91%, 92%, and 100%, for drugs tested against MABC, MAC, and M. kansasii strains, respectively. Categorical agreement above 91% was obtained for most drugs tested against MABC, except to cefoxitin (76.5%). For drugs tested against MAC and M. kansasii , categorical agreement above 92% and 100% was observed, respectively. TTC showed to be a promising colorimetric indicator of growth to be used in DST for NTM, allowing an easier reading of results. [ABSTRACT FROM AUTHOR]

Summary: Silver(I)-assisted reduction of Blue tetrazolium chloride (3,3'-(3,3'-dimethoxy[1,1'-biphenyl]-4,4'-diyl)-bis(2,5-diphenyl-2H-tetrazolium) dichloride, BTC) by semicarbazide hydrochloride (SCH) was investigated in water-chloroform medium. The obtained colored products can be used for the visual detection and sensitive liquid-liquid extraction- spectrophotometric determination of Ag(I). Under the optimum conditions the calibration curve (which can be best approximated by a third-order polynomial; R2=0.9990) has two linear segments. For Ag(I) concentrations up to 0.011 g/ml, the linear regression equation had an intercept that was statistically indistinguishable from zero. The limit of detection, limit of quantitation, and molar absorptivity coefficient at λmax=573 nm were 0.6 ng/ml, 2 ng/ml and 1.2×106 l/(molcm), respectively. The regression equation of the second linear segment (0.021- 0.028 g/ml) was A = 200γ - 3.5 (R2=0.9989), where γ is the concentration in µg/ml. The relative standard deviation at the 22 ng/ml level (n = 5) was 3.8%. The effect of concomitant ions was studied, and the analysis of real samples tested the applicability of the developed procedure. [ABSTRACT FROM AUTHOR]

A liquid-liquid extraction-chromogenic system for vanadium(IV, V) containing 2,3-dihydroxynaphtahlene (DN), 2,3,5-triphenyl-2H-tetrazolium chloride (TTC), water and chloroform was studied in detail. When the vanadium is in the oxidation state of IV, the extracted species are aggregates containing three 1:2:1 (V:DN:TTC) ion-pair units composed of triphenyltetrazolium cations (TT+) and chelate anions {[VIVO(DN)(DNH)]− (I) and/or [VIV(OH)(DN)2]− (II)}. When the initial oxidation state of vanadium is V and the DN concentration is high, vanadium(V) is reduced by DN to a lower oxidation state, V(IV). However, at low DN concentration, vanadium(V) can enter the organic phase as a part of an ion-pair consisting of TT+ and [VVO2(DN)]− (III). The ground-state equilibrium geometries of the anions I, II, and III were optimized by quantum chemical calculations using BLYP/6-31++G⋆. The following characteristics were determined under the optimum conditions for VIV extraction: absorption maximum λmax = 333 nm, molar absorptivity ε333= 2.1x104 dm3 mol−1 cm−1, Sandell’s sensitivity SS = 2.4 ng cm−2, and fraction extracted E = 98%. The conditional extraction constant was calculated by two independent methods. The calibration graph was linear in the range 0.1-3.1 μg cm−3 (R2=0.9994) and the limit of detection was 0.03 μg cm−3.

The complex formation in the vanadium(V) / 4-(2-thiazolylazo)orcinol (TAO) / 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) liquid-liquid extraction-chromogenic system was studied. The chloroform-extracted complex has a composition of 2:2:2 under the optimum conditions (pH 4.8–5.2, extraction time 3 min, concentration of TAO 3.4´10–4 mol dm–3, and concentration of TTC 9.4´10–4 mol dm–3) and could be regarded as a dimer (D) of two 1:1:1 species (S) presented by the formula (TT+)[VO2(TAO)]. The constant of extraction was calculated by two methods and some analytical characteristics were determined. The wavelength of maximum absorption (lmax), molar absorptivity (el) and fraction extracted (E) were found to be l = 545 nm, e545 = 1.97´104 dm3 mol–1 cm–1, and E = 97.9 %. The ground-state equilibrium geometries of the complexes S and D were optimized by quantum chemical Hartree-Fock calculations using 3-21G* basis functions. The bonding and interaction energies were calculated as well.

