Preparation of Schiff's reagent. Histochemical detection of polysaccharides

SCHIFF REACTIVE

Aqueous solution of fuchsinsulfur solution (forms I or II). Serves for qualitative detection of aldehydes. When interacting Sh.r. with the aldehyde RCHO a purple-violet dye (form III) is formed.

The reaction is very sensitive (for example, 1 mcg of formaldehyde can be detected). At the same time aromatic. hydroxyaldehydes, many unsaturated ones, do not give coloring. Some (eg.), unsaturated compounds, inorg. and org. bases, a number of salts capable of hydrolysis, and all compounds that oxidize sulfur dioxide, lead to the formation of a pink color. Therefore, the appearance of a light pink color cannot be considered positive. Aldehyde test.
Sh.r. obtained by passing SO 2 into a 0.025% aqueous solution of fuchsin until discolored. When determining aldehyde groups, it is necessary to preliminarily. bring the acidity to pH 3 by adding to the Sh. to-you or a buffer mixture. Usually a control experiment is carried out. When heated The solution of fuchsin-sulfur may make you blush.
The method underlying the use of Sh. R. was discovered by G. Schiff in 1864.

Lit.: Guben-Weil, Methods of organic chemistry, trans. from German, 4th ed., vol. 2. Methods of analysis, M., 1963, p. 431-32; Identification of organic compounds, trans. from English, M., 1983, p. 195-96; Mazor L., Methods of organic analysis, trans. from English, M., 1986, p. 122-24.

Ya. A. Kutseva.

Chemical encyclopedia. - M.: Soviet Encyclopedia. Ed. I. L. Knunyants. 1988 .

See what "SCHIFF REACTIVE" is in other dictionaries:

- (H. Schiff; synonym fuchsinous acid) a reagent for the qualitative determination of the aldehyde group of organic compounds, discovered by Hugo Schiff. Contents 1 Receipt 2 Storage ... Wikipedia

- (N. Schiff, 1834 1915, German biochemist) a reagent for the qualitative determination of the aldehyde group of organic substances, which is a solution of fuchsinous acid; used for histochemical detection of carbohydrates and... ... Large medical dictionary

Schiff's reagent- fuchsinous acid A reagent intended for the qualitative determination of aldehyde groups; used in the Feulgen reaction for DNA detection, as well as in a number of other tests (for glycogen, etc.). [Arefyev V.A., Lisovenko L.A. English Russian... ... Technical Translator's Guide

Schiff's reagent Schiff's reagent, fuchsulfuric acid. A reagent intended for the qualitative determination of aldehyde groups; used in the Feulgen reaction when identifying DNA, as well as in a number of other tests (for... ... Molecular biology and genetics. Dictionary.

An aqueous solution of fuchsin, decolorized with sulfur dioxide. In the presence of aldehydes, this solution turns blue.

Schiff's reagent.

What it is?

Schiff's reagent is a solution that combines chemically with aldehydes to form a bright red product. Strictly speaking, ketones also react, but for all practical purposes they can be ignored. Many fabric components can be dyed this way. Schiff's reagent is made from pararosaniline treated with sulfurous acid (H2SO3). This is the reason for the destruction of the chromophore by the addition of a sulfonic acid group to the central carbon. Sulfurous acid is formed by dissolving sulfur dioxide (SO2) in water.

In some, usually older texts, you will find Schiff's reagent referred to as leukofuchsin . Console leuko means white and refers to the loss of color in a solution. However, leucobase refers to reduced compounds, and its color is restored by oxidation. Schiff's reagent does not have the original color of pararosaniline. Rather, the new color compound is produced by a chemical combination with an aldehyde. For this reason it is usually now referred to as Schiff's reagent or fuchsulfuric acid.

There doesn't seem to be much difference regarding the source of the sulfurous acid. Because it is simply sulfur dioxide dissolved in water. There are several ways to get it. Four procedures that were recommended:

· Sulfur dioxide gas from the cylinder is slowly bubbled through the pararosaniline solution until the solution begins to change color. The container is then carefully closed and kept in a dark place, usually until the morning or longer.

