ch 16 histo

• Staining


  The process whereby tissue components are made visible in microscopic sections by direct interaction with a dye or staining solution
• Color compound


  Used to produce a contrast between different tissues and cellular components based on their varying affinities for most dyes and stains
• Histologic stain


  The purified form of a coloring agent or crude dye that is generally applied in an aqueous solution
• Mordant


  A chemical compound that reacts with the stain to form an insoluble, colored precipitate on the tissue and make the staining reaction possible
• Hematoxylin and Eosin (H&E) staining


  • Great majority of routine histology is done with this because it is quick, cheap and informative
  • It involves the use of two contrasting stains, e.g., hematoxylin which stains the nuclear detail, and eosin which brings out the cytoplasmic detail of the cell and the tissue's architecture
• Staining of paraffin sections


  1. Paraffin wax removal with xylene

  2. Replacement of xylene with decreasing grades of alcohol

  3. Replacement of alcohol with water before staining
• Direct staining


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


  The 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
• Progressive staining


  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


  The 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)


  The 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


  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


  A 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


  The 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)
• Intravital staining


  Intravital staining of 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
• Supravital staining


  A method of staining living cells by applying the stain directly to the tissue or cell preparation, without injecting it into the body
• 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
• Vital staining


  • The usual purpose is to reveal cytological details that might otherwise not be apparent; however, staining can also reveal where certain chemicals or specific chemical reactions are taking place within cells or tissues
  • The nucleus of a living cell is resistant to vital stains, and therefore is not demonstrated
• Intravital staining


  Staining of living cells 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 for intravital staining

  • lithium
  • carmine
  • India ink
• Supravital staining


  A method of staining used in microscopy to examine living cells that have been removed from an organism, differs from intravital staining which is done by injecting or otherwise introducing the stain into the body
• Hematoxylin and Eosin (H&E) staining


  The cornerstone of tissue-based diagnosis, stains thin tissue sections so that pathologists can visualize tissue morphology, using hematoxylin to stain cell nuclei (and other parts) blue and eosin to stain other structures pink or red
• Hematoxylin and Eosin (H&E) staining


  • Provides exceptional detail of tissue structure and the makeup of the cells
  • The term lesion is used by pathologists to indicate any area of damage, infection, inflammation, tumor, necrosis or otherwise abnormal tissue
• Frozen section staining


  Frozen sections mounted on the slides may be stained as in paraffin sections although the duration of staining is usually shorter, sections may be mounted in an aqueous medium directly from water if necessary
• Staining methods commonly employed for frozen sections

  • Hematoxylin-Eosin method
  • Thionine method
  • Polychrome Methylene Blue method
  • Alcoholic Pinacyanol method (used also for supravital staining of mitochondria and primarily for color sensitization in photography)
• Stains on the skin should be avoided not only because they are signs of poor technique but because stains are health hazards
• Stains may be effectively removed from the skin by prompt topical application of 0.5% acid alcohol, followed by rinsing with tap water
• Failure of staining may be due to paraffin, fixative, or decalcifying solution that has not been thoroughly washed out and removed
• Early fixation in alcohol before paraffin embedding may have been incorrect, for which no remedy can be made
• Hematoxylin must not be used too soon after preparation to ensure complete ripening, impurities found in the dye or in the water solvent will affect not only the solubility of the dye but even the intensity of the staining reaction, necessitating purification and filtering of the dye
• Stains that have already been deteriorated should be replaced
• If, after staining, sections are fuzzy and do not appear clear under the microscope, xylol should be replenished
• There may be water in the absolute alcohol, moisture in the coverslip, or too much egg albumin on the slide, thereby obliterating the image of the stained tissue
• Stains may be saved and used again for as long as they have not lost their staining properties
• Formation of precipitate in staining solution and poor staining results signify loss of staining property and hence, the stain should be discarded and replaced with a fresh solution
• Failure of sections to remain on the slide during staining could have been due to a dirty or oily slide
• Colloid ionization of sections

  Paraffin ribbons containing air bubbles, torn or inadequately infiltrated sections are likely to float from the slide when deparaffinized and stained, they are more firmly attached by coating the slide with dilute (thin) celloidin solutions, a process known as collodionization
• Re-staining of old sections

  The slide is usually immersed in xylene for 24 hours, or gently heated until the mounting medium begins to bubble, the coverslip may then be removed, the section is placed in xylene for up to 24 hours to remove the remaining balsam and then brought down to water, placed in a 0.5 potassium permanganate solution for 5-10 minutes, rinsed in tap water and subsequently immersed in 5% oxalic acid for 5 minutes or until the section is decolorized, after washing it again in running tap water for another 5 minutes, the section may then be re-stained with the appropriate staining technique
• Histochemical staining (Histochemistry)


  The process whereby various constituents of tissues are studied through chemical reactions that will permit microscopic localization of a specific tissue substance, such as chemical ions, molecules, and biopolymers