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay is a popular tool in estimating the metabolic activity of living cells. The test is based on enzymatic reduction of the lightly colored tetrazolium salt to its formazan of intense purple-blue color, which can be quantified spectrophotometrically. Under properly optimized conditions the obtained absorbance value is directly proportional to the number of living cells. Originally, the MTT assay was devised for use in eukaryotic cells lines and later applied for bacteria and fungi. As the mechanism of MTT reduction was studied in detail mostly considering eukaryotic cells, the lack of information resulted in generating a vast variety of MTT based protocols for bacterial enzymatic activity evaluation. In the presented article the main aspects of the MTT assay applicability in bacterial research were summarized, with special emphasis on sources of inaccuracies and misinterpretation of the test results. [ABSTRACT FROM AUTHOR]

The MTT assay was the first widely accepted method to assess cytotoxicity and cell viability. However, there is controversy on whether this indicator is a useful tool. In this work we intend to expand the interpretability of the MTT study by its combination with widely used cellular biology techniques. We propose complementary approaches to the colorimetric assay, based on the use of measurements in three different settings: confocal microscopy, multi-well plate assay and flow cytometry. Using confocal microscopy, we confirmed that MTT uptake and reduction by cells is a time-dependent process, and that formazan accumulates in round-shaped organelles. Quantitative measurements with a multi-well fluorimeter combined with nuclear staining result in a useful method, yielding a ratio between formazan production and cell number that informs about the average cell metabolic state. We also found that flow cytometry is a suitable technique to measure MTT reduction in large cell populations. When assaying the effect of an oxidizing agent such as paraquat (PQ), this approach allows for the distinction of subpopulations of cells with different reducing power. Finally, we prove that it is feasible to monitor MTT reduction in an in vivo model, the Drosophila larvae, without affecting its survival rate. Formazan accumulates exclusively in the larval fat body, confirming its lipid solubility. The methods explored in this work expand the MTT potential as a useful tool to provide information of the physiological state of cells and organisms. [ABSTRACT FROM AUTHOR]

There is scientific evidence that beach sands are a significant contributor to the pathogen load to which visitors are exposed. To develop beach quality guidelines all beach zones must be included in microbiological evaluations, but monitoring methods for beach sand quality are relatively longstanding, expensive, laborious and require moderate laboratory infrastructure. This paper aimed to evaluate the microorganism activity in different beach zones applying and comparing a classical method of membrane filtration (MF) with two colorimetric screening methods based on fluorescein (FDA) and tetrazolium (TTC) salt biotransformation to evaluate a new rapid and low-cost method for beach sand microbiological contamination assessments. The colorimetric results can help beach managers to evaluate rapidly and at low cost the microbiological quality of different beach zones in order to decide whether remedial actions need to be adopted to prevent exposure of the public to microbes due to beach sand and/or water contamination. [ABSTRACT FROM AUTHOR]

Exposure to air pollutants is significantly associated with health risks ranging from bronchial reactivity to morbidity and mortality. However, the precise mechanisms are not always fully understood. The aim of this study was to investigate the effects of sulphur dioxide (SO2) on cell viability and cytokine production of A549-human pulmonary epithelial cells. Test atmospheres of SO2 were generated using a direct dilution method and calibrated by ion-chromatography. Test atmospheres were delivered to lung cells cultured on porous membranes (0.4 µm) using Harvard Navicyte horizontal diffusion chamber systems. The cytotoxic endpoints were investigated using the MTS (tetrazolium salt; Promega), NRU (neutral red uptake; Sigma) and ATP (adenosine triphosphate; Promega) assays. Expression of inflammatory markers including tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) were evaluated using double-antibody immunometric assays. Dose-dependent effects of SO2 were observed in A549 cells using all in vitro assays at test concentrations (10-200 ppm). The ATP assay appeared to be the most sensitive test (IC50 = 48 ± 2.83 ppm) that may related to the impaired metabolic activity of the cells following SO2 exposure. After analysis of TNF-a, no statistically significant differences were observed between control and exposed cells. However, the IL-6 production in A549 cells was significantly reduced in a dose-dependent manner (P

