Azure B staining of DNA-aldehyde molecules of acid-hydrolysed tissue sections

This communication presents a method for the preparation of azure B-SO2 with trichloracetic acid (TCA) and potassium metabisulphite for in situ demonstration of DNA-aldehyde molecules following acid hydrolysis of tissue sections. The shelf-life of such a dye-reagent is slightly more than that of the control, prepared with N HCl and potassium metabisulphite. The slightly increased shelf-life of the experimental dye-reagent has been considered to be due to a somewhat higher pH as compared with that of the control. The in situ absorption characteristics of nuclei stained for DNA-aldehyde molecules with either an aqueous solution of azure B or with TCA-azure B-SO2 show peak-absorption at 600 nm in both cases. This phenomenon has been interpreted as due to the fact that azure B does not contain any primary amino group in its molecules and, therefore, the mode of binding of DNA-aldehydes with this dye is different from that with dyes that contain primary amino group. The implications of some of the findings have been discussed.     

In situ demonstration of DNA with Schiff-type dyes prepared with meta-phosphoric or tartaric acid

A new method for the preparation of azure A-SO2 and safranine-SO2 For use in Feulgen procedure has been described herein. The method involves the use of m-phosphoric acid or tartaric acid in place of N HCl in the preparation of these eye-reagents which exhibit enhanced pH producing increased staining intensity of the nuclei as compared with those of the controls, prepared with N HCl. Possible explanation for the increased staining intensity as well as the reason for the shorter shelf-life of these eye-reagents have been offered.     

Feulgen type staining with Hoffmann's violet-SO2 under exposure to UV rays.

The paper contains an account of the use of Hoffmann's violet-SO2 under exposure to UV rays during staining acid-hydrolysed DNA of mammalian tissue nuclei. Preparations stained with Hoffmann's violet-SO2 without exposure to UV rays reveal extremely pale violet nuclei but when stained under the influence of UV rays show a considerably faster reaction resulting in a very much deeper staining of the nuclei. Sections after staining with this dye-reagent require n-butanol as differentiating reagent. Possible interpretation for the increase in staining ability of this dye-reagent under exposure to UV rays has been elucidated and the reason for considering the reaction as Feulgen type has been discussed.     

Staining of DNA-phosphate groups with a mixture of azure A and acridine yellow.

This paper deals with staining of DNA-phosphate groups with a mixture of an equal parts of aqueous solution of azure A and acridine yellow in a 1:1 proportion and also embodies a study of the absorption properties of the stained nuclei. It also embodies results of sequential staining of nuclei stained first with azure A followed by staining with acridine yellow and vice versa, after extraction of RNA with cold phosphoric acid. The results indicate that the absorption peaks of nuclei differ from those of nuclei stained for DNA-aldehyde molecules with azure A-SO2 or acridine yellow-SO2. The in vitro absorption characteristics of an aqueous solution of azure A and those of an aqueous solution of acridine yellow are also presented herein. The conclusion obtained from this study is that all the phosphate groups of DNA do not take part in the staining process when staining is carried out with azure A or acridine yellow alone after after RNA has been extracted. This is because the nuclei stained with these dyes sequentially show the presence of acridine yellow-DNA and azure A-DNA complex.     

Shelf-life of citric acid-toluidine blue O-SO2 and its influence on Feulgen staining.

The paper reports on the preparation of a dye-SO2 reagent employing a Schiff-type dye, toluidine blue O. The method is to replace 5 ml of N HCl per 100 ml of the dye solution by citric acid. The usual potassium metabisulphite is then added. The pH of this new modified dye-SO2 reagent is 2.5 as against 1.6 for the hydrochloric acid-toluidine blue O-SO2. The shelf-life of this newly developed dye-SO2 reagent is four weeks with appreciable reduction of staining intensity after this period as compared with that of a freshly prepared dye-reagent with N HCl. A possible interpretation for the observed phenomenon has been suggested.     

Preparation of Schiff-type dye-SO2 reagents with phosphoric acid and their cytochemical properties

A new method employing phosphoric acid in place of hydrochloric acid for the preparation of azure A-SO2 and toluidine blue O-SO2 has been described in this communication. It has been found that o-phosphoric acid in a weak concentration can be a suitable substitute for hydrochloric acid in the preparation of these dye-SO2 reagents. These dye-reagents produce excellent blue nuclei in human uterine tumour. Toluidine blue O-SO2 fortified with an amino aicd, glycine also produces excellent blue chromosomes in the squash preparations of grasshopper testes fixed in acetic acid-alcohol. The shelf-life of these two dye-reagents has also been presented herein.     

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.     

Propionic acid-metabisulphite-Schiff reagent for quick Feulgen staining

This communication presents a method for the preparation of Schiff reagent in which N hydrochloric acid has been replaced by a low concentration of propionic acid. The result of using such a Schiff reagent indicates that the dye-reagent thus prepared is extrafast in action on acid-hydrolysed mammalian tissue sections. The intensity of nuclear colouration is also increased considerably, since the pH of the dye-reagent is 6.0. It is, therefore advocated that this newly developed Schiff reagent be used at 5 degrees C.     

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.     

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.     

Hydrochloric acid-sodium thiosulphate-Schiff reagent and its cytochemical properties.

Cytochemical properties of modified Schiff reagent, prepared with 5 ml of N HCl and 1.0 g of sodium thiosulphate, the latter replacing potassium metabisulphite, have been presented in this communication. It has been seen that such a modified Schiff reagent has a slightly elevated pH as compared with that of the conventional Schiff reagent. It has also been found that this modified Schiff reagent has a much longer shelf-life and that the staining intensity produced by this dye-reagent is more than those of the usual Schiff reagent prepared with N HCl and potassium metabisulphite (potassium pyrosulphite, K2S2O5). Possible mechanism as to why the staining intensity of the nuclei is more with this modified Schiff reagent as well as the reason as to why its shelf-life is increased compared with those of the conventional Schiff reagent have been discussed.     

