A rapid procedure for the staining of chromosomes in fixed animal materials

This paper presents a very simple and reliable procedure for the staining of animal chromosomes employing dyes, such as pyronin G, acridine red, rhodamine B, rhodamine 3GO, belonging to aminoxanthene group and brilliant cresyl blue and methylene violet 3RD, belonging to quinone-imine group. The procedure has been tried on sections of the grasshopper and mouse testes fixed in Dutt's modification of Nawaschin mixture. The method is to deparaffinise sections and then to stain with aqueous solution of these dyes for 2--3 minutes, rinsed with water and dehydrated through grades of ethanol, keeping for 15--30 seconds in each grade with several dips. Preparations are then cleared in xylene and mounted. Stained preparations following this procedure revealed excellent colouration of the chromosomes at all the various stages of mitosis and meiosis, particularly in the case of the grasshopper. Mouse chromosomes stained with these dyes following the same method revealed perfect colouration of the fully condensed chromosomes at all stages of mitosis and meiosis but not of the very early stages, except the sex chromosome. Moreover, grasshopper testis sections when treated with cold concentrated phosphoric acid for varying time-periods and then stained with these dyes also revealed excellent colouration of the chromosomes. The implications of these findings have been discussed.     

Combining the gold chloride and toluidine blue stains to investigate chick embryo neural tissue

Toluidine blue and gold chloride stains are both well‐known for staining nervous tissue. Toluidine blue is a general method to stain neurons and glia, used because of the speed of action. Gold sublimate is a delicate procedure that requires extreme purity of reagents and materials, and it stains astrocytes and neurons. The aim of this study was to combine these two special staining methods for embryonic neural tissue and to obtain a repeatable and easily manageable new staining method with improved overall visualization of neural tissue. Fertilized Broiler hatching eggs were incubated at 36°C and on day 8 prepared for histology. Sections were cut at 7 μm thickness and slides were stained for 30 s using a 0.1% Toluidine blue solution. These slides were rinsed with dH₂O followed by placing them in gold sublimate for 4 h at room temperature in the dark. Rinsing with dH₂O and transferal to a 5% sodium thiosulfate solution was done, followed by another rinsing and mounting of slides. Results showed that the combination of the two methods offers a fast, reliable method with which chick embryonic neural tissue can be evaluated.     

Use of tris-buffer-fortified thionine-SO2 for detection of DNA-aldehyde following Feulgen procedure

This communication presents informations on the use of tris-buffer along with N HCl and potassium metabisulphite for the preparation of thionine-SO2 in staining DNA-aldehyde molecules of acid hydrolysed mammalian liver sections. It has been found that thionine, containing tris-buffer, N HCl and potassium metabisulphite, stains DNA-aldehyde molecules with better result than is possible with the control dye-SO2 reagent that does not contain this buffer. The absorption spectra of nuclei stained with this dye-reagent prepared with tris-buffer have also been presented. Further, it has been found that nuclei stained with the freshly prepared dye-SO2 reagent is bluish-violet, whereas those stained with an old dye-reagent is sky blue in colour. The reason for the slightly enhanced nuclear colouration with the experimental dye-reagent over the control has been considered to be due to slightly increased pH in the former as compared with that of the latter. The mechanism of staining with thionine-SO2 has been considered to be of Feulgen type.     

Use of aqueous solutions of two basic dyes for the demonstration of DNA

This paper presents informations as to the ability of aqueous solutions of two basic dyes, such as Dahlia and Victoria blue, belonging to aminotriarylmethane group for the staining of DNA-aldehyde molecules as well as DNA-phosphate groups. It has been found that sections of rat tissues stained with aqueous solutions of these dyes after acid hydrolysis followed by drying between folds of filter paper and treatment in n-butanol for a minute and then by a very brief treatment in a mixture consisting of equal parts of n-butanol and absolute ethanol reveal well-stained nuclei. Tissue sections after acid hydrolysis when stained with aqueous solutions of these dyes and then treated with SO2 water do not reveal any colouration of the nuclei. Since both the dyes are without any primary amino group in their molecules, it has been concluded that the imino group of Dahlia and the tertiary amino group of Victoria blue with cold concentrated phosphoric acid and then stained with any of these dyes also exhibit well-stained nuclei. The absorption spectra of nuclei stained with these dyes for DNA-aldehyde molecules as well as DNA-phosphate groups reveal positions of the peaks of maximum absorption at the same wavelength, which, however, are different in the case of nuclei stained with the two dyes. The implications of these findings have been discussed.     