Rapid and simple method for the determination of minimum biofilm eradication concentration (MBEC) using a colorimetric microbial viability assay based on reduction of a tetrazolium salt WST-8 and the biofilm formation method on 96-pegs on the lid of a microtiter plate was developed. The biofilms formed on the 96 pegs were challenged by antibiotics, and the MBEC was then determined from the microbial viability of the biofilms formed on the 96 pegs, assessed by the WST-8 colorimetric assay. The MBECs obtained by the proposed and conventional methods favorably agreed. The most effective inhibitors of S. aureus and P. aeruginosa biofilms were vancomycin and ciprofloxacin, respectively. In addition, we clarified that Staphylococcus aureus biofilm was maximally suppressed by a combination of vancomycin, daptomycin, and teicoplanin. The proposed method yields similar performance to conventional methods, but is faster and more easily implemented. Therefore, the proposed method alleviates the tediousness and time-consuming nature of conventional biofilm susceptibility assay. [ABSTRACT FROM AUTHOR]

The complex formation in the vanadium(V) / 4-(2-thiazolylazo)orcinol (TAO) / 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) liquid-liquid extraction-chromogenic system was studied. The chloroform-extracted complex has a composition of 2:2:2 under the optimum conditions (pH 4.8-5.2, extraction time 3 min, concentration of TAO 3.4 x 10-4 mol dm-3, and concentration of TTC 9.4 x 10-4 mol dm-3) and could be regarded as a dimer (D) of two 1:1:1 species (S) presented by the formula (TT+)[VO2(TAO)]. The constant of extraction was calculated by two methods and some analytical characteristics were determined. The wavelength of maximum absorption (λmax), molar absorptivity (ελ) and fraction extracted (E) were found to be λ = 545 nm, ε545 = 1.97 x 104 dm³ mol-1 cm-1, and E = 97.9%. The ground-state equilibrium geometries of the complexes S and D were optimized by quantum chemical Hartree-Fock calculations using 3-21G* basis functions. The bonding and interaction energies were calculated as well. [ABSTRACT FROM AUTHOR]

[Display omitted] • Neuro-2a cells were immobilised on electrodes of different materials. • Their viability was assessed using electrochemical methods. • Carbon and carbon/polyaniline electrodes provided the best results. • Light microscopy proved the presence of immobilised and living cells on electrodes. • The system was able to detect toxicity in fish extracts. Marine toxins are potent toxic compounds that may reach humans and poison them. Therefore, their detection in seafood is crucial to prevent intoxication cases. Colorimetric cell-based assays (CBAs) have been developed to analyse marine neurotoxins, such as ciguatoxins (CTXs) and tetrodotoxins (TTXs), and are based on the toxicological effect of these toxins on the cells. Cell viability can be quantified by measuring the mitochondrial activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). With the purpose of moving forward in the development of cell-based biosensors (CBBs) for neurotoxins, Neuro-2a cells were immobilised on electrodes of different materials (carbon, carbon/polyaniline, carbon/poly- l -lysine, carbon/poly(3,4-ethylenedioxythiophene) and gold) and their presence and viability were assessed by the detection of MTT formazan crystals with cyclic voltammetry (CV). Best results in terms of oxidation potential and current intensity were achieved with carbon and carbon/polyaniline electrodes. Light microscopy also proved the presence of immobilised and living cells on electrodes. Cell density, incubation time and MTT concentration were optimised. Appropriate electrochemical responses were obtained incubating 100,000 cells/electrode for 2 h and using 0.86 mg/mL MTT. The system was able to detect toxicity when exposed to CTX1B and TTX standard solutions as well as Seriola dumerili and Lagocephalus sceleratus fish extracts containing these toxins. [ABSTRACT FROM AUTHOR]

