1. Dr. Samah Kotb Nasr Eldeen

3.  Several types of staining processes are used to color tissues for microscopical examination.  Staining methods depend on the metallic impregnation or the production of a colored substance from colorless reagents.

4.  The mechanism of how some of these techniques work is not yet known.  There are physical and chemical theories for staining mechanism.

5. Theories for staining mechanism

6. A. Physical theory: this depends on: 1.Osmosis. 2.Solubility. 3.Absorption. 4.Adsorption. 5.Permeability of the tissues.

7. B. The chemical theory: this is depends on the pH. 1.basic. 2.Acidic. 3.Neutral. 4.Amphoteric “ acts as a base in acidic solution and as an acid in a basic solution.

9. 1) Production of colored chemical substances: Divided into: (a) stable method ex: Pearls and iron pigments “ferric salts”.

10. ( b) Unstable method: This is applied in enzyme histochemistry In both methods we use colorless chemical substances and end up after the reaction with colored production; this is a pure chemical mean “chemical theory”.

11. 2) Metallic impregnation: Silver technique and other like osmium tetroxide and mercuric chloride. The metallic impregnation with silver techniques divided into: (a) Argentophilic: “direct” This is when dealing with or demonstrating substances which do not need an outside reducer i.e. direct impregnation for substances like melanin.

12. (b) Argyrophilic: “indirect” This is when using outside reducer as light (sun or artificial light), photographic developer or hydroquinone to demonstrate substances like fibrils and calcium.

13. Factors affect staining

14. 1.The mode of action of dyes. 2. Solvents ex: alcoholic or aqueous solutions. 3. Low or high temperature during the reaction. 4. Simple or multiple combination of dyes. 5. Strong or weak dye solutions. 6. The covering power of the dye.

15. 7. The time. 8.The type of the tissue. 9.The fixative used. 10.Type of section. 11.The thickness of section. 12.The temperature during drying paraffin sections. 13. The make up of the dye (in the factory).

16. Preparation of the stains

17. 1.Clean dry glassware. 2.The correct solvent. 3.Flasks and pipettes for silver solution should marked and kept separately.

18. 4. Silver and osmic tetroxide solutions should be kept in dark bottles in a cool place. 5. Dilute ammonia for staining should be freshly prepared and the stock kept in the fridge.

19. 6.Constituents of stains should be dissolved in the order giving in the formulae. 7. Alcoholic solutions of stain should be kept in glass-stoppered bottles or containers to avoid evaporation which cause precipitation of the stain.

20. 8. All dye solution used to demonstrate bacteria should be filtered immediately before used. 9. Appropriate batch of dyes should be used. 10. Controls of stains and test slides should be applied.

21. Solvents 1.Water: tap or distilled water. 2.Ethyl alcohol (ethanol). 3.Methyl alcohol (methanol).

22. 4. Acetone. 5. Phenol. 6. Buffer solutions. 7. Aniline water.

23. Staining process Equipment 1.Coplin jars.

24. 2. Staining dishes.

25. 3. Staining racks + staining sets.

26. 4. Staining machines.

27. Staining procedures 1.Removal of wax by xylene. 2.Removal of xylene by alcohol; taking by descending grades of alcohol. 3.Removal of pigments. 4.Application of staining solution.

28. 5. Dehydration; taking through ascending grades of alcohol. 6. Clearing with xylene. 7. Mounting or cover slipping. 8. Labelling. 9. Examine under the microscope.