Microscopic analysis of crude drugs is done mainly for the determination of (a) the size, shape and relative position of different cells and I tissues; (b) the chemical nature of the cell walls, and (c) the form and nature of cell contents. In order to achieve a successful microscopic analysis certain techniques and methods are employed to prepare the material prior to examination. The type of technique used depends on the nature and aim of the analysis.
General method for studying tissue distribution
A general idea of the distribution of tissues in a plant organ can be obtained by mounting sections or powders of the drug in water or dilute glycerol and examining them under the microscope using low and high powers.
Staining with Colour reagents Colour reagents can be used to stain and locate the position of different tissues in plant organ. Some of the common colour reagents used in microscopic analysis are mentioned below:
This reagent stains true celluloses blue and pectic substances yellow. Cell walls containing a mixture of these substances in different proportions stain blue, violet or brown. Lignified or suberised cell walls tum yellow or brown with this reagent.
Phloroglucinol and Hydrochloric acid:
When mounted in phloroglucinol followed by concentrated hydrochloric acid lignified walls of cells stain pink or red.
Tincture of Alkanna:
Suberised and cutinised cell walls stain red with this reagent.
Clearing, Bleaching and Defatting
In most cases structures in plant tissues are obscured due to presence of various cell contents, colouring matters and pigments. Prior to microscopical examinations these tissues must be cleared, bleached or defatted by the use of suitable chemical reagents. Some of them are described below
Chloral hydrate solution:
When heated or boiled slowly, 75 percent chloral hydrate solution dissolves and clears out starch, proteins, chlorophyll, resins and oils from cells and causes shrunken cells to expand. This is a good reagent for examination of cellular structures and calcium oxalate crystals.
It is a good reagent for general defatting of plant tissues. A mixture of equal parts of ether and alcohol removes fats, oils, resins, tannins and even chlorophyll from cells and tissues.
Chlorinated Soda solution:
This solution is useful for bleaching dark-coloured sections of barks ‘and for removing · chlorophylls from leaves. Under normal condition, this solution does not remove starch and lignin.
Isolation of Tissue elements
For detailed study of internal structures of plants it often becomes necessary to separate and isolate individual cells of a tissue or a specific element in a tissue. This is achieved by first dissolving or softening the cementing material (middle lamella) between the cells using some chemical reagents. The macerated or softened tissue is then mechanically separated (often under a microscope) by a pair of needles. Some of the reagents used for disintegration of the middle lamella and isolation of tissues are described below.
Nitric acid and Potassium chlorate (Schultz method):
The plant part in small pieces is immersed in 50 percent nitric acid and minute quantities of potassium chlorate are added at intervals to maintain an evolution of gas. The process is continued until the tissue is soft enough to be teased. The tissue is then washed free from acid.
Chromic acid and Nitric or Sulphuric acid:
The reagent consists of a mixture of equal parts of 10 percent chromic acid and 10 percent nitric or sulphuric acid. It is mainly used for the separation of sclerenchymatous tissues and lignified hairs.
Solution of Potash:
A 5 percent solution of potash is used to digest the material by boiling on a water bath until the tissues become very soft. This reagent is very useful for the separation of secretory cells (like vittae), latex vessels and lignified elements.