The possibility of application of the ternary ion-association complex of gallium(III), 4-(2-pyridyl azo)resorcinol (PAR) and 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) for extraction-spectrophotometric determination of gallium(III) was studied. The liquid-liquid extraction system Ga(III)-PAR-TTC-HO-CHCl was applied for this purpose. The effect of the foreign ions on the extraction was investigated. Based on the obtained results, a sensitive, relatively simple, and inexpensive method for determination of gallium(III) in a model mixture was developed, which can be implemented in industrial, biological, medical, and pharmaceutical samples. [ABSTRACT FROM AUTHOR]

Extracorporeal photopheresis (ECP) is accepted as a second-line therapy for the treatment of acute and chronic steroid-refractory graft versus host disease (GvHD), cutaneous T-cell lymphoma and solid organ transplantation. ECP should be validated: we compared in parallel apoptosis and proliferation analysis of patient lymphocytes treated with 8-MOP ECP using respectively Annexin V/7-aminoactinomycin D (7-AAD) and CFSE with a tetrazolium salt (WST-1) method. Using WST-1 assay we found a significant decrement (p < 0.01) of metabolic activity at 4 days between ECP-treated and untreated cells. This finding was confirmed by the significant decrease of cell proliferation and increase of cell death observed by CFSE and 7AAD-Annexin V, respectively. Accordingly, once validated against a reference method, WST-1 could represent a rapid and easy assay for routinely quality control of ECP. [ABSTRACT FROM AUTHOR]

Complex formation and liquid-liquid extraction (LLE) were studied in systems containing vanadium(V), 5-methyl-4-(2-thiazolylazo)resorcinol (TAO), tetrazolium salt (TZS), water and chloroform. The following three TZSs were used: 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 3-(2-naphtyl)-2,5-diphenyl-2H-tetrazolium chloride (Tetrazolium violet, TV), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT). Concentration of the reagents (TAO and TZS), pH of the aqueous medium, and shaking time were subjects of optimization experiments. Formation of ternary complexes with a composition of 2:2:2 was demonstrated by a set of different methods. Some key characteristics concerning the analytical application of the studied LLE-chromogenic systems were established as well. [ABSTRACT FROM AUTHOR]

Microbiological assays have been used to evaluate antimicrobial activity since the discovery of the first antibiotics. Despite their limitations, microbiological assays are widely employed to determine antibiotic potency of pharmaceutical dosage forms, since they provide a measure of biological activity. The aim of this work is to develop, optimize and validate a rapid colorimetric microplate bioassay for the potency of neomycin in pharmaceutical drug products. Factorial and response surface methodologies were used in the development and optimization of the choice of microorganism, culture medium composition, amount of inoculum, triphenyltetrazolium chloride (TTC) concentration and neomycin concentration. The optimized bioassay method was validated by the assessment of linearity (range 3.0 to 5.0μg/mL, r=0.998 and 0.994 for standard and sample curves, respectively), precision (relative standard deviation (RSD) of 2.8% and 4.0 for repeatability and intermediate precision, respectively), accuracy (mean recovery=100.2%) and robustness. Statistical analysis showed equivalency between agar diffusion microbiological assay and rapid colorimetric microplate bioassay. In addition, microplate bioassay had advantages concerning the sensitivity of response, time of incubation, and amount of culture medium and solutions required. [ABSTRACT FROM AUTHOR]