Electron-irradiation-induced flattening of negatively stained 2D protein crystals

The thickness of negatively stained 2D crystalline arrays of the bladder membrane does not vary significantly during air drying and exposure to high vacuum. High-dose electron irradiation reduces the thickness to about 60% of the native value. These results, together with the fact that the same behaviour has been observed on another 2D system (gap junctions), indicate that the flattening induced by an electron beam on 2D crystals may be general. The implications for 3D reconstruction of negatively stained objects are discussed.     

Demonstration of DNA with an extra-sensitive Schiff's reagent and a Schiff-type dye-reagent, toluidine blue O-SO2

This paper describes a method for the preparation of Schiff's reagent as well as a Schiff-type dye-reagent, toluidine blue O-SO2 for use in Feulgen procedure. The method involves replacement of the usual N HCl by N H2SO4 and the usual amount of potassium metabisulphite. Following this method of preparation, an extra-sensitive Schiff's reagent is obtained which requires only 4-5 min for optimum nuclear colouration even when staining is performed at 5 degrees C. This Schiff's reagent produces perfect Feulgen staining up to 6 months after preparation. Toluidine blue O-SO2, prepared with N H2SO4 and potassium metabisulphite, also produces perfect Feulgen type staining of the DNA-aldehyde molecules of acid-hydrolysed mammalian tissue sections. Toluidine blue O-SO2 when shaken with activated charcoal and filtered produces very satisfactory result. The shell-life of this dye-reagent is just a week. The suitability of the use of N H2SO4 for the preparation of Schiff's reagent as well as a Schiff-type dye-reagent, toluidine blue O-SO2, has been discussed.     

Chromoxane cyanine R. I. Physical and chemical properties of the dye and of some of its iron complexes

Chromoxane cyanine R (Colour Index No. 43820, Mordant blue 3; also known as eriochrome cyanine R and solochrome cyanine R) is a valuable biological stain. The dyestuff is supplied as a powder containing a little less than 50% by weight of the monosodium salt of the dye, mixed with colourless crystalline and amorphous fillers. The tetrabasic colour acid was prepared and purified for study of the chemical and spectral properties of the dye. Chromoxane cyanine R is an acid-base indicator, with five different colours corresponding to the colour acid and the four anions. The most conspicuous colour change, from yellow to blue, occurs with ionization of the phenolic hydroxyl group at pH 11–12. The dye is assayed by measuring the absorbance of a strongly alkaline solution at 585 nm, with reference to a standard solution prepared from the purified colour acid. Spectrophotometric evidence has been found for the existence of three dye-metal complexes in solutions of the dye containing added ferric chloride at pH 1·5 (the pH of iron-dye solutions most useful in histological staining). These have the postulated compositions [Fe₂H(dye)]-(red), [FeH₂(dye)]- (red), and [Fe₂(dye)]^2- (blue). The first two are probably simple carboxylate complexes of low stability. Increase in pH or molar iron:dye ratio promotes formation of the more stable blue complex, which is a metal chelate. Other blue complexes have been described by other investigators in solutions less acid than those that are useful in microtechnique. The production of blue and various shades of red in tissues stained by solutions containing iron(III) and chromoxane cyanine R probably involves reactions of both the red and the blue complexes of the dye.     

Detection of DNA in mammalian tissues following removal of RNA with cold phosphoric acid.

The paper deals with the staining of nuclei in mammalian tissue sections with night blue, belonging to diphenylnaphthylmethane group and is devoid of any primary amino group in its molecules but is provided with a secondary amino group and tertiary amino groups. Staining of the DNA-phosphate groups with an aqueous solution of night blue depends upon selective removal of RNA from formalin-fixed mammalian tissues by the use of cold concentrated phosphoric acid for 20 min or 75% phosphoric acid in the cold for 2 h. Moreover, sections from which RNA has been extracted can be hydrolysed in 6N HCl at room temperature for 15 min and then can be stained with the aqueous solution of the dye. Sections of tissues after only acid hydrolysis and staining also reveal very satisfactory staining of the nuclei. Possible mechanism of staining has been suggested.     