· Buy a ready-made solution of sulfurous acid. Pararosaniline dissolves in it without high temperature. The container is then carefully closed and kept in a dark place, usually until the morning or longer.

· Sodium or potassium metabisulfite or sulfite (commercial examples of which are apparently composed primarily of metabisulfite) are added to the pararosaniline solution. Hydrochloric acid is added, producing sulfur dioxide in solution. The container is then carefully closed and kept in a dark place, usually until the morning or longer.

Reagents that decompose in solution to produce sulfur dioxide, such as sodium hydrosulfite (dithionite) or thionyl chloride, are added to the pararosaniline solution. The container is then carefully closed and kept in a dark place, usually until the morning or longer.

Schiff's reagent should be colorless or very pale yellow. However, pararosaniline may contain other dyes, especially if they are from a basic fuchsin sample that is a mixture. Other dyes are usually homologues of pararosaniline, such as rosaniline and magenta II, but produce deep amber solutions and can dye aldehydes brownish red. These darker products can be removed by adding a small amount of activated charcoal powder, shaking the solution for a minute and filtering. Sometimes this treatment does not work and the solution remains brown. Unfortunately, if enough charcoal is added to completely remove all the brown color by bleaching, the final clear solution may only produce a very pale color. This usually means that a sample of basic fuchsin was used which contains large quantities of one of the other dyes. If possible, buy samples of basic fuchsin listed as suitable for Schiff's reagent, or buy pararosaniline. As stated above, Schiff's reagent combines with aldehydes to produce a bright red product. In histology, these aldehydes are attached to, or formed from, tissue structures, turning a very bright red color. For all aldehydes in tissues, the mechanism is the same. The aldehyde condenses with Schiff's reagent to form a new compound, attaching to the tissue. The process converts the chromophore and produces color. Fuchsinous acid. Schiff's reagent Aldehyde Regular product formula. Alternative product formula. Note the converted double bonds in the product (highlighted in red) due to aldehyde addition. The new red product comes in two formulas. Most explanations are given by the first one - this is the final product. However, some texts give an alternative representation involving one aldehyde. In either case, the colored product consists of a Schiff's reagent, an aldehyde, and the reagent to which the aldehyde is attached. Kiernan reported that recent research indicates that the product is distinct from the above and exists as a tautomer of the two products presented below. To make Schiff type reagents, other dyes can be used. They are often referred to as pseudo-Schiff reagents. They are usually colored, not completely bleached, although the color and clarity of the solution often varies. It is possible that residual color compounds in these solutions may stain tissues through an ionic mechanism. To remove this stain, it is common practice to treat the sections with acidic alcohol after staining. Any color remaining after treatment with acidic alcohol is a positive reaction from the pseudo-Schiff reagent. None of these pseudo-Schiff reagents have achieved anywhere near the popularity of the pararosaniline Schiff reagent, and this solution remains the standard. The reason is the bright and clear red color of the pararosaniline Schiff reagent. Other dyes produce weak color, although few are truly effective. Solutions that have become more common are made with a fluorescent dye, acriflavine (see below), for example. They provide a fluorescent positive result which can be very useful for demonstrating materials such as mushrooms, for example, which may be present in small quantities. Culing provides a list of many of these dyes, several of which are listed here. For more information, see Kasten's writings listed below Culing. Name CI No. Color Name CI No. Color Sour fuchsin violet Acriflavine Crystal Violet Blue-violet. Methyl violet purple. Methylene blue Safranin O Toluidine blue When tissues are removed from the Schiff reagent and washed, they are inevitably transferred to the washing liquid. If tap water is used, the reagent quickly changes color and the water becomes very red. Initially, there was some concern that this re-staining Schiff reagent would behave like a base dye and stain tissue, giving false positive results. For this reason, sulfite rinses were recommended. They dissolve sulfurous acid, and are used to wash off Schiff's reagent for a few minutes, dissolving it well enough before washing the fabrics in water, ensuring that there is no chance of non-aldehyde staining. Experience over the years has shown that these rinses are not necessary and a good wash in tap water is satisfactory as long as the sections are not left in Schiff's reagent, which restores color. If pseudo-Schiff reagent is used, the sections should be treated with 1% hydrochloric acid in 70% ethanol for 5-10 minutes after this wash. Some of them are colored, cannot be completely bleached and can dye by an