A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay based on the reduction in a tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2 H-tetrazolium, monosodium salt ( WST-8) with 2-methyl-1,4-napthoquinone as the electron mediator was developed. The proposed method was useful to measure the proliferation of 18 kinds of moulds and seven kinds of yeasts, including representative pathogens such as Aspergillus spp., Candida spp. and Cryptococcus spp. Linear relationships between the absorbance and viable fungal cell density were obtained for all fungi, suggesting that the absorbance change reflected the fungal proliferation. In addition, the minimum inhibitory concentrations ( MICs) against a variety of different pathogenic moulds and yeasts for amphotericin B, itraconazole and 5-flucytosine were determined by susceptibility testing using the proposed method and compared with those obtained using the conventional broth microdilution method. There was an excellent agreement between the results obtained using the WST-8 colorimetric method and those obtained using the conventional Clinical and Laboratory Standard Institute method. The WST-8 colorimetric assay is a useful method for rapid determination of accurate MICs for a variety of different fungi. Significance and Impact of the Study A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay based on reduction in a tetrazolium salt ( WST-8) was developed. The WST-8 colorimetric method was useful to measure the proliferation of a variety of different fungi. In the antifungal susceptibility testing, there was a good agreement between the MICs determined after 24 h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The proposed method was superior to conventional methods in terms of its rapidity towards a variety of different fungi. [ABSTRACT FROM AUTHOR]

Exposure to air pollutants is significantly associated with health risks ranging from bronchial reactivity to morbidity and mortality. However, the precise mechanisms are not always fully understood. The aim of this study was to investigate the effects of sulphur dioxide (SO2) on cell viability and cytokine production of A549-human pulmonary epithelial cells. Test atmospheres of SO2 were generated using a direct dilution method and calibrated by ion-chromatography. Test atmospheres were delivered to lung cells cultured on porous membranes (0.4 μm) using Harvard Navicyte horizontal diffusion chamber systems. The cytotoxic endpoints were investigated using the MTS (tetrazolium salt; Promega), NRU (neutral red uptake; Sigma) and ATP (adenosine triphosphate; Promega) assays. Expression of inflammatory markers including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were evaluated using double-antibody immunometric assays. Dose-dependent effects of SO2 were observed in A549 cells using all in vitro assays at test concentrations (10-200 ppm). The ATP assay appeared to be the most sensitive test (IC50 = 48 ± 2.83 ppm) that may related to the impaired metabolic activity of the cells following SO2 exposure. After analysis of TNF-α, no statistically significant differences were observed between control and exposed cells. However, the IL-6 production in A549 cells was significantly reduced in a dose-dependent manner (p

Complex formation and liquid-liquid extraction in a system containing iron(III), 4-nitrocatechol (4NC), 2,3,5-triphenyl-2H-tetrazolium chloride (TTC), water, and chloroform were studied. The effect of some experimental parameters (pH, shaking time, concentration of reagents) was investigated, and the optimum conditions for Fe(III) extraction as an ion-association complex, (TT+)3[Fe3+(4NC)3]3-, were found. The following key constants were calculated: constant of distribution (Log KD=1.49±0.02), constant of association (Log β=11.3±0.1), and constant of extraction (Log Kex=12.8±0.1). Some additional characteristics concerning the application of 4NC and TTC for extractive-spectrophotometric determination of Fe(III) were estimated as well: absorption maximum (λ=435 nm), apparent molar absorptivity (ε=2.7×104 l mol-1 cm-1), Sandell's sensitivity (SS=2.0 ng cm-2), recovery factor (R %=94.3±0.2), limit of detection (LOD=0.11 μg cm-3), and limit of quantification (LOQ=0.37 mg cm-3). Beer's law was obeyed for Fe(III) concentrations up to 2.0 μg ml-1 with a correlation coefficient of 0.9990. [ABSTRACT FROM AUTHOR]

Abstract: The minimum inhibitory concentrations (MICs) obtained from the susceptibility testing of various bacteria to antibiotics were determined by a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone as an electron mediator and compared with those obtained by the broth microdilution methods approved by the Clinical and Laboratory Standard Institute (CLSI). Especially for drug-resistant bacteria, the CLSI method at an incubation time of 24h tended to give lower MICs. The extension of incubation time was necessary to obtain consistent MICs for drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococi (VRE) and multi-drug resistant Pseudomonas aeruginosa (MDRP) in the broth microdilution method. There was excellent agreement between the MICs determined after 24h using the WST-8 colorimetric method and those obtained after 48–96h using the broth microdilution method. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of consistent MICs for drug-resistant bacteria. [Copyright &y& Elsevier]