Chromoxane cyanine R. II. Staining of animal tissues by the dye and its iron complexes

The staining properties of chromoxane cyanine R (Colour Index No. 43820, Mordant blue 3; also known as eriochrome cyanine R and solochrome cyanine R) have been studied. Used alone, the dye imparted its red colour to nuclei, cytoplasm and collagen. The dye was extracted by mild alkali but not by acids. Stainability required ionized amino groups in the tissue, and there was also evidence for non-ionic binding of the dye. The colours obtained by staining with mixtures of chromoxane cyanine R and ferric chloride varied with the molar iron: dye ratio and with the pH. Useful staining was seen only between pH 1 and 2. The tissues were coloured either all blue (when Fe: dye was high), or both red and blue (when Fe: dye was low). Lower pH favoured the deposition of red, higher pH the deposition of blue colour. The red was mainly in cytoplasm, blue in nuclei and myelin. Collagen fibres were red or purple, depending on pH and iron: dye ratio. Red colours were differentiated by acid and changed to blue, but not extracted, by mild alkali. The red substance in the stained sections was clearly not the free dye, so it was probably an iron-dye complex. From the effects of various differentiating agents, it was deduced that the red and blue dye-metal complex molecules were bound to the tissue by the dye moiety, not by interposition of iron atoms. Staining by the complexes of iron(III) with chromoxane cyanine R did not involve nucleic acids or other polyanions or the amino groups of proteins. There was evidence for only non-ionic binding of both red and blue complexes. It is suggested that the red colour in sections stained by solutions with low iron: dye ratio is due to a simple carboxylate complex, [Fe₂H(dye)]^?. The blue colour would then result from withdrawal of a proton from the red complex to give [Fe₂(dye)]^2-. The bases that remove the protons may be arginine-rich nucleoproteins of nuclei and phospholipid bases of myelin. Techniques are described for informative simultaneous staining in two colours, and for the selective staining of either nuclei or myelin.     

Nanocomposite polymer electrolyte based on rice starch/ionic liquid/TiO2 nanoparticles for solar cell application

Nanocomposite polymer electrolyte (NCPE) was prepared using solution cast technique. Rice starch (RS), lithium iodide (LiI), 1-methyl-3-propylimidazolium iodide (MPII) as ionic liquid and TiO₂ nanopowder (RS:LiI:MPII:TiO₂) were introduced to prepare the sample. The conductivity of 3.63 × 10⁻⁴ S/cm was achieved by introducing 30 wt.% of 1-methyl-3-propylimidazolium iodide (MPII) as ionic liquid and 2 wt.% of TiO₂. Temperature-dependent conductivity and dielectric behavior were analyzed in this work. Dye sensitized solar cell was fabricated using the nanocomposite film for this sample and analyzed.     

Specific staining of nuclei with aqueous solutions of celestin blue B and gallocyanine

This paper presents methods for specific staining of nuclei with aqueous solutions of celestin blue B and gallocyanine in tissue sections from which RNA has been extracted selectively with concentrated phosphoric acid at 5 degrees C for 20 min or by hydrolysis in 6 N HCl at 28 degrees C for 15 min. It has been found that pH of the freshly prepared celestin blue B dye solution is 3.0 and that of an aqueous solution of gallocyanine is 2.8. These pHs can be lowered to 1.5 with concentrated sulphuric or nitric acid and at this pH staining of the nuclei is possible. But with concentrated sulphuric or nitric acid and at this pH staining of the nuclei is possible. But if the pHs are lowered with concentrated hydrochloric or phosphoric acid, effective use of these dyes is not possible. It has been suggested that some dispersion of the two dyes takes place with concentrated sulphuric or nitric acid which are used to lower the pH. Staining of the nuclei is also possible with an aqueous solution of celestin blue B at pH 3.0 but the same is not possible with gallocyanine at pH 2.8. The absorption spectra of nuclei stained with an aqueous solution of celestin blue B at pH 1.5 and 3.0 are fairly identical, the peak of maximum absorption being at 620 nm. Those of nuclei stained with an aqueous solution of gallocyanine reveal irregular peaks. Possible implications of these findings have been discussed.     

