120 resultados para Microplate immunocapture
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Pós-graduação em Microbiologia Agropecuária - FCAV
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A microplate assay was modified for the detection of antimicrobial activity in plant extracts. The aim was to develop an in vitro assay that could rapidly screen plant extracts to provide quantitative data on inhibition of microbial growth. A spectrophotometric assay using a microplate with serial dilutions of the plant extract and the bacteria was developed. Two bacteria, Staphylococcus aureus and Escherichia coli, were used for this study. Essential oils, oregano (Origanum vulgare) and lemon myrtle (Backhousia citriodora), and three active components carvacrol, thymol and citral were evaluated. The reproducibility of the assay was high, with correlation coefficients (r aureus and E. coli between 0.9321 and 0.9816. Similarly, r and 0.9814. This assay could also be used to measure antimicrobial activity in plant extracts which vary in pH and color.
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The present work describes the optimization of a short-term assay, based on the inhibition of the esterase activity of the alga Pseudokirchneriella subcapitata, in a microplate format. The optimization of the staining procedure showed that the incubation of the algal cells with 20 μmolL−1 fluorescein diacetate (FDA) for 40 min allowed discrimination between metabolic active and inactive cells. The shortterm assay was tested using Cu as toxicant. For this purpose, algal cells, in the exponential or stationary phase of growth, were exposed to the heavy metal in growing conditions. After 3 or 6 h, cells were subsequently stained with FDA, using the optimized procedure. For Cu, the 3- and 6-h EC50 values, based on the inhibition of the esterase activity of algal cells in the exponential phase of growth, were 209 and 130 μg L−1, respectively. P. subcapitata cells, in the stationary phase of growth, displayed higher effective concentration values than those observed in the exponential phase. The 3- and 6-h EC50 values for Cu, for cells in the stationary phase, were 443 and 268 μgL−1, respectively. This short-term microplate assay showed to be a rapid endpoint for testing toxicity using the alga P. subcapitata. The small volume required, the simplicity of the assay (no washing steps), and the automatic reading of the fluorescence make the assay particularly well suited for the evaluation of the toxicity of a high number of environmental samples.
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A generic optical biosensing strategy was developed that relies on the absorbance enhancement phenomenon occurring in a multiple scattering matrix. Experimentally, inserts made of glass fiber membrane were placed into microplate wells in order to significantly lengthen the trajectory of the incident light through the sample and therefore increase the corresponding absorbance. Enhancement factor was calculated by comparing the absorbance values measured for a given amount of dye with and without the absorbance-enhancing inserts in the wells. Moreover, the dilution of dye in solutions with different refractive indices (RI) clearly revealed that the enhancement factor increased with the ΔRI between the membrane and the surrounding medium, reaching a maximum value (EF>25) when the membranes were dried. On this basis, two H2O2-biosensing systems were developed based on the biofunctionalization of the glass fiber inserts either with cytochrome c or horseradish peroxidase (HRP) and the analytical performances were systematically compared with the corresponding bioassay in solution. The efficiency of the absorbance-enhancement approach was particularly clear in the case of the cytochrome c-based biosensor with a sensitivity gain of 40 folds and wider dynamic range. Therefore, the developed strategy represents a promising way to convert standard colorimetric bioassays into optical biosensors with improved sensitivity.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
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CLSI method M27-A3 is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this study, we developed a microdilution method and added the alamarBlue reagent to test the responses of Paracoccidioides brasiliensis and Paracoccidioides lutzii against amphotericin B and itraconazole antifungals. The test proved to be sensitive, practical, and inexpensive and can be used to monitor the activity of low-growth microorganisms and their response to various drugs. © 2013, American Society for Microbiology.
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The endemic stingless honey-making bee Melipona (Melikerria) insularissp.n. on Coiba and Rancheria Islands in Pacific Panama is described, together with the proposed sister species, M. ambigua sp.n. from northeast Colombia. The Coiba Island group and Panama mainland were surveyed, yielding one meliponine endemic (M. insularissp.n.) and six meliponine genera and species. The poor Coiba fauna of amphibians and birds corresponds to the poor social bee fauna and suggests habitat barriers generally precluded recolonization from the mainland during glacial periods. Many animals became extinct, yet some remain as relicts. Melipona insularissp.n. was isolated on accreted terranes of Coiba rainforest in the Panama microplate. Morphology suggests that M. insularissp.n. is not a direct descendant of the San Blas-E. Panama endemic Melikerria, M. triplaridis. A phylogenetic hypothesis corroborates disjunct distributions. Rainforest endemics such as Peltogyne purpurea (Fabaceae) and Ptilotrigona occidentalis (Apidae, Meliponini) also occur as relictual, disjunct populations in Central and South America. These may have been isolated before accelerated biotic exchange began 2.4 Ma. Our work supports the geological findings of both a volcanic arc and the San Blas massif providing a substantial bridge for Melikerria from Colombia and Panama in Eocene to Miocene times. We suggest there have been taxon cycles permitting recolonization during glaciations, whereby colonies of M. insularissp.n. were able to recolonize Rancheria, a 250 ha island, 2 km from Coiba. However, rafting colonies nesting in trees, carried on vegetation mats, may have produced founding populations of Melipona in Central America and on oceanic islands such as Coiba.
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Biogenic particle fluxes from highly productive surface waters, boundary scavenging, and hydrothermal activity are the main factors influencing the deposition of radionuclides in the area of the Galapagos microplate, eastern Equatorial Pacific. In order to evaluate the importance of these three processes throughout the last 100 kyr, concentrations of the radionuclides 10Be, 230Th, and 231Pa, and of Mn and Fe were measured at high resolution in sediment samples from two gravity cores KLH 068 and KLH 093. High biological productivity in the surface waters overlying the investigated area has led to 10Be and 231Pa fluxes exceeding production during at least the last 30 kyr and probably the last 100 kyr. However, during periods of high productivity at the up welling centers off Peru and extension of the equatorial high-productivity zone, a relative loss of 10Be and 231Pa may have occurred in these sediment cores because of boundary scavenging. The effects of hydrothermal activity were investigated by comparing the 230Thex concentrations to the Mn/Fe ratios and by comparing the fluxes of 230Th and 10Be which exceed production. The results suggest an enhanced hydrothermal influence during isotope stages 4 and 5 and to a lesser extent during isotope stage 1 in core KLH 093. During isotope stages 2 and 3, the hydrothermal supply of Mn was deposited elsewhere, probably because of changes in current regime or deep water oxygenation. A strong increase of the Mn/Fe ratio at the beginning of climatic stage 1 which is not accompanied by an increase of the 230Thex concentration is interpreted to be an effect of Mn remobilization and reprecipitation in the sediment.
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Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading. (C) 2003 Elsevier B.V. All rights reserved.