Abstract: A method for the determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone (NQ) was developed. Measurement conditions were optimized for the microbiological determination of water-soluble vitamins, such as vitamin B6, biotin, folic acid, niacin, and pantothenic acid, using microorganisms that have a water-soluble vitamin requirement. A linear relationship between absorbance and water-soluble vitamin concentration was obtained. The proposed method was applied to determine the concentration of vitamin B6 in various foodstuffs. There was good agreement between vitamin B6 concentrations determined after 24h using the WST-8 colorimetric method and those obtained after 48h using a conventional method. The results suggest that the WST-8 colorimetric assay is a useful method for the rapid determination of water-soluble vitamins in a 96-well microtiter plate. [Copyright &y& Elsevier]

Abstract: We developed a microwell plate, high-throughput, screening method aimed at quantitating the tolerance of a panel of Gram-positive and Gram-negative bacteria to metals (Frankia sp., Escherichia coli, Cupriavidus metallidurans, Rhizobium leguminosarum, and Streptomyces scabies). Microbial viability was quantified using MTS; a tetrazolium salt converted to a water-soluble formazan through microbial reduction. In this paper, we present the stepwise development of the method, highlighting the main elements underlying its reliability, and compare results obtained with literature. We conclude the method is well suited to efficiently screen bacteria, including those that are filamentous and slow-growing, without the need for large amounts of inoculum which may not always be available. The method allows testing of compound gradients with sufficient replicates to generate statistically robust results, and is transposable to other types of cell proliferation assays such as those for antimicrobial susceptibility, and chemoresistance. [Copyright &y& Elsevier]

Abstract: A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microplate format. In general, XTT reduction is an extremely slow process which takes almost 24h to produce a detectable signal. Menadione could drastically induce this reduction to an almost equal extent within a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cell concentration in the presence of menadione. The standardized protocol used 200μM of XTT and 60μM of menadione in 250μl of cell suspension grown either in aerobic or anaerobic conditions. The cell suspension of M. bovis BCG and M. tuberculosis were incubated for 40min before reading the optical density at 470nm whereas M. smegmatis was incubated for 20min. Calculated Signal/Noise (S/N) ratios obtained by applying this protocol were 5.4, 6.4 and 9.4 using M. bovis BCG, M. tuberculosis and M. smegmatis respectively. The calculated Z′ factors were >0.8 for all mycobacterium bacilli indicating the robustness of the XTT Reduction Menadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying 10 standard anti-tubercular agents on M. tuberculosis, M. bovis BCG and M. smegmatis. The Minimum Inhibitory Concentration (MIC) values were found to be similar to reported values from Colony Forming Unit (CFU) and REMA (resazurin microplate assay) assays. Altogether, XRMA is providing a novel anti-tubercular screening protocol which could be useful in high throughput screening programs against different physiological stages of the bacilli. [Copyright &y& Elsevier]

Abstract: The applicability of a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt [WST-8]} via 2-methyl-1,4-naphthoquinone (2-methyl-1,4-NQ) as an electron mediator for determining the susceptibility of various bacteria to antibiotics and screening antimicrobial substances was investigated. The measurement conditions, which include the effects of the concentration of 2-methyl-1,4-NQ, were optimized for proliferation assays of gram-negative bacteria, gram-positive bacteria, and pathogenic yeast. In antimicrobial susceptibility testing, there was excellent agreement between the minimum inhibitory concentrations determined after 8h using the WST-8 colorimetric method and those obtained after 22h using conventional methods. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of the susceptibility of various bacteria to antibiotics. In addition, the current method was applied to the screening of bacteriocin-producing lactic acid bacteria and its efficiency was demonstrated. [Copyright &y& Elsevier]

