Plant Anatomy: Workshop - Panyuta O.O. 2019

Appendices
Microchemical Reactions

Plant Cells contain A wide variety of Organic compounds, which are identified using Special Methods of chemical analysis that allow for a fairly precise determination of their quantity at a specific stage of plant development.

In plant anatomy, it is often necessary to determine merely the presence of a particular organic compound. In such cases, microchemical reactions are employed, allowing the presence of various substances in cells to be detected through color reactions, namely: CARBOHYDRATES, Proteins, Alkaloids, Fatty acids, Tannins, and many Other Compounds.

Microchemical reactions make it possible to quickly determine the presence of a given substance using a staining method, which is based on the fact that specific substances take on a characteristic color when treated with certain Reagents.

Qualitative tests for carbohydrates

Molisch's test (the Podobedov-Molisch reaction) is used for the qualitative identification of carbohydrates. A few drops of Cell sap are squeezed from the test material into a test tube, and two drops of a 10% alcoholic solution of alpha-naphthol are added. Then, 2 or 3 ml of concentrated sulfuric acid is carefully poured down the inner walls of the test tube so that it does not mix with the liquid and settles at the bottom. Soon, a red-violet ring forms at the interface of the two liquids, with the color intensity increasing rapidly. All carbohydrates yield this reaction.

Selivanov's test. 50 ml of cell sap is squeezed from the plant material under investigation and heated for 10 minutes in a test tube within a boiling Water bath with 10 ml of resorcinol and 10 ml of 1 N Hydrochloric acid solution. If hexoses are present in the test material, a bright red color develops.

Diphenylamine test. To 50 ml of squeezed cell sap placed in a small flask, add 1 ml of water, 0.5 ml of a 20% solution of diphenylamine in 96% alcohol, and 1 ml of 25% hydrochloric acid. The mixture is heated for 5 minutes in a boiling water bath. After the color appears, heating is continued for another 5 minutes. The formation of an intense blue color indicates the presence of organic compounds known as ketoses.

Fehling's solution is used as a reagent for glucose, as well as for reducing sugars such as fructose and maltose.

To test for sugar, the specimen is dropped into a test tube or placed on a Microscope slide with a specific amount of Fehling's solution diluted with twice the volume of water. The specimen and the solution are then heated. If sugar is present, a brick-red precipitate of cuprous oxide will form after some time.

The presence of sugar can also be detected using Fehling's solution in an alternative manner. In this case, the specimen is first placed in a saturated solution of copper sulfate, then washed with water, and treated with a boiling solution of the following composition: 100 ml of water, 10 g of Rochelle salt (potassium sodium tartrate), and 11 g of caustic potash (potassium hydroxide). In some instances, Rochelle salt may be omitted; the specimens are then placed directly into a hot potassium hydroxide solution after the copper sulfate Treatment.

Chlor-zinc-iodine is used to remove chemically bound water from Cellulose. This process converts cellulose into amyloid.

Phosphoric acid is used in combination with a solution of iodine in potassium iodide. This reagent is suitable for identifying cellulose. Under its influence, cellulose is transformed into amyloid and stains purple.

Microchemical reactions for proteins

To determine the presence of proteins, the following tests are used: the xanthoproteic reaction, the biuret test, Millon's test, the Raspail reaction, and the sulfur test.

Xanthoproteic reaction. Concentrated nitric acid is added to the test solution and heated over a spirit lamp. A yellow color subsequently appears, which turns orange upon The addition of an ammonium hydroxide or alkali solution.

Biuret reaction. This involves an aromatic ring with which nitric acid forms a yellow nitro compound. This reaction is characteristic of all proteins except protamines, whose molecules lack a benzene ring. The reaction is driven by the presence of -COOH groups in the protein molecule, which can produce coloration even with non-protein substances if they contain such a group. The biuret reaction is characteristic of all proteins, peptones, and Peptides.

Strong alkali is added to a protein solution, followed by a few drops of a low-concentration copper sulfate solution. After a short time, a violet color develops. Peptones and Polypeptides yield a red tint.

This same reaction can be performed differently: 2.5 ml of a 33% sodium hydroxide solution is added to 10 ml of the protein-containing solution, mixed thoroughly, and 1 ml of a 0.2% copper sulfate solution is carefully layered down the inner wall of the test tube.

A violet ring forms at the interface between the two contacting liquids.

Millon's test. Millon's reagent is added to the protein solution. To prepare this reagent, 1 ml of metallic mercury is dissolved in 9 ml of nitric acid (specific gravity 1.52). An equal volume of water is then added to this solution.

The protein solution mixed with Millon's reagent is left for 20-30 minutes or gently warmed. A flesh-red color subsequently develops. This reaction is characteristic of almost all proteins containing a benzene ring with a hydroxyl group. Certain protamines and gelatin do not react with Millon's reagent because their molecules lack Tyrosine.

Raspail reaction. Thin sections are prepared from wheat seeds, which have been previously soaked in water until swollen. The prepared sections are placed on a microscope slide in a drop of sugar solution, to which sulfuric acid is added, and then gently warmed. A red color appears after a short time. When viewed under a microscope, the sections appear pink.

Test for sulfur. Sulfur is a constituent of protein compounds. To verify this, an equal volume of a 40% sodium hydroxide solution and a few drops of a concentrated lead acetate solution are added to a protein solution. The mixture is carefully heated to boiling. Initially, a white precipitate forms, which gradually turns brown and eventually black. This reaction is due to the presence of sulfur within the protein molecule. Sodium hydroxide is used because it breaks down the protein, releasing sulfur in the form of hydrogen sulfide, which reacts with lead to form a black precipitate of lead sulfide. This reaction is characteristic of all proteins except gelatin and protamines.

