Brilliant Cresyl Blue Research Articles

Factorial design in the optimization of preconcentration procedure for lead determination by FAAS

The present paper proposes a preconcentration procedure for lead determination using flame atomic absorption spectrometry (FAAS). It is based on lead(II) ions extraction as brilliant cresyl blue (BCB) complex and its sorption onto Diaion HP-2MG, a methacrylic ester copolymer. The optimization step was carried out using factorial design and the variables studied were pH, shaking time and reagent concentration. In the established experimental conditions, lead can be determinate with a limit of detection of 3.7 μg L −1 lead ( N = 20) and a relative standard deviation of 7% for a lead concentration of 100 μg L −1. The accuracy was confirmed by analysis of a certified reference material, the stream sediment furnished by National Research Centre for Certified Reference Materials (NRCCRM), China (GBW 07310). Effect of other ions in the procedure proposed was also studied. The method was applied for lead determination in real samples of water, tea, soil and dust. Tests of addition/recovery in the experiments for lead determination in water samples revealed that the proposed procedure could be applied satisfactorily for analysis of these samples.

Potential pitfalls of comparative measurements of reticulocytes with flow cytometry and microscopy in prematures and infants

Reticulocyte counting by flow cytometry (Bayer H*3, ADVIA 120) in blood of prematures, infants and children > 1 year of age was compared with microscopic counting under research conditions (9000 counted red blood cells per slide). While in children >1 year a good concordance of both methods was observed, 2.3-2.4-fold higher values were obtained in neonates by microscopy (Brilliant Cresyl Blue stain, 0.5%). However, another laboratory found good agreement between H*3-counting and microscopy in samples also obtained from neonates using the same methods. Despite very similar results for all age groups in comparative flow cytometry measurements in both laboratories, counting of smears from neonates differed, showing an approximately 2.3-fold larger amount of reticulocytes in our laboratory. The reason for these observations was a greater enlargement (1250-fold) used routinely in our laboratory compared with 800-fold in the other one. Thus very mature reticulocytes frequently found in neonates could only be detected using a 1250-fold enlargement. Similarly, the low concentration of the colouring matter used in the H*3 (0.0005% oxazin or 0.001% ADVIA 120) is obviously not sufficient for detection of mature reticulocytes. Therefore, it is important to consider this phenomenon and to standardise microscopic enlargement, especially for comparisons in multicentre studies.

Supplementation with cysteamine during maturation and embryo culture on embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue test.

Our previous studies have shown that the addition of 100 mircroM cysteamine to the in vitro maturation (IVM) medium increased the embryo development of prepubertal goat oocytes. The aim of the present study was to evaluate the effect of adding different concentrations of cysteamine to the IVM medium and to the in vitro embryo culture medium (IVC) on the embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue (BCB) test. Oocytes were exposed to BCB and classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) or oocytes without blue cytoplasm or growing oocytes (BCB-). In Experiment 1, oocytes were matured in a conventional IVM medium supplemented with 100 microM, 200 microM or 400 microM cysteamine. In Experiment 2, oocytes were matured with 400 microM cysteamine and following in vitro fertilization (IVF) were cultured in SOF medium supplemented with 50 microM and 100 microM cysteamine. In Experiment 1, BCB+ oocytes matured with 100 microM and 200 microM cysteamine showed higher normal fertilization and embryo development rates than BCB- oocytes. Oocytes matured with 400 microM cysteamine did not present these differences between BCB+ and BCB- oocytes. In Experiment 2, the addition of 50 microM and 100 microM cysteamine to culture medium did not affect the proportion of total embryos obtained from BCB+ oocytes (35.89% and 38.29%, respectively) but was significantly different in BCB- oocytes (34.23% and 29.04%, respectively, P < 0.05). In conclusion, the addition of 400 microM cysteamine to the IVM improved normal fertilization and embryo development of BCB- oocytes at the same rates as those obtained from BCB+ oocytes. The proportions of morulae plus blastocyst development were not affected by the treatments.

