PCG 204 Chemomicroscopy PDF

CHEMOMICROSCOPY  Chemomicroscopy is the application of chemical reagents in the microscopical examination of crude drugs. It aims to determine the chemical nature of the cell wall, the form and chemical nature of the cell contents.  It is also the microchemical testing of crude drugs...

CHEMOMICROSCOPY  Chemomicroscopy is the application of chemical reagents in the microscopical examination of crude drugs. It aims to determine the chemical nature of the cell wall, the form and chemical nature of the cell contents.  It is also the microchemical testing of crude drugs using chemical reagents to produce precipitate and colour reactions for the purpose of identifying the chemical nature of the plants.  This study of crude drugs offer important diagnostic evaluation for the identification of both entire and powdered crude drugs and detection of adulterants in them.  The chemical microscopical examination of the components of the plant cells and tissues is a series of processes which involves disintegrating, clearing, staining, mounting and examining them under the microscope. DISINTEGRATING AGENTS  This is the process of chemical softening and disintegration of the plant cell wall components to dissolve the pectic substances of the middle lamella, thereby separating adjacent cells.  1.) Sodium or potassium hydroxide solution: it is used for separation of non-lignified tissues or lignified cells which occur singly  2.) Nitric Acid: It is used for the separation of the epidermis of leaves and stems of fresh or dried crude drugs. The leaves or the stems of the drugs under study are cut into small pieces and the material is boiled gently with dilute acid until the epidermis of the sample can be easily separated.  3.) Sodium Hypochlorite Solution: This is useful as a powerful bleaching agent to remove the dark color of many barks, as well as to remove chlorophyll from leaves. On prolonged contact, it may remove starch and lignin from the microscopical mount, which should be avoided when performing the experiments.  4.) Caustic Alkali: This includes both aqueous and alcoholic solutions of sodium or potassium hydroxide in 2%–5% concentration of water or alcohol. A 5% aqueous solution is mostly used as a clearing agent, which rapidly dissolves starch and proteins. CLEARING AGENTS  1.) Chloral Hydrate Solution: It is effectively used as a clearing and bleaching agent to dissolve starch, protein, chlorophyll, resins, and other materials and causes shrunken cells to expand. It does not dissolve calcium oxalate, so it can be used effectively for detecting these crystals.  2.) Ether–Ethanol Reagent: This consists of a mixture of equal parts of ether and alcohol and is used effectively for the removal of oil, fats, volatile oil, resins, tannins, and chlorophyll, among others. It is mostly used as a defatting agent in the case of oily seeds, such as Linseed and Strophanthus.  3.) Phenols Cresol and liquefied phenol: are used as clearing agents. The advantages of using them over chloral hydrate are that they cause no destruction of starch but cause good penetration and produce less swelling and also render starch particles so transparent as to be practically invisible, while the tissues themselves are clearly defined.  4.) Nitric Acid: It is prepared by diluting nitric acid. It is used for the separation of the epidermis of leaves and stems of fresh or dried crude drugs. The leaves or the stems of the drugs under study are cut into small pieces and the material is boiled gently with dilute acid until the epidermis of the sample can be easily separated. For this testing, a small portion of the sample under study is removed and an attempt is made to strip the epidermis off from the specimen on the slide. When this can be easily accomplished, the boiled leaves or stems are taken out in a beaker with water.  4.) Hydrochloric Acid: It is a powerful clearing reagent. A test solution may dissolve many cell contents, including calcium oxalate. HCI vapor may cause damage to the microscope; to prevent this, the slide should be removed as soon as possible from the microscope stage.  5.) Hydrofluoric Acid: This is more preferable than nitric acid for softening the hard woody herbal drugs, such as olive stone, coconut cells, peach stone, and hard woods. It acts by dissolving out any minerals and silica present in the cell wall but it does not attack the middle lamella so that the cells of the softened tissue do not fall apart. For this purpose, hard woody drugs are boiled with water for a few hours. Then they are cooled and transferred to commercial hydrofluoric acid for 1–4 weeks depending on the hardness of the drugs. The soaking is continued until they are soft enough to cut into pieces for microscopical studies. This process may be continued up to 6 weeks and, in that case, the hydrofluoric acid solution can be changed once during the whole operation. After this operation is completed, the samples are washed with distilled water several times and stored in a mixture of equal parts of glycerin and 30% alcohol. After a week or so, they will be useful for sectioning. The maceration is carried out in dishes coated with paraffin wax as it is corrosive and should not come into contact with the skin.  6.) Ether–Ethanol Reagent: This consists of a mixture of equal parts of ether and alcohol and is used effectively for the removal of oil, fats, volatile oil, resins, tannins, and chlorophyll, among others. It is mostly used as a defatting agent in the case of oily seeds.   7.) Sodium hydroxide or Potassium hydroxide: A 5% aqueous solution is mostly used as a clearing agent, which rapidly dissolves starch and proteins. STAINING AGENTS These are reagents for staining the entire crude drugs and microchemical analysis of powdered plants.  