Appendix XI U. Microscopic Examination of Herbal Drugs

The microscopic examination of herbal drugs is carried out on the powdered drug (355) (2.9.12) unless otherwise prescribed in the monograph.

Chloral hydrate solution R is the most commonly prescribed reagent. However, certain features are not visible or not easily seen after mounting in this reagent. In this case, other reagents are used, for example, a 50 per cent V/V solution of glycerol R, which makes it possible to visualise starch granules. It may also be necessary to prescribe specific reagents in a monograph, for example: lactic reagent R which is used to show the presence of various features, 10 per cent V/V alcoholic solution of phloroglucinol R and hydrochloric acid R, which are used to identify the presence of lignin in cells or tissues, ruthenium red solution R, which is used to show the presence of mucilage in cells or glycerol R used to show the presence of starch and inulin.

Examination under polarised light (between crossed nicol prisms) is used to identify starch granules (black cross phenomenon), calcium oxalate crystals (refringence) or lignified structures.

MOUNTING IN CHLORAL HYDRATE SOLUTION

Place 2-3 drops of chloral hydrate solution R on a glass microscope slide. Disperse a very small quantity of the powdered drug in the liquid and cover the preparation with a cover slip. Heat the preparation very gently to boiling on a hot plate or a micro gas burner. Maintain gentle boiling for a short time. Make sure that the quantity of mounting fluid is sufficient. If necessary, add more fluid using a tapered glass pipette. Allow to cool and then examine under a microscope. Repeat the heating until the starch granules and the water-soluble contents of the cells are no longer visible. Examine under a microscope.

Chloral hydrate tends to crystallise as long needles. To avoid this, proceed as follows: after heating, remove the cover slip; to the preparation add 1 drop of a 10 per cent V/V mixture of chloral hydrate solution R in glycerol R; place a clean cover slip on the preparation; examine under a microscope.

MOUNTING IN A 50 PER CENT V/V SOLUTION OF GLYCEROL

Place 2 drops of a 50 per cent V/V solution of glycerol R on a glass microscope slide. Disperse a very small quantity of the powdered drug in the liquid and cover the preparation with a cover slip. Examine under a microscope.

MOUNTING IN A 10 per cent V/V ALCOHOLIC SOLUTION OF PHLOROGLUCINOL AND HYDROCHLORIC ACID

Place a very small quantity of the powdered drug on a glass microscope slide. Add 1-2 drops of a 10 per cent V/V alcoholic solution of phloroglucinol R. Mix and allow the solvent to evaporate almost completely. Add 1-2 drops of hydrochloric acid R and cover the preparation with a cover slip. Examine immediately under a microscope. The red colour indicates the presence of lignin.

MOUNTING IN LACTIC REAGENT

Place 2-3 drops of lactic reagent R on a glass microscope slide. Disperse a very small quantity of the powdered drug in the liquid and cover the preparation with a cover slip. Heat the preparation very gently to boiling. Maintain gentle boiling for a short time. Make sure that the quantity of mounting fluid is sufficient. If necessary, add more fluid using a tapered glass pipette. Allow to cool and then examine under a microscope. Lignified structures stain bright yellow; structures containing cellulose remain colourless. Starch granules stain more or less violet; certain secretions (e.g., essential oils, resins, oleoresins) stain orange and cork stains red.

MOUNTING IN RUTHENIUM RED SOLUTION

Place 2 drops of ruthenium red solution R on a glass microscope slide. Disperse a very small quantity of the powdered drug in the liquid and cover the preparation with a cover slip. After about 1 minute, allow a drop of distilled water R to be taken up between the slide and the cover slip. Examine under a microscope. The mucilage stains violet red.