Developmental competence of heifer oocytes selected using the brilliant cresyl blue (BCB) test

The aim of this study was to evaluate the usefulness of the brilliant cresyl blue (BCB) test in the selection of more competent heifer oocytes for in vitro embryo production (IVEP). IVEP from selected BCB heifer oocytes was compared to IVEP from morphologically selected heifer (control group) and cow oocytes. BCB staining determines the activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme synthesized in growing oocytes but with less activity in grown oocytes. Six hundred and fifty seven heifer cumulus–oocyte complexes (COC) were classified morphologically as Grade 1–3 and exposed to 26 μM of BCB and classified as: blue (or grown) oocytes (BCB+) or unstained oocytes or growing oocytes (BCB−). Grade 1–3 heifer oocytes showed significantly different percentages of BCB+ oocytes (78.6, 66.2, and 51.1%, respectively; P<0.05). The diameter of BCB+ oocytes was significantly higher than BCB− oocytes (152.6±5.8 μm and 147±5.9 μm, respectively; P<0.001). The percentage of BCB+ oocytes reaching the blastocyst stage was significantly higher than those of BCB− and control heifer oocytes (12.3, 1.6, and 5.2%, respectively; P<0.05), but lower than those of cow oocytes (30.0%; P<0.05). In conclusion, heifer oocytes selected by the BCB test (BCB+) are larger and more competent for IVEP than control heifer oocytes. However, fewer heifer oocytes selected using the BCB test develop to blastocyst stage compared to cow oocytes.

Decolourisation of industrial dyes by solid-state cultures of Pleurotus pulmonarius

A solid-state system to obtain rapid in vivo industrial dye decolourisation by the white-rot fungus Pleurotus pulmonarius is proposed. When cultivated on glucose\\ammonium tartrate–corncob solid-state medium, P. pulmonarius decolourised structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes). Amido black, congo red, trypan blue, methyl green, remazol brilliant blue R (RBB), methyl violet, ethyl violet and brilliant cresyl blue were completely decolourised after 6 days of cultivation, while methylene blue and Poly R-478 were partially decolourised. The capability of fungal culture to decolourise industrial dyes appears to be due to high titres of laccase (480 U/ml) produced in response to the presence of soluble phenolics.

Effect of the addition of glutathione and glucose to the culture medium on embryo development of IVM-IVF prepubertal goat oocytes.

Our previous studies have shown that larger and more competent oocytes can be selected using the brilliant cresyl blue (BCB) test. The objective of this study was to assess, in BCB-selected oocytes, the effect on the embryo development of prepubertal goat oocytes of the addition to in vitro culture (IVC) medium of either glutathione (GSH) alone or GSH in combination with glucose. Oocytes were exposed to 26 mM BCB and were classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocyteswithout blue cytoplasm or growing oocytes (BCB-). Oocytes were matured in TCM-199 with 100 microM cysteamine. Presumptive zygotes were cultured in synthetic oviductal fluid (SOF) in the presence or absence of 1 mM glutathione (experiment 1) for 7 days (8 days post-insemination, p.i.). In experiment 2 we tested the addition to culture of 2.78 mM glucose at day 5 p.i. BCB+ oocytes showed higher percentages of nuclear maturation than the BCB- and control groups (82.6%, 55.7% and 74.7%, respectively). The percentage of polyspermic oocytes was higher in BCB- than BCB+ oocytes. Supplementation of SOF medium with 1 mM GSH did not affect embryo development but the percentage of total embryos developed after culture was higher in BCB+ oocytes than in BCB- oocytes independently of the GSH supplementation. Glucose, alone or with GSH, added at 5 days p.i. did not affect embryo development. In conclusion, prepubertal goat oocytes were unable to develop beyond the 8-cell stage embryo under the culture conditions in this study.

Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation.

The aim of this study was to assess the effect of oocyte selection using the brilliant cresyl blue (BCB) test plus the addition of cysteamine to the in vitro maturation (IVM) medium to improve the in vitro embryo development of prepubertal goat oocytes. The oocytes were exposed to 26 microM BCB and classified according to their cytoplasm coloration: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). The oocytes were matured in a conventional IVM medium supplemented with cysteamine 100 microM. The control group consisted of oocytes not exposed to BCB and matured without cysteamine. The IVM-oocytes were inseminated and cultured in synthetic oviductal fluid (SOF) for 7 days. The normal fertilisation rate (oocytes showing 2 pronuclei and 1 sperm tail) of BCB+ oocytes (40%) was higher than those of BCB- (21%) and control oocytes (22%). The percentage of morulae plus blastocysts was higher (P < 0.05) in the BCB+ group than in the BCB- group (23.8 vs. 5.1%, respectively). In conclusion, the integration of the BCB test and the addition of cysteamine in the protocol of in vitro embryo production from prepubertal goat oocytes has improved the developmental rates of embryo development.