AutoIHC‐scoring: a machine learning framework for automated Allred scoring of molecular expression in ER‐ and PR‐stained breast cancer tissue

In prognostic evaluation of breast cancer Immunohistochemical (IHC) markers namely, oestrogen receptor (ER) and progesterone receptor (PR) are widely used. The expert pathologist investigates qualitatively the stained tissue slide under microscope to provide the Allred score; which is clinically used for therapeutic decision making. Such qualitative judgment is time‐consuming, tedious and more often suffers from interobserver variability. As a result, it leads to imprecise IHC score for ER and PR. To overcome this, there is an urgent need of developing a reliable and efficient IHC quantifier for high throughput decision making. In view of this, our study aims at developing an automated IHC profiler for quantitative assessment of ER and PR molecular expression from stained tissue images. We propose here to use CMYK colour space for positively and negatively stained cell extraction for proportion score. Also colour features are used for quantitative assessment of intensity scoring among the positively stained cells. Five different machine learning models namely artificial neural network, Naïve Bayes, K‐nearest neighbours, decision tree and random forest are considered for learning the colour features using average red, green and blue pixel values of positively stained cell patches. Fifty cases of ER‐ and PR‐stained tissues have been evaluated for validation with the expert pathologist's score. All five models perform adequately where random forest shows the best correlation with the expert's score (Pearson's correlation coefficient = 0.9192). In the proposed approach the average variation of diaminobenzidine (DAB) to nuclear area from the expert's score is found to be 7.58%, as compared to 27.83% for state‐of‐the‐art ImmunoRatio software.     

Tribological properties of solid lubricants (graphite,Sb2S3, MoS2) for automotive brake friction materials

The effect of three different solid lubricants (graphite, Sb₂S₃, and MoS₂) in the brake friction material on various aspects of friction characteristics was investigated. Three friction material specimens were produced based on an experimental formulation, and they contained 10 vol.% graphite, 7 vol.% graphite + 3 vol.% Sb₂S₃, and 7 vol.% graphite + 3 vol.% MoS₂, respectively, fixing the composition of other ingredients. Tribological properties of the friction materials were obtained using a brake dynamometer. Results showed that the friction materials containing Sb₂S₃ and graphite improved friction stability and fade resistance. The friction materials with Sb₂S₃ and MoS₂, on the other hand, exhibited disadvantages in terms of wear resistance, anti-fading, and DTV (disk thickness variation) generation.     

Negative staining-carbon film technique: New cellular and molecular applications

Two techniques are presented which extend the original negative staining-carbon film technique into new areas of cellular and molecular application. These relate (1) to the production of negatively stained specimens of single-layer plasma membrane split from intact cells during the overall procedure that are negatively stained from the cytoplasmic face and (2) to the production of negatively stained specimens directly from glycerol-containing protein solutions, membrane or viral suspensions. In both cases in vacuo drying onto mica from glycerol is performed, prior to deposition of a carbon film. (For the cellular technique, freshly cleaved mica is firstly rendered positively charged by immersion in Alcian blue.) This is followed by release of the carbon film plus adsorbed membrane or protein by floating onto water, with subsequent negative staining. Selected preliminary applications using human erythrocyte membrane and the high molecular weight (native) human erythrocyte tripeptidyl peptidase-II complex are given and considered speculation as to the future application of the techniques is provided.     

Formation and tribological properties of hollow sphere-like nano-MoS2 precipitated in TiO2 particles

MoS₂ hollow spheres with an average diameter of 165 nm were prepared from Na₂MoO₄ and CH₃CSNH₂ at 82 °C. A simple method was used to obtain smaller hollow spheres (70 nm) without any complicated step, except for the addition of TiO₂. The tribological properties of MoS₂/TiO₂ in rapeseed oil were studied using a four-ball tribometer under 350 N at 0.383 m/s. The effects of load and sliding velocity were also investigated. Wear was significantly alleviated by the produced lubricating film as lubricated with MoS₂/TiO₂, which was composed of MoO₃, Fe₂O_3, Fe₂(SO₄)₃, TiO₂ (trace), and carbon-containing compounds. The tribological properties were also improved because of the decrease in the size of MoS₂ and the synergistic effect between MoS₂ and TiO₂.     