ionic mechanism. Acidic alcohol will remove any such staining. After treatment with acidic alcohol, sections should be washed with tap water as usual. Although Schiff's reagent is used to detect aldehydes, they are not usually found free in tissues, and must be produced in some way. There are three ways this can be done. The first, and by far the most common, is the oxidation of some tissue components. This produces aldehyde groups, allowing the material to which the component is attached to be exposed. The second way is to treat the sections with acids to convert some of the deoxyribose into DNA and aldehydes and then stain them with Schiff's reagent. The third is to apply the aldehyde directly to the tissue, usually a protein, then demonstrate the aldehyde with Schiff's reagent. This latter can be seen easily with glutaraldehyde fixation for light microscopy. Glutaraldehyde has two aldehyde groups and, if molecules are attracted to the tissue of one of them, the other remains unbound when fixation is completed. Because of this, with any procedure using Schiff's reagent, single aldehydes will react with it, producing a deep pink background. So, before Schiff's reagent can be applied to these sections, the aldehydes must be blocked so they cannot react. For any precision application using this reagent, two controls must be used. The first is a slice to which aldehyde blocking has been applied. The second is a section in which Schiff's reagent was applied to the tissue without any pretreatment. The first cut thus blocks any pre-existing aldehydes, while the second highlights the sites where they were located. In addition, of course, sections with established negative and positive controls for the target tissue element must be used. If these last two controls do not stain as expected, then the Schiff's reagent, and no others, must be examined to ensure that it has not deteriorated and is suitable for use. Histochemistry: Theoretical and Applied Ed. 3 Pearse, A. G. E., 1968, 1972, v.1, pp.448 Churchill Livingstone, Edingburgh, London, UK Histological and histochemical methods: Theory and practice Ed. 3 Kiernan. J. A., 1999, pp.203 Butterworth Heinemann Oxford, UK. Handbook of histopathological and histochemical techniques Ed. 3 Culling C. F. A., 1974, pp. 249, 250 Butterworth London, UK. citing: Kasten, F.H., 1958 Stain Technology, vol.33, pp.39and Kasten, F.H., 1959 Histochemie, vol.1, pp.466 Histopathologic technique and practical histochemistry Ed.2 Lillie, R. D., 1954 Blakiston New York, USA. http://stainsfile. info/StainsFile/stain/schiff/schiffwhatis. htm Periodic acid–Schiff reaction Periodic acid – Schiff The PAS reaction is used to demonstrate the presence of 1,2 glycols and is therefore an important technique in carbohydrate histochemistry and the histological demonstration of many structures. Solutions 1% aqueous periodic acid Reagent Schiffa Gemalaun Mayer Fixing and wiring Most fasteners and wiring methods are satisfactory. Glutaraldehyde fixation requires blocking aldehydes before painting. Method 1. Bring sections to water through xylene and ethanol. 2. Place in periodic acid for 10-30 minutes. 3. Rinse well in tap water. 4. Rinse in distilled water. 5. Place in Schiff's reagent for 10-30 minutes. 6. Wash with distilled water. 7. Wash well in tap water for approximately 10 minutes. 8. Finish with Mayer’s hemalaun for 2 minutes. 9. Wash well with tap water until Mayer’s hemalaun turns blue. 10. Dehydrate with ethanol, clear with xylene and embed in a resinous medium. Expected results Notes · Glutaraldehyde fixatives release free aldehyde groups by attracting them to tissues. They cause a complete false positive reaction. This must be stopped from reacting by an appropriate procedure such as aniline-acetic blocking. · The tap water in step 7 is needed for the red color to develop. Within certain limits, longer wash times produce a darker color. · Initially, it was recommended that Schiff's reagent be washed by dissolving sulfurous acid (sulfite rinse). While water returned the color to the Schiff reagent, it was assumed that the water washed out could lead to false positive results. It is now known that this is not the case if the Schiff's reagent is removed quickly and the sections do not remain in the water contaminated with it for long. The following list, also compiled by Culling, gives CHIC-positive materials commonly encountered, although it is not exhaustive: · Accumulations of actinomycetes. · Basement membranes. · Cerebrosidae. Areolar connective tissue collagen. · Compound lipids. · Glycogen. · Kerasin (Gaucher disease). · Kidney tubules. · Megakaryocyte granules. Mucins: § intestinal tract, § pepsin glands, § glands of the cervix, § salivary glands, § conjunctiva, § bronchial glands, § follicles and ovarian cysts, § secretion of the prostatic glands, § amyloid bodies, · Capsule of the eye lens. · Pancreatic zymogenic granules. · Paneth cell granules (rats, Guinea pigs, rabbits). · Phospholipids. · B-cells of the pituitary gland. · Renal hyaline casts. · Rods of the retina. · Roussel's corpuscles. · Starch. · Thyroid colloid. citing: Hotchkiss, R. D., 1948 Arch. Biochem, vol 16, pp 131 http://stainsfile. info/StainsFile/stain/schiff/pas-standard. htm Last updated August 2003.