New scheme for electrochemical detection of DNA based on electrocatalytic oxidation of NADH

A new electrocatalytic scheme for the detection of DNA is described. This scheme relies on the different interactions of single- and double-stranded oligonucleotides with the organic dyes methylene blue, thionin, azure A, azure B, toluidine blue O, brilliant cresyl blue and neutral red, which in addition can act as NADH catalysts. This catalytic activity can be used for amplifying differentiation between duplex and single-stranded DNA. Pyrolytic graphite electrodes modified with adsorbed dsDNA–organic dye complexes gave higher electrocatalytic NADH oxidation currents than those measured with electrodes modified with adsorbed ssDNA–organic dye. This difference is maximum for brilliant cresyl blue. These results suggest that the proposed scheme may be useful for the detection of DNA hybridisation and damage.

Surface-enhanced and STM-tip-enhanced Raman Spectroscopy at Metal Surfaces

The strong electromagnetic field enhancement, occurring at illuminated metal structures with sub-wavelength dimensions, is exploited to develop Raman spectroscopy with exceedingly high sensitivity and lateral resolution, possibly down to single molecule detection. After reviewing Surface Enhanced Raman Spectroscopy (SERS) on single crystalline surfaces and colloids we present recent results on the Tip Enhanced Raman Spectroscopy (TERS) approach, where a metal tip is used as an external enhancing unit. In this way the electromagnetic and chemical surface enhancement are physically separated: the former is confined to the tip, the latter to the metal-adsorbate system. So far, TERS has been reported only for a few molecules exhibiting large Raman cross sections, such as sulphur or dye molecules. Here, we present combined SERS and TERS studies for the CN- ions and TERS for Brilliant Cresyl Blue adsorbed at smooth thin gold films.

Electrochemical Polymerization of Brilliant Cresyl Blue and Properties of the Polymer

AbstractThe electrochemical polymerization of brilliant cresyl blue (BCB) has been carried out using cyclic voltammetry in the potential region between −0.20 V and 1.15 V (vs. Ag/AgCl with saturated KCl solution). The electrolytic solution consisted of BCB (0.1 mol/L) and Na2HPO4(0.2 mol/L) with pH 2.1. BCB can not be polymerized at pH &lt; 1. There are an anodic peak and a cathodic peak on the cyclic voltammogram of poly (BCB) at pH ≤ 4.3. Both peak potentials shift towards more negative with increasing pH value, and their peak currents decrease with increasing pH value. Poly (BCB) depicts a good electrochemical reversibility, fast charge transfer ability and stability in aqueous solutions with pH ≤ 4.3. The visible spectrum, FTIR spectrum and Raman spectrum of poly (BCB) are different from those of BCB.

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme.

The ability of a Brazilian strain of Pleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase), P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorization in vivo was tested using three dyes--Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability of P. pulmonarius to decolorize industrial dyes.

Selection of prepubertal goat oocytes using the brilliant cresyl blue test

Brilliant cresyl blue stain allows us to determine the activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme synthesized in growing oocytes but with decreased activity in oocytes that have finished their growth phase. The objective of this study was to evaluate the utility of the brilliant cresyl blue (BCB) test as an indirect measure of oocyte growth, in order to select competent prepubertal goat oocytes for in vitro embryo production. Oocytes were exposed to BCB diluted in PBS and were classified according to their cytoplasm coloration: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocytes without a blue cytoplasm or growing oocytes (BCB−). After exposure to different BCB concentrations, we evaluated in vitro maturation (IVM), in vitro fertilization (IVF) and embryo development parameters. We defined matured oocytes as those oocytes that reached the metaphase II (MII) stage after being cultured for 27 h. Oocytes showing two pronuclei at 20 h post-insemination were classified as normally fertilized oocytes. We assessed embryo development 8 days post-insemination and recorded the percentage of total embryos, morale and blastocysts. The mean percentage of BCB+ oocytes was 29.4%. Mean diameter of BCB+ oocytes (136.6±6.3 μm) was higher ( P<0.001) than that of BCB− oocytes (125.5±10.2 μm). The percentage of BCB+ oocytes reaching the MII stage (81.4%) was higher ( P<0.05) than that of BCB− (52.5%) and control oocytes (72.4%). Normal fertilization rate of BCB+ oocytes was also higher (23.5%) than that of BCB− (8.2%; P<0.0001) and control oocytes (11.9%; P<0.05). The percentages of total embryos undergoing development to ≥8-cell and the morula plus blastocyst stages were higher ( P<0.05) in the group of BCB+ (41.3 and 12.0%, respectively) than in BCB− oocytes (21.3 and 3.6%, respectively). In conclusion, the BCB test is a useful way to select more competent prepubertal goat oocytes for in vitro embryo production.