Microspectrophotometric studies of Romanowsky stained blood cells. I. Subtraction analysis of a standardized procedure

This paper describes a microspectrophotometric study of blood smears stained by a simple, standardized Romanowsky technique, using only the dyes azure B and cosin. Absorbance spectra are presented for twenty-two classes of cellular object, and for the two dyes in solution, together with tabulations of spectral maxima, and suitable wavelengths for use in automated image processing. The colours of objects stained with azure B/eosin are discussed in terms of absorbance spectra. By a spectral subtraction technique, it is shown that the differential colouration of various cell structures may be explained satisfactorily in terms of the varying proportions of only four dye components. These are the monomers and dimers of azure B and eosin. Polymerization was found to occur both in solution and on binding to biopolymers. A similar analysis of a conventional Romanowsky stain would present much greater difficulties, due to the greater number of dye components, which, however, contribute little to the colours observed.     

The role of carbamino compounds in histological staining

The effects of high CO₂ concentration on tissue staining with buffered eosin, methylene blue and azure A were investigated to determine if carbamino compound formation could alter dye binding. Only in the case of eosin staining were the differences in results with high and low CO₂ concentrations compatible with the formation of carbamino compounds. The differences observed with basic dyes were statistically significant but could not be accounted for by carbamino compound formation.     

搅拌棒萃取-气相色谱-质谱法分析地表水中合成麝香及紫外线吸收剂

建立了搅拌棒萃取-溶剂解吸结合气相色谱-质谱法测定水样中16种合成麝香及2种紫外线吸收剂。实验优化了萃取温度、萃取时间、萃取体积、pH值、离子强度、溶剂解吸条件等参数，最终确定使用50 mL水样在40 ℃下搅拌提取4 h后，采用1.2 mL溶剂超声解吸后直接浓缩分析。该方法的线性范围为2~120 μg/L，线性相关系数大于0.996，各化合物的加标回收率在68.5%~126%之间，相对标准偏差范围为2.3%~14.9%，方法检出限在0.003~0.038 μg/L之间。应用该方法分析北京市内不同采样点的7个地表水样品，结果表明，佳乐麝香的检出率最高，浓度在0.003~0.337 μg/L之间。该方法操作简单、有机溶剂使用量少，可以实现分析测试快速、绿色的目的，能够用于实际水样中合成麝香和紫外线吸收剂的同时准确分析。     

Acridine orange--its use in the specific staining of DNA in mammalian tissue sections

This paper reports on a new method for the use of acridine orange (AO) in an aqueous solution at pH 4.5 for staining DNA of rat tissue sections from which RNA has been extracted selectively with cold phosphoric acid. Not only this, AO can also be used as dye-SO2 reagent, prepared with NHCl and potassium metabisulphite, for staining DNA-aldehyde molecules of acid-hydrolysed tissue sections. AO samples, manufactured by the National Aniline Division as well as by G. T. Gurr have been used with equal success. Studies of stained sections under light microscope reveal the presence of specifically stained yellowish-orange nuclei. Those sections under fluorescent microscope with proper exciter and barrier filters reveal nuclei of maroon colour. The in situ absorption spectra of nuclei stained with AO-SO2 following acid-hydrolysis of tissue sections as well as those of nuclei stained with an aqueous solution of the dye following extraction of RNA have been presented herein. The mode of binding in the former case has been considered to be due to binding of the teritary amino group of the dye molecules with the DNA-aldehyde molecules and in the latter case to be due to electrostatic binding between the positively charged dye molecules with negatively charged phosphate groups of DNA. Implications of all these findings have been discussed.     

5-氯水杨酸工业品的高效液相色谱分析

本文研究了用反相高效液相色谱测定5-氯水杨酸工业品及其杂质水杨酸,3-氯水杨酸和3,5-二氯水杨酸含量的方法。样品在ODS柱上进行分离,流动相为甲醇-稀高氯酸水溶液,紫外226nm检测,方法简便快速,结果准确可靠。     

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.     

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.     

Improved staining procedures for photographic documentation of phenolic deposits in semithin sections of plant tissue

Several modifications of the toluidine blue O and safranin O staining procedures and a differential staining method with safranin O/azure II are described. These modifications include prestaining oxidation with sodium hypochlorite and/or poststaining treatment with iodine/potassium iodide. Greatly enhanced contrast of phenolic vacuolar inclusions is achieved by prestaining oxidation. Additionally, poststaining treatment with iodine results in a characteristic colour change of all stained tissue constituents except phenolic vacuolar inclusions. In particular, cellulose walls and cytoplasm exhibit a marked shift in colour from violet to brown (toluidine blue O) or from red to orange (safranin O). It is concluded that the methods presented can be recommended for superior photographic documentation of vacuolar polyphenol deposition in semithin sections of glycol-methacrylate-embedded plant tissue.     

光纤pH传感器的设计

设计开发了一个用于环境水质 p H值监测的光纤传感系统。采用5发光二极管作光源 ,硅光电二极管作检测器 ,大芯径、大数值孔径的 Y型分叉光纤束作传感介质 ;染料指示剂苯酚红以两种方法固定 ,设计了相应的两种探头结构。系统中提出了双 L ED双光束补偿的设计思想 ,对两种探头的传感机理做了归一性推导。该系统体积小 ,价格低廉 ,性能稳定 ,响应灵敏度可达 0 .0 1 p H。