Abstract: A colorimetric cell proliferation assay for measuring neutralizing antibodies to influenza viruses in human sera is described. Following a 90-min incubation, the serum–virus mixture was transferred to Madin-Darby canine kidney cells cultured in 96-well plates. After further incubation for three days, a tetrazolium salt was added to the wells. Cellular mitochondrial dehydrogenases cleave the tetrazolium salt to formazan, and the resulting color change is read by a spectrophotometer. The absorbance values correlate directly to the number of viable cells in the assay well and thus also to the neutralizing activity of influenza-specific antibodies present in the serum. With the few hands-on manipulations required, this assay allows simultaneous testing of a considerable number of sera, offers opportunities for automation, and is suitable for use under biosafety level-3 conditions. The test was used to study the antibody response after the administration of seasonal, inactivated, trivalent influenza vaccine. Antibody titers determined by the neutralization test in pre- and post-vaccination serum pairs were compared with those obtained by the hemagglutination inhibition assay. The neutralization test yielded higher pre- and post-vaccination titers and a larger number of significant increases in post-vaccination antibody titer than the hemagglutination inhibition test. This new test format could serve as a valuable laboratory tool for influenza vaccine studies. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Exposure to airborne contaminants is significantly associated with human health risks, ranging from bronchial reactivity to morbidity and mortality due to acute intense or long term low level repeated exposures. However, the precise mechanisms that derive such effects are not always understood. Although inhalation studies are technologically complicated, correct hazard characterisation is essential for comparable risk assessment of inhaled materials. The aim of this study was to investigate the comparative in vitro cytotoxicity of selected gaseous contaminants in human lung cells. The cytotoxicity of nitrogen dioxide (NO2), sulphur dioxide (SO2) and ammonia (NH3) was investigated in A549- human pulmonary type II-like epithelial cell lines cultured on porous membranes in Snapwell inserts. A dynamic direct exposure method was established by utilizing the horizontal diffusion chamber system (Harvard Apparatus Inc, USA) for delivery of test atmospheres. Test atmospheres were generated using a dynamic direct dilution method and the concentration monitored by appropriate analytical methods. A diversified battery of in vitro assays including the MTS (tetrazolium salt; Promega), NRU (neutral red uptake; Sigma) and ATP (adenosine triphosphate; Promega) assays was implemented. Airborne IC50 (50% inhibitory concentration) values were calculated based on the most sensitive assay for each test gas including NO2 (IC50 =11±3.54ppm; NRU)>SO2 (IC50 =48±2.83ppm; ATP)> and NH3 (IC50 =199±1.41ppm; MTS). However, all in vitro assays revealed similar toxicity ranking for selected gaseous contaminants. Identical toxicity ranking was achieved using both in vitro and published in vivo data. This comparison suggests that results of in vitro methods are comparable to in vivo data and may provide greater sensitivity for respiratory toxicity studies of gaseous contaminants. [Copyright &y& Elsevier]

Abstract: A new method to assay alkaline and acid phosphatases assay using ascorbic acid 2-phosphate (AsA-P) and ditetrazolium salt nitroblue tetrazolium chloride (NBT) was developed. AsA-P is hydrolyzed in the presence of phosphatase to yield ascorbic acid. In turn, the ascorbic acid reduces NBT directly or indirectly, opening the tetrazole ring to produce an insoluble formazan as a colored precipitate. The proposed method for alkaline phosphatase was compared with a conventional method in which 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is used in combination with NBT in the dot blots of a dilution series of β-lactoglobulin. AsA-P reduced NBT more effectively than BCIP in the presence of alkaline phosphatase. AsA-P could be also used as the chromogenic substrate for an acid phosphatase assay in the presence of phenazinium methylsulfate and NBT. [Copyright &y& Elsevier]

A simple and sensitive method for the detection and enumeration of microbial s-triazine-degrading microorganisms in soil was designed. The procedure is based on the ability of some microbes to use s-triazines, such as simazine, atrazine, and cyanuric acid, as sole nitrogen source. It employs the respiration indicator 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) to detect metabolic activity and the most-probable-number (MPN) enumeration in microtiter plates. The method was used to identify simazine- and cyanuric acid-degrading activities in agricultural soils treated with the herbicide simazine. The MPN-TTC method showed that the number of simazine- and cyanuric acid-degrading microorganisms increased four weeks after the herbicide simazine had been applied. [ABSTRACT FROM AUTHOR]