Role of G6PD Activity on Sex Ratio and Developmental Competence of Bovine Embryos under Oxidative Stress.

Inactivation of the X-linked gene in the female embryo is one of the major events during mammalian early embryogenesis. Before this inactivation, enzyme activity encoded by the X-linked gene is different between male and female embryos. In the present study, we demonstrated a possibility that there may be a different response to specific culture conditions in vitro between male and female early embryo. Glucose-6-phosphate dehydrogenase (G6PD) is one of the X-linked enzymes and its activity is semi-quantitated by brilliant cresyl blue (BCB). In experiment 1, the relationship between semi-quantitated G6PD activity and the sex ratio of the embryos was examined. In experiment 2, the relationship between semi-quantitated G6PD activity and the developmental competence of embryos was examined. No relationship was found between G6PD activity and sex in 8-cell, 16-cell and blastocyst stage embryos. However, G6PD activity was high in female embryos and low in male embryos at the morula stage. When morula stage embryos were categorized by BCB and cultured under high oxygen tension (5% CO2, 95% air), the developmental competence of embryos having high G6PD activity was higher than those having low enzyme activity. However, when the categorized morula stage embryos were cultured under low oxygen tension (5% CO2, 5% O2, 90% N2), there was no difference in the developmental rates among the categories. These results indicate that G6PD activity is high in female morula stage embryos compared with that in male embryos, and that the activity of this enzyme strongly affects the developmental competence of morula stage embryo when they are cultured under high oxygen tension.

Use of superswelling acrylamide/maleic acid hydrogels for monovalent cationic dye adsorption

Acrylamide/maleic acid (AAm/MA) superswelling hydrogels prepared by irradiation with γ radiation were used in experiments on swelling, diffusion, and adsorption of some water-soluble monovalent cationic dyes such as basic red 5 (BR-5), basic violet 3 (BV-3), and brilliant cresyl blue (BCB). The AAm/MA hydrogel containing 60 mg MA and irradiated at 5.20 kGy was used for swelling and diffusion studies in water and aqueous solutions of the monovalent cationic dyes. For this superswelling hydrogel the swelling studies indicated that swelling increased in the following order: BR-5 > water > BV-3 ≥ BCB. The diffusion of water and the dyes within the hydrogels was found to have a non-Fickian character. The uptake of the cationic dyes to the AAm/MA superswelling hydrogels was studied by the batch adsorption technique at 25°C. The uptake of dyes within the hydrogel increased in the following order: BR-5 > BV-3 > BCB. In the experiments of the adsorption equilibrium, S-type adsorption in Giles' classification system was found. The binding ratio of the hydrogel/dye systems was gradually increased with the increase of the MA content in the AAm/MA hydrogel and the irradiation dose. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 79: 1809–1815, 2001

Phenothiaziniums as Putative Photobactericidal Agents for Red Blood Cell Concentrates

The antibacterial activities of Methylene Blue and several of its congeners were measured against Yersinia enterocolitica, a Gram-negative pathogen known to exhibit significant growth at 4°C and thus constituting a threat to red blood cell concentrates which are stored at this temperature. None of the derivatives was highly active in dark conditions, as expected, but on illumination using a lamp emitting light in the waveband 615-645 nm, considerable bactericidal activity was noted using similar photosensitizer concentrations to those used elsewhere to inactivate blood-borne viruses. Two novel compounds in this area, the phenothiazinium New Methylene Blue N and the phenoxazini-um Brilliant Cresyl Blue, exhibited bactericidal activity at lower concentrations than both of the established phenothiaziniums, Methylene Blue and Toluidine Blue O and the recently published blood photovirucidal agent 1,9-Dimethyl Methylene Blue. The photoactivity of these compounds was undiminished in the presence of red blood cells.