Preparation,characterization, electrical conductivity and dielectric studies of Na2SO4 and V2O5 composite solid electrolytes

Composite solid electrolytes (1 − x) Na₂SO₄–(x) V₂O₅ were prepared and characterized by various techniques such as XRD, FT-IR, DTA and SEM. AC impedance spectroscopy revealed that the contribution of grain is strong enough over the grain boundary. Arrhenius plot of the Na₂SO₄ shows a sharp increase in conductivity at 523 K due to the structural phase transition (phase V → I). Composites show the enhanced ionic conductivity than the pristine Na₂SO₄ over the entire temperature range. The maximum conductivity σ = 0.003 S cm⁻¹ at 773 K with the lowest activation energy of 0.28 eV was observed for the x = 0.4 sample. The enhanced value of dielectric constant and dielectric loss in the case of composites was obtained because of increase of conductivity, resulted from the increase of space charge polarization and charge motion.     

Effect of TiB2 particles on sliding wear behaviour of Al–4Cu alloy

Dry sliding wear of Al–4Cu–xTiB₂ (x = 0, 2.5, 5, 7.5 and 10 wt.%) in situ composites have been studied in the peak-aged condition using a pin-on-disc wear testing machine at different loads. The composites were prepared by the reaction of a mixture of K₂TiF₆ and KBF₄ salts with molten alloy. The results indicate that TiB₂ particles markedly improve the wear performance of the Al–4Cu alloy. The wear resistance increases with increase in the amount of TiB₂. The load bearing capacity of the alloy during wear increases in presence of TiB₂ particles. Study of the wear surfaces and debris of both alloy and composites using the scanning electron microscope suggests that the improvement in wear resistance is mainly due to the formation of finer debris.     

工程数字化系统在番禺4—2／5—1调整项目组的应用

工程数据中心系统的成功上线,标志着中海石油工程建设领域一个全新时代的开始。本文首先分析了工程数字信息化的必然性;其次,概述了番禺4-2／5—1调整项目的情况;最后,就工程数字化系统在番禺4—2／5—1调整项目组的应用进行了深入的探讨。     

Preparation of a new red dye from Janus black and its use in the staining of DNA-aldehyde molecules

This communication presents a method for the preparation of a new red dye from an aqueous solution of Janus black by adding NHC1 and sodium thiosulphate to it. This new red dye when used on acid-hydrolysed tissue sections reveals the presence of red nuclei when sections after staining are dried between folds of filter paper, differentiated in n-butanol, cleared in xylene and mounted. Similarly stained sections when treated with SO2 water show partial leaching of the dye from the nuclei. Tissue sections when treated with cold concentrated phosphoric acid for 20 min and then stained with an aqueous solution of Janus black reveal the presence of orange-red nuclei. The new red dye obtained from Janus black does not respond to treatment under UV rays. The in vitro absorption data of the red dye indicate peaks at 210, 270 and 545 nm. The in situ absorption spectra of nuclei stained with the new red dye following Feulgen procedure reveal the peak of maximum absorption at 560 nm and those of nuclei treated with cold concentrated phosphoric acid and then stained with this red dye reveal peak at 530--540 nm. Some relevant points raised out of this investigation have been discussed.     

Effect of CuO as a dopant in TiO2 on ammonia and hydrogen sulphide sensing at room temperature

The heterogeneous nanocomposites of CuO doped TiO₂ nanoparticles were synthesized using sol gel method by varying the concentration of CuO as 0.1, 0.5 and 1 mol% for the sensing of ammonia and hydrogen sulphide. The substitutional doping of CuO in TiO₂ matrix was confirmed by the X-ray diffraction. Average crystallite size of the doped nanocomposites was found to reduce with increase in concentration of CuO. The 0.1 mol% CuO doped TiO₂ nanocomposites showed highest sensitivity to ammonia (97%) with response time of 2 s, while 1 mol% was selective to H₂S gas (77%) with response time of 45 s for 50 ppm of each gas at room temperature.