Observações sobre o comportamento do teste do "nitroblue tetrazolium" relativamente à toxoplasmose adquirida, forma linfoglandular

O teste de "nitroblue tetrazolium", apesar de inespeclfico, tem-se mostrado útil no sentido de facilitar diagnóstico diferencial relativo a diversas afecções e, sobretudo, de indicar se determinado processo mórbido é presumivelmente bacteriano. Diante da conveniência de analisar o comportamento dessa prova no que concerne a doenças das mais diferentes naturezas, foi efetuada investigação referente à toxoplasmose adquirida, forma linfoglandular. Quanto à casuística considerada, composta de 26 indivíduos, ficaram detectados valores variáveis de 6% a 50%, mas habitualmente superiores a 17% na vigência de infecção com até quatro meses de duração, de acordo com a época de início das manifestações clínicas. Em face a evoluções já mais prolongadas, as cifras registradas comumente mostraram-se inferiores à por último citada. Nenhum tipo de comprometimento estava presente associadamente, o que permitiu avaliação isenta de ponderáveis influências falseadoras. O estudo efetuado retrata contribuição dotada do intuito de possibilitar eventuais aplicabilidades, do exame em questão, também no âmbito do reconhecimento das enfermidades parasitárias talvez em vigor em determinadas circunstâncias.

Susceptibility of Mycobacterium tuberculosis to first-line antimycobacterial agents in a Brazilian hospital: assessing the utility of the tetrazolium (MTT) microplate assay

We conducted a cross-sectional, hospital-based study between January 2006-March 2008 to estimate the resistance of Mycobacterium tuberculosis to first-line drugs in patients with tuberculosis at a Brazilian hospital. We evaluated the performance of the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT) microplate assay compared with the Bactec-MGIT 960™ system for mycobacteria testing. The prevalence of resistance in M. tuberculosis was 6.7%. Multidrug-resistance [resistance to rifampicin (RMP) and isoniazid (INH)], INH-resistance and streptomycin (SM)-resistance accounted for 1%, 3.8% and 3.8% of all resistance, respectively, and all isolates were susceptible to ethambutol (EM). The resistance was primary in four cases and acquired in three cases and previous treatment was associated with resistance (p = 0.0129). Among the 119 M. tuberculosis isolates, complete concordance of the results for INH and EM was observed between the MTT microplate and Bactec-MGIT 960TM methods. The observed agreement for RMP was 99% (sensitivity: 90%) and 95.8% for SM (sensitivity 90.9%), lower than those for other drugs. The MTT colourimetric method is an accurate, simple and low-cost alternative in settings with limited resources.

Determination of myocardial infarction size in rats by echocardiography and tetrazolium staining: correlation, agreements, and simplifications

Triphenyltetrazolium chloride (TTC) staining and echocardiography (ECHO) are methods used to determine experimental myocardial infarction (MI) size, whose practical applicability should be expanded. Our objectives were to analyze the accuracy of ECHO in determining infarction size in rats during the first days following coronary occlusion and to test whether a simplified single measurement by TTC correctly indicates MI size, as determined by the average value for multiple slices. Infarction was induced in female Wistar rats by coronary artery occlusion and MI size analysis was performed after the acute (7th day) and chronic periods (after 4 weeks) by ECHO matched with TTC. ECHO and TTC showed similar values of MI size (% of left ventricle perimeter) in acute (ECHO: 33 ± 11, TTC: 35 ± 14) and chronic (ECHO: 38 ± 14, TTC: 39 ± 13 periods), and also presented an excellent correlation (r = 0.92, P < 0.001). Although measurements from different heart planes showed discrepancies, a single measurement acquired from the mid-ventricular level by TTC was a good estimate of MI size calculated by the average of multiple planes, with minimal disagreement (Bland-Altman test with mean ratio bias of 0.99 ± 0.07) and close to an ideal correlation (r = 0.99, P < 0.001). In the present study, ECHO was confirmed as a useful method for the determination of MI size even in the acute phase. Also, the single measure of a mid-ventricular section proposed as a simplification of the TTC method is a satisfactory prediction of average MI extension.

Tetrazolium test for evaluating triticale seed viability

Freshly harvested triticale seeds are usually dormant, making the immediate evaluation of the physiological potential of seed lots difficult. We evaluated different triphenyl tetrazolium chloride (TTC) test methods for rapidly determining the viability of four seed lots of x.Triticosecale Wittmack cultivar IPR111. The test variables were: Preconditioning, (i) placing whole seeds between moistened paper towels or (ii) directly soaking the seeds in water, both procedures being conducted at 20 ºC for 18 hours; Post-conditioning seed preparation, (i) longitudinal bisection of the seed through the embryo with one half being stained and the other discarded or (ii) longitudinal bisection with both halves being stained; Staining for three and four hours, in the dark, with 0.1%, 0.5% or 1.0% (w/v) TTC according to the preconditioning method described above, (i) both halves of each seed were placed on filter paper moistened with TTC and maintained at 40 ºC or (ii) one half of each seed was immersed in 5 mL of TTC solution in a 100 mL glass beaker at 30 ºC. The best results were obtained by preconditioning seeds between moistened paper towels at 20 ºC for 18 hours and staining on filter paper with 1.0% (w/v) TTC for three hours at 40 ºC.