The Feulgen reaction indicates the presence of DNA, so it can primarily be used to state the presence or absence of nuclei in cells, their size, shape, location, etc. In addition, the intensity of staining can give indirect indications of quantitative changes in DNA.

Schiff's reagent - fuchsinous acid - is a characteristic reagent for aldehydes. The Feulgen reaction with this reagent on DNA is also based on the ability of the Schiff reagent to interact with aldehyde groups. However, due to the fact that the aldehyde groups in the DNA molecule are bound, they must first be freed. This is achieved by hydrolysis of DNA with a weak acid, as a result of which purine bases (adenine, guanine) are separated from the DNA molecule and the remainder of the DNA molecule with free aldehyde groups is formed:

The change occurs at the first carbon atom of deoxyribose; a potential aldehyde group is revealed at the site of purine base abstraction. At. This converts the furanose form of deoxyribose into a non-cyclic sugar form. During the reaction, one molecule of fuchsinous acid is added to the two aldehyde groups of deoxyribose.

Fuchsinous acid (leukofuchsin) is obtained from an acidic solution of basic fuchsin (parafuchsin) in 1 N. hydrochloric acid when saturated with sulfur dioxide. Sulfur dioxide is formed by the interaction of hydrochloric acid with sodium bisulfite, which are added to an acidic solution of basic fuchsin:

HCl+NaHSO 3 =NaCl+SO 2 +H 2 O

When sulfur dioxide is passed into a fuchsin solution, two double bonds are broken, the quinoid group disappears in one of the three aromatic rings of fuchsin, and with it the lilac-red color characteristic of fuchsin. Fuchsinous acid is a fragile, colorless compound.

The production of fuchsinous acid from basic fuchsin proceeds as follows: fuchsin, interacting with hydrochloric acid, forms a complex compound, which then reacts with sulfurous acid according to the following scheme:

Thus, during the formation of fuchsinous acid, two groups (-SO 2 H) are added to fuchsin.

When reacting with aldehydes, the quinoid group is restored, since fuchsinous acid is an unstable compound and decomposes to form sulfurous acid.

When one molecule of fuchsinous acid reacts with two molecules of DNA residues having exposed aldehyde groups, one molecule of a colored compound is obtained.

When preparing material for the Feulgen reaction, fixation in alcohol and acidic fixatives is usually recommended. The most commonly used is Carnoy's fixative (p. 45). Such a fixative precipitates nucleoproteins, which leads to successful morphological pictures. However, prolonged residence of the material in the fixative causes the destruction of the bond between nucleic acids and proteins, leads to a gradual extraction of nucleic acids and gives a distorted picture. Therefore, the stay in the fixator should be limited as much as possible.

Reagents

1) Schiff's reagent (fuchsinous acid).