Test for fats

Sudan III can be used as a reagent for fats, causing them to stain pink. This same reagent is applied directly to tissue sections when it is necessary to separate the cuticle from The Cell wall. The cuticle stains a more intense red with Sudan III than the cutinized cell wall.

Alkanin serves as a specific reagent for Lipids. This substance stains the roots of alkanet and certain other plants of the borage family (Boraginaceae) a red color. To prepare this reagent, small roots of alkanet are placed in alcohol to obtain an alcoholic extract, which is then filtered. The resulting alcoholic infusion contains alkanin, which stains lipids, resins, the cuticle, and cork red. Lipids react instantly, whereas cork and the cuticle take somewhat longer to stain.

Osmic acid also reacts with lipids, producing a black or brown coloration.

Test for Alkaloids

Alkaloids are precipitated using so-called alkaloidal reagents, the most important of which are:

Potassium hexacyanoferrate(II) (yellow prussiate of potash). When treated with acidified solutions of this salt, most alkaloids form crystalline precipitates.

Saturated aqueous solution of picric acid forms precipitates containing alkaloids.

Iodine in potassium iodide. Upon treatment with this solution, alkaloids form brownish-red precipitates.

Bromine and potassium bromide. First, a 10% aqueous solution of potassium bromide is prepared and then saturated with bromine.

When exposed to this solution, alkaloids form spherical orange and brown precipitates.

Phosphomolybdic acid. A 10% aqueous solution is prepared, causing alkaloids to form white and yellow precipitates.

Double salt of mercuric iodide and potassium iodide (Mayer's reagent). Dissolve 49.8 g of potassium iodide in 1 L of water and add 13.5 g of mercuric iodide. Treatment with this reagent causes alkaloids to form white or yellowish, predominantly amorphous precipitates.

When performing microchemical tests for alkaloids, it is necessary to use multiple reagents because other substances can yield similar precipitation reactions.

Test for Volatile Fatty Acids

Plants contain various organic acids. The presence of some can be identified through color reactions.

A solution of formic acid, neutralized with sodium hydroxide, yields a white precipitate with silver nitrate that rapidly darkens upon heating and is reduced to metallic silver.

In the presence of nitric acid, formic acid or its salts readily reduce chromic acid. To carry out this test, take 3–4 mL of a 0.5% solution of K₂Cr₂O₇ in concentrated nitric acid and add a single drop of chromic acid or several of its crystals. A blue-violet color develops rapidly in the cold.

Formate salts react with lead acetate to form a crystalline precipitate. Upon heating, it dissolves, and upon cooling, it recrystallizes into small, shiny needles.

The presence of butyric acid is determined as follows: heating butyric acid salts with sulfuric acid produces a characteristic rancid butter odor. If the salts are warmed with a few drops of alcohol and concentrated sulfuric acid, the scent of ethyl butyrate emerges, reminiscent of pineapple.

Test for Tannins

Tannins give color reactions with numerous compounds. To detect tannins in a plant, oak bark or another plant sample is soaked to obtain an infusion. After filtering, a few drops of a 1% ferric chloride or iron alum solution are added. It must first be verified that the infusion has a neutral reaction. If tannins are present, a blue or greenish coloration appears.

Other tests for tannins involve the formation of precipitates. For instance, in the potassium dichromate test—where tissue sections are placed on a microscope slide in a drop of potassium dichromate solution (1:10)—tannins precipitate as a dense gray or red-brown sediment. Adding lead acetate or salts of other heavy metals to a tannin solution likewise causes precipitation.

Barium chloride is used to separate calcium sulfate crystals from calcium oxalate crystals. Treating tissue sections with a barium chloride solution leaves calcium oxalate crystals unchanged while dissolving calcium sulfate crystals. This same reagent is suitable for detecting sulfur. Horseradish ROOT sections serve as the optimal preparation. When these sections are treated with barium chloride, the formation of barium sulfate crystals can be observed. Sulfur from both mineral and organic compounds present in the studied samples is incorporated into these crystals.

The phloroglucinol test is employed to detect lignified cell walls and to identify pentoses.

To test for pentoses, a purée made from crushed apples is prepared so that it contains 10 mg of pentoses. An equal volume of 36% hydrochloric acid is added to the solution, followed by a millet-seed-sized piece of phloroglucinol. The mixture is heated in a boiling water bath for a few minutes. After a short time, a violet-red color develops, indicating the presence of pentoses.

Phloroglucinol is also utilized to identify SHOOT lignification. Thin sections of the shoots are placed in a drop of phloroglucinol solution, and a drop of hydrochloric acid is added. Lignified elements stain pink or red.

The phloroglucinol test makes it possible to detect changes occurring in the wood parenchyma and vessels.

Reaction with aniline sulfate. This reagent is used to identify lignified cell walls. Upon its application, lignified walls turn a bright yellow color.

Chloral hydrate is the best clearing agent for various fluids and microscopic preparations. It provides much better clarification than Javelle water. Clearing occurs because proteins dissolve under the Influence of the reagent. It is prepared as follows: 5 g of regular crystalline chloral hydrate is dissolved in 2 mL of water; this same reagent is used for clearing pollen grains and ovules.