Viability of barley seeds by the tetrazolium test

The tetrazolium test is used to control seed quality of various plant species since it allows a rapid evaluation of viability. Freshly harvested barley seeds show dormancy that can make the germination test ineffective for an immediate evaluation. Therefore, the development of more efficient methods, such as the tetrazolium test, is necessary. The objective of this research work was to study various procedures for performing the tetrazolium test on barley seeds. Five lots of cv. BRS 195 barley seeds were used and subjected to the following treatments: two different methods of seed preconditioning (direct immersion in H2O and between sheets of moistened paper towels); two types of preparation for staining (longitudinal cross-section of the seed through the embryo with immersion of one half in a 2,3,5 triphenyl tetrazolium chloride solution or placing both halves on top of filter paper moistened with the tetrazolium salt solution); two methods of staining (on top of filter paper and direct immersion in the tetrazolium salt solution). Three concentrations of the tetrazolium salt solution (0.1%, 0.5%, and 1.0%) were used. It was concluded that the tetrazolium test on barley seeds may be accomplished with preconditioning by direct immersion in H2O and staining by immersing in a 0.1% or 0.5% concentration of tetrazolium salt solution or staining on top of filter paper moistened with such solution at a 1.0% concentration.

Optimising tetrazolium test procedures to evaluate the physiological potential of peanut seeds

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Criteria for peanut seed pre-conditioning for the tetrazolium test

The staining pattern of peanut (Arachis hypogaea L.) seeds with a tetrazolium solution was evaluated to determine the adequate conditions of seed quality evaluations. Three seed lots with different vigour levels and similar moisture content were pre-conditioned using the combination of five periods of time (4, 8, 12, 16 and 20 hours) and four temperatures (20, 25, 30 and 35 degrees C) and then exposed to a 1.0% tetrazolium solution for three hours. The moisture content levels of seeds conditioned for 8 hours at the temperatures of 25, 30 or 35 degrees C, for 12 hours at 20 or 25 degrees C and for 16 hours at 20 degrees C, were above 30%. In this range of moisture level, the staining in tetrazolium was clear and uniform, reflecting an appropriate conditioning of the seeds. However, under a practical point of view, the period of 16 hours at 20 degrees C showed to be the best option, since it allows to start the pre-conditioning an late afternoon of one day and submit the seeds for staining in the following morning.

Use of reduced concentrations of tetrazolium solutions for the evaluation of the viability of peanut seed lots

The possibility of reducing the concentration of the working solution used in the tetrazolium test for peanut seeds (Arachis hypogaea L.) with or without seedcoats was studied. Tetrazolium solutions of different concentrations (0.05%, 0.075% and 0.1%) were tested at the temperatures of 35 and 40 degrees C, for determining the time needed for the seeds to reach proper staining. The efficiency of the selected treatments in evaluating the viability potential of the seeds was determined by comparing the results of the tetrazolium tests with those obtained by standard germination (using sand and rolled paper towel as substrata) and seedling emergence in the field tests. Staining the seeds without seedcoat in 0.05% tetrazolium solution for three hours at 40 degrees C yielded efficient results. on the other hand, reduced concentrations can be employed in the staining process of seeds with seedcoat; however, this method requires a higher consumption of tetrazolium salt, longer staining time as well as a higher ability and availability of time for embryo evaluation, since the cross-cutting of seeds is much more difficult in the presence of the seedcoat and the occurrence of damage to the outer surface of the cotyledons cannot be determined.

Tetrazolium test for assessment of seed viability of atemoya (Annona cherimola Mill. x A. squamosa L.)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of a New Tetrazolium-Based Assay to Study the Production of Superoxide Radicals by Tobacco Cell Cultures Challenged with Avirulent Zoospores of Phytophthora parasitica var nicotianae1

The relationship between the production of reactive oxygen species and the hypersensitive response (HR) of tobacco (Nicotiana tabacum L.) toward an incompatible race of the Oomycete Phytophthora parasitica var nicotianae has been investigated. A new assay for superoxide radical (O2−) production based on reduction of the tetrazolium dye sodium,3′-(1-[phenylamino-carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) has enabled the quantitative estimation of perhydroxyl/superoxide radical acid-base pair (HO2·/O2−) production during the resistant response. Tobacco suspension cells were inoculated with zoospores from compatible or incompatible races of the pathogen. Subsequent HO2·/O2− production was monitored by following the formation of XTT formazan. In the incompatible interaction only, HO2·/O2− was produced in a minor burst between 0 and 2 h and then in a major burst between 8 and 10 h postinoculation. During this second burst, rates of XTT reduction equivalent to a radical flux of 9.9 × 10−15 mol min−1 cell−1 were observed. The HO2·/O2− scavengers O2− dismutase and Mn(III)desferal each inhibited dye reduction. An HR was observed in challenged, resistant cells immediately following the second burst of radical production. Both scavengers inhibited the HR when added prior to the occurrence of either radical burst, indicating that O2− production is a necessary precursor to the HR.