Preparation of Schiff's reagent. Grind 1 g of basic fuchsin in a mortar and dissolve in 200 ml of boiling distilled water; cool to 50°C. Add 20 ml of 1 N to the cooled solution. hydrochloric acid * and cool to 25°C. Add 1 g sodium bisulfite or metabisulfite. Shake the resulting mixture with activated carbon (from 1 to 3 minutes) and filter. Pour into a dark vessel or wrapped in dark paper, close it with a ground-in stopper and place in the dark for 12 or more hours until the main fuchsin discolors and fuchsulfurous acid forms.

* (1 n. hydrochloric acid corresponds to a 10% solution prepared from concentrated hydrochloric acid (sp. 1.19))

2) Sulfurous water.

Preparation of sulfurous water. To 200 ml of distilled water add 20 ml of 1 N. hydrochloric acid and 1 g sodium bisulfite.

3) 1 n. hydrochloric acid.

4) Media for dehydrating objects and encasing them in Canada balsam (pp. 66-70).

5) Canada balsam.

Carrying out the reaction

* (See footnote on page 118.)

1. Immerse the preparations in 1 N. hydrochloric acid for a few seconds.

2. Transfer them to 1 n. preheated to 60° C. HCl and place in a thermostat at a temperature of 60° C (or in a water bath at the same temperature) for 5-10 minutes *.

* (The duration of hydrolysis depends on the fixative used. When using Carnoy's fixative and formalin, the optimal time is 8 minutes.)

3. Rinse the preparations with cold 1 N. HCl.

4. Place them in Schiff's reagent for 1 hour.

5. Wash off excess Schiff's reagent with sulfurous water (3 shifts of 3-5 minutes each).

6. Rinse the preparations in tap water for 5-10 minutes, replacing the water that is beginning to turn pink.

7. Bring the preparations to the balm through 96%, 100% alcohol, a mixture of alcohol with xylene and xylene.

8. Observe the appearance of a crimson color.

Reaction results (Table 20)

At the tip of the onion root, the kernels acquire a crimson-violet color. The nuclei of the extension zone and especially the suction zone appear somewhat less bright than the nuclei of the fission zone. The nucleoli, cytoplasm and cell membranes remain unstained.

In the corn grain, the nuclei of the root, bud and all other tissues of the embryo, as well as the cells of the aleurone layer, are painted in a color characteristic of the Feulgen reaction. The flattened and stretched nuclei of endosperm cells are stained paler.

(N. Schiff, 1834-1915, German biochemist)
a reagent for the qualitative determination of the aldehyde group of organic substances, which is a solution of fuchsinous acid; used for the histochemical detection of carbohydrates and deoxyribonucleic acid.

View value Schiffa Reactive in other dictionaries

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(N. Schiff, 1834-1915, German biochemist)

a reagent for the qualitative determination of the aldehyde group of organic substances, which is a solution of fuchsinous acid; used for the histochemical detection of carbohydrates and deoxyribonucleic acid.

• - a reagent for detecting glucose in urine, which is an aqueous solution of copper sulfate, sodium citrate and sodium carbonate...

  Medical encyclopedia

• - a reagent containing phloroglucinol, vanillin and alcohol, used in the qualitative determination of hydrochloric acid in the gastric...

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• - a mixture of ethyl alcohol, glacial acetic and hydroiodic acids, used in forensic medicine to detect blood in stains...

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• - a reagent containing 2.5 g of oxalic acid, ammonium oxalate and glacial acetic acid per 150 ml of their aqueous solution...

  Medical encyclopedia

• - a reagent for the qualitative and quantitative determination of aldehydes, the simplest reducing sugars and some other substances in biological fluids...

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• - a mixture of solutions of sodium tungstic and sodium molybdate, to which are added successively phosphoric, hydrochloric acid and, after boiling, lithium sulfate, as well as a few drops of bromine water...

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• - a, m. chem. A substance that causes a certain characteristic reaction when combined with another substance and thereby makes it possible to detect the presence of the latter in a given composition...

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• - ...

  Russian word stress

"Schiff reagent" in books

Test No. 2.