Development of an integrated single-fiber SERS sensor

An integrated fiberoptic surface-enhanced Raman scattering (SERS) sensor based on a single-fiber system is described and evaluated for remote and in situ measurements. The heart of the sensor consists of a unique single optical fiber designed to induce the SERS effect through the incorporation of alumina nanoparticles and silver coatings onto the probe tip. The unique design based on single-fiber for excitation/emission (SFEE) is practical for the fabrication of in situ sensors in microenvironments. Since the single fiber carries both the laser excitation and SERS signal radiation, optical parameters of the remote tip are simple and consistent. Depending on the tip size, the fiberoptic SERS sensor allows in situ measurements at microscale environments. Details of sensor tip fabrication and optical system design are described. Two detection schemes can be used: a “dip-and-dry” mode of detection and an in situ sensing mode are demonstrated for several compounds, including cresyl fast violet (CFV), brilliant cresyl blue (BCB), p-aminobenzoic acid (PABA) and 3-aminopyrene (3AP). To illustrate the effectiveness of this new SERS sensor for remote in situ analysis, SERS spectra of aqueous dye samples (CFV and BCB) are presented. A quantitative calibration curve for CFV in groundwater is established and a limit of optical detection of approximately 50 ppb CFV in groundwater is determined.

Flow injection catalytic determination of ruthenium with spectrophotometric detection

A new flow injection catalytic method was described for the determination of ruthenium(III) based on its catalytic effect on the oxidation of brilliant cresyl blue (BCB) by periodate in acidic media. The reaction was followed spectrophotometrically by measuring the absorbance of the dye at 635 nm. Under optimum conditions, ruthenium can be determined in the range of 1.0–100.0 ng ml−1 with a relative standard deviation of 1.1% and with a limit of detection of 0.70 ng ml−1. The influence of reagent and manifold variables were studied and optimized. The method was tested for the determination of ruthenium(III) in some synthetic mixtures.

Brilliant cresyl blue as a new red region fluorescent probe for determination of nucleic acids

A fluorescence quenching method was developed for determination of microamounts of nucleic acids by using brilliant cresyl blue (BCB) as a new red region fluorescent probe. In aqueous hexylmethylene tetramine solution, BCB showed maximum excitation and emission wavelengths at 626 and 670 nm, respectively, and the fluorescence of BCB could be greatly quenched by DNA (or RNA). Under optimal conditions, the calibration graphs are linear over the range of 0.02–0.80 μg/ml for SM DNA and 0.25–1.5 μg/ml for yeast RNA. The corresponding detection limits are 7 ng/ml for SM DNA and 25 ng/ml for yeast RNA, respectively. SM DNA can be determinated in the presence of 40% (w/w) RNA, and the relative standard deviation of six measurements is 2.5% for 500 ng/ml SM DNA. The result of the determination of golden staphylococcus DNA by this method was satisfactory.

A new surface-enhanced Raman scattering substrate based on silver nanoparticles in sol-gel

We report for the first time the development of a new surface-enhanced Raman scattering (SERS)-active silver chloride silica–glass substrate prepared by using in situ precipitation of silver chloride particles in sol–gel films as a precursor for nanoparticles of silver. The controlled precipitation of silver chloride was achieved by the reaction of silver nitrate with trichloroacetic acid, which leads to a slow release of chloride ions. Silver chloride particles were reduced to silver nanoparticles by FeSO4·7H2O. The sol–gel films prepared exhibit good optical properties and induce a strong SERS effect for several model compounds, including cresyl fast violet and brilliant cresyl blue. Optimization studies of substrate preparation and coating conditions were performed and are discussed in detail. The performance of this new substrate compares favorably with those of previously developed silver-coated alumina substrates. The SERS spectra of other compounds of environmental and biological interest, such as 1−aminonaphthalene, 2−aminopyrimidine, pyridine and p$-aminobenzoic acid, were investigated to illustrate the usefulness of this novel type of SERS substrate for use in chemical and biological analysis. Copyright © 1999 John Wiley & Sons, Ltd.

Electropolymerized Azines: Part II. In a Search of the Best Electrocatalyst of NADH Oxidation

A comparative investigation of catalytic activity of different polyazines in NADH electrooxidation is reported. The structure of azine monomers taken for electropolymerization was varied systematically changing both the second heteroatom and the substituents of aromatic rings. It was found that the monomer structure affects catalytic activity of the resulting polymer in the following way: (i) additional substitution of benzene ring by alkyl group reduced the catalytic activity, (ii) polymerized phenoxazine Brilliant Cresyl Blue was a better electrocatalyst than the corresponding phenothiazine (o-Toluidine Blue), (iii) ring substitution with only tertiary nitrogen atoms as ligands provides higher catalytic activity, (iv) higher redox potential of the polymer also provides higher catalytic activity.