Principles of staining

• Staining


  process whereby tissue components are made visible in microscopic sections by direct interaction with a dye or staining solution
• Because different parts of the cell are biochemically different, they take up specific stains to varying degrees.
• Certain parts of cells and tissues that are acidic in character (e.g. nucleus) have greater affinity for basic dyes
• basic constituents (e.g. cytoplasm) take more of the acid stains
• Paraffin wax is poorly permeable to most staining solutions and should therefore be removed from the section prior to staining.
• If an alcoholic stain is to be used, there is no more need to replace the alcohol with water. After deparaffinization with xylene, the section is transferred to decreasing grades of alcohol, and in such instances, the term "Sections to Alcohol" is used, and the staining procedure is subsequently done unless the tissue has been fixed in mercuric chloride solution, in which case, the section is taken “to water”.
• HISTOLOGICAL STAINING


  process whereby the tissue constituents and general relationship between cell and tissue are demonstrated in sections by direct interaction with a dye or staining solution, producing coloration of the active tissue component.
• Direct Staining


  process of giving color to the sections by using aqueous or alcoholic dye solutions
• Indirect Staining


  process whereby the action of the dye is intensified by adding another agent or a MORDANT which serves as a link or bridge between the tissue and the dye, to make the staining reaction possible. By itself, the dye may stain only weakly, if at all.
• The mordant combines with a dye to form a colored "lake", which in turn combines with the tissue to form a "tissuemordant-dye-complex" that is rendered insoluble in ordinary aqueous and alcoholic solvents.
• ACCENTUATOR


  not essential to the chemical union of the tissue and the dye. It does not participate in the staining reaction, but merely accelerates the reaction.

  Examples are potassium hydroxide in Loeffler's methylene blue and phenol in carbol thionine and carbol fuchsin
• PROGRESSIVE STAINING


  process whereby tissue elements are stained in a definite sequence, and the staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained
• REGRESSIVE STAINING


  tissue is first overstained to obliterate the cellular details, and the excess stain is removed or decolorized from unwanted parts of the tissue, until the desired intensity of color is obtained
• DIFFERENTIATION (DECOLORIZATION)


  selective removal of excess stain from the tissue during regressive staining in order that a specific substance may be stained distinctly from the surrounding tissues.
• Differential Staining


  uses more than one chemical stain to better differentiate between various microorganisms or structures/cellular components of a single organism
• Metachromatic staining


  technique entails the use of specific dyes which differentiate particular substances by staining them with a color that is different from that of the stain itself (metachromasia)
• Metallic Impregnation


  process where specific tissue elements are demonstrated, not by stains, but by colorless solutions of metallic salts which are thereby reduced by the tissue, producing an opaque, usually black deposit on the surface of the tissue or bacteria.
• Vital staining


  selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of the dye particle (cytoplasmic phagocytosis), or by staining of pre-existing cellular components (true vital staining), as in the staining of mitochondria by Janus green
• INTRAVITAL STAINING


  living cells is done by injecting the dye into any part of the animal body (either intravenous, intraperitoneal or subcutaneous), producing specific coloration of certain cells, particularly those of the reticulo-endothelial system. Common dyes used are lithium, carmine and India ink.
• Supravital staining


  method of staining used in microscopy to examine living cells that have been removed from an organism
• Neutral red


  probably the best vital dye
• Janus green


  especially recommended for mitochondria
• Trypan blue


  -one gram of dye is dissolved in 100 ml. of sterile distilled water to be used immediately; it is dangerous to allow the suspension to stand for more than one hour, because it is likely to become toxic to the cell
• Hematoxylin and Eosin (H&E) staining


  corner stone of tissue-based diagnosis. The process stains thin tissue sections so that pathologists can visualize tissue morphology