1.) Phloroglucinol in conc. Hydrochloric acid: It stains all lignified walls pink or red. It is not only useful for staining sections but also for vegetable powders.  2.) Iodine water: Iodine is the most important of all the reagents used in microchemical testing. Iodine colors starch blue and imparts a yellow color to aleurone grains.  3.) Caustic soda solution: A 50% solution of soda or potash is used for the test of suberized cell walls, which become yellow when mounted in the reagent.  4.) Iodinated zinc chloride: This is used to stain cellulose walls to a blue or violet color. When added, it slowly stains cellulose walls and lignified or suberized walls to yellow or brown and starch grains to blue.  5.) Acetic acid: It is a very useful reagent in microchemical analysis. The reagent is most useful for the analysis of calcium carbonate or calcium oxalate present in drug preparations.  6.) Sudan red: It is used for staining suberized or cuticular cell walls to an orange red or red color. Also testing fixed oils, volatile oils and fats to red colour  7.) Picric acid solution: It is used to stain aleurone grains and animal fibers. This stains animal fibers yellow, while cellulose fibers remain uncolored and protein yellow.  8.) Ruthenium red: It stains many gums, pectin, and mucilage giving it a pink colour.  9.) Safranin: It is used for staining lignified cell walls cuticular cell walls to red colour.  10.) Ferric chloride: The solution is a microchemical reagent for tannins and gives bluish black or green colour.  11.) Chlorine-zinc-iodine solution: This is used to stain cellulose walls to a blue or violet color, lignified or suberized walls to yellow or brown, and starch grains to blue.  12.) Sulfuric acid: Strong sulfuric acid is the best reagent used for the identification of suberized tissues. In this examination, the section is mounted in acid, which dissolves almost all of the structures except the suberized cells.   13.) Cuoxam: This is an ammoniacal solution of copper oxide that dissolves cellulose walls.  14.) Fehling solution: The solution is a microchemical reagent for reducing sugars giving it a red colour.  15.) Million’s reagent: This reagent is a microchemical reagent for proteins giving it a red colour and also for aleurone grains.  16.) Dragendroff’s reagents: The solution is a microchemical reagent for alkaloid giving it a reddish brown colour. MOUNTANT  They are reagents used to fix the tissue or section on the slide (holding the specimen in place) and to prevent drying of fixed tissue e.g glycerin or a mixture of glycerin and water. Glycerol is also used as a mountant.  Sections could be temporarily or permanently mounted.  Water –soluble mounting media that remain liquid e.g glycerol, glycerol-water mixture  Permanent mountant: Canada Balsam, Xylene, Clear nail polish PREPARATION OF CRUDE DRUG SAMPLE FOR MICROSCOPICAL EXAMINATION Preparing a crude drug sample for microscopical study necessarily depends on the form and nature of the sample. In order to observe the details of the structure, the specimens have to be placed in the proper optical path of a microscope in which the form and condition of the sample play a major role. The main factors affecting the visibility of the object are sample thickness and the presence of other pigments or obscuring materials. The sample thickness can be controlled by proper sectioning of the material or by the selection of a particular portion of powdered material. Preparing a crude drug sample for microscopical study necessarily depends on the form and nature of the sample. In order to observe the details of the structure, the specimens have to be placed in the proper optical path of a microscope in which the form and condition of the sample play a major role. The main factors affecting the visibility of the object are sample thickness and the presence of other pigments or obscuring materials. For surface tissues of leaves and flowers, representative pieces of the sample to be examined are selected and cut to a suitable length, cross or transverse sections are prepared by cutting with a razor blade or by microtome at a right angle to the longitudinal axis of the material. PROCEDURE FOR MICROSCOPICAL EXAMINATION OF A PLANT SAMPLE  With the help of a brush/ forcep, transfer the leaf, stem or plant part section taken from water to a clean glass slide  To the section on the glass slide add the staining solution to it and allow to stay for 2 – 3 minutes.  This is ready for mounting. Take a clean cover slip with the help of forceps and gently place the cover slip on the section.  If any bubbles are seen, slightly lift the cover slip and add a drop of water and replace the cover slip till the air bubble is removed.  With the help of a blotting paper, wipe off excess water present outside the cover slip. The slide is ready for observation.  PERMANENT STAINING (DOUBLE STAININING)  Take a clean watch glass, add safranin solution to it and transfer a thin uniform section to this sol. and allow to stand for 10 minutes.  Take one watch glass containing 50% alcohol, transfer the section from safranin to 50% alcohol, keep for 5 minutes  Transfer the section into a watch glass containing water, keep for 5 minutes to remove the stain from the cellulose part.  Transfer this safranin section to a watch glass containing dilute haematoxyline and treat for 2 minutes.  Transfer to a watch glass containing water for washing  For mounting, select a thick glass slide and a thin cover slip. Place the section in the centre of the slide and add few drops of clove oil. This makes the section clear, as it removes unwanted debris.  After 5 minutes, dry the section with a blotting paper.  To this section, now ad few drops of Canada balsam dissolved in xylol.  Slightly warm the slide or keep for drying in sun in a dust-free place. Natural drying takes longer time (days). The solvent evaporates and the balsam fixes the section.  Label the slide accordingly.