Tetrazolium test for seed viability and germinability of Melocactus ernestii Vaupel subsp. ernestii and Melocactus zehntneri (Briton & Rose) Luetzelb. (Cactaceae)

Despite its ornamental value, some Melocactus species (Cactaceae) are threatened by several factors and only sexual propagation is possible. Thus, artificial seed banks are an appropriate method for their ex situ conservation. Suitable methods to germinate and to monitor viability are necessary for the seeds of these species. This work explored the use of tetrazolium test for monitoring seed viability of two Melocactus species. There was a correlation between the percentage of stained embryos, considering different cover area or tone stain, and the germination percentage. Unviable embryos did not stain. The embryos curved shape can explain the partial stain on the most of cases.

Evaluation Of The Cytotoxicity And Phototoxicity Of Caryocar Brasiliense Supercritical Carbon Dioxide Extract.

Caryocar brasiliense Camb (Pequi) is a typical Brazilian Cerrado fruit tree. Its fruit is used as a vitamin source for culinary purposes and as a source of oil for the manufacture of cosmetics. C. brasiliense supercritical CO2 extracts exhibit antimicrobial activity against the bacteria Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus and also possess antioxidant activity. This study was designed to evaluate the in vitro cytotoxicity and phototoxicity of the supercritical CO2 extract obtained from the leaves of this species. In vitro cytotoxicity and phototoxicity of C. brasiliense supercritical CO2 extracts were assessed using a tetrazolium-based colorimetric assay (XTT) and Neutral Red methods. We found that the C. brasiliense (Pequi) extract obtained by supercritical CO2 extraction did not present cytotoxic and phototoxic hazards. This finding suggests that the extract may be useful for the development of cosmetic and/or pharmaceutical products.

Cardioprotective effect of ornitho-kinin in an anesthetized, open-chest chicken model of acute coronary occlusion

The generation of bradykinin (BK; Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) in blood and kallidin (Lys-BK) in tissues by the action of the kallikrein-kinin system has received little attention in non-mammalian vertebrates. In mammals, kallidin can be generated by the coronary endothelium and myocytes in response to ischemia, mediating cardioprotective events. The plasma of birds lacks two key components of the kallikrein-kinin system: the low molecular weight kininogen and a prekallikrein activator analogous to mammalian factor XII, but treatment with bovine plasma kallikrein generates ornitho-kinin [Thr6,Leu8]-BK. The possible cardioprotective effect of ornitho-kinin infusion was investigated in an anesthetized, open-chest chicken model of acute coronary occlusion. A branch of the left main coronary artery was reversibly ligated to produce ischemia followed by reperfusion, after which the degree of myocardial necrosis (infarct size as a percent of area at risk) was assessed by tetrazolium staining. The iv injection of a low dose of ornitho-kinin (4 µg/kg) reduced mean arterial pressure from 88 ± 12 to 42 ± 7 mmHg and increased heart rate from 335 ± 38 to 402 ± 45 bpm (N = 5). The size of the infarct was reduced by pretreatment with ornitho-kinin (500 µg/kg infused over a period of 5 min) from 35 ± 3 to 10 ± 2% of the area at risk. These results suggest that the physiological role of the kallikrein-kinin system is preserved in this animal model in spite of the absence of two key components, i.e., low molecular weight kininogen and factor XII.

Viability of fibroblasts cultured under nutritional stress irradiated with red laser, infrared laser, and red light-emitting diode

Phototherapy is noninvasive, painless and has no known side effect. However, for its incorporation into clinical practice, more well-designed studies are necessary to define optimal parameters for its application. The viability of fibroblasts cultured under nutritional stress irradiated with either a red laser, an infrared laser, or a red light-emitting diode (LED) was analyzed. Irradiation parameters were: red laser (660 nm, 40 mW, 1 W/cm(2)), infrared laser (780 nm, 40 mW, 1 W/cm(2)), and red LED (637 +/- 15 nm, 40 mW, 1 W/cm(2)). All applications were punctual and performed with a spot with 0.4 mm(2) of diameter for 4 or 8 s. The Kruskal-Wallis test and analysis of variance of the general linear model (p <= 0.05) were used for statistical analysis. After 72 h, phototherapy with low-intensity laser and LED showed no toxicity at the cellular level. It even stimulated methylthiazol tetrazolium assay (MTT) conversion and neutral red uptake of fibroblasts cultured under nutritional stress, especially in the group irradiated with infrared laser (p = 0.004 for MTT conversion and p < 0.001 for neutral red uptake). Considering the parameters and protocol of phototherapy used, it can be concluded that phototherapy stimulated the viability of fibroblasts cultured under nutritional deficit resembling those found in traumatized tissue in which cell viability is reduced. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3602850]