h&e, pap and fite faraco

• H & E Staining

  1. Xylene 1

  2. Xylene 2

  3. Drain

  4. Ethanol 95%

  5. Ethanol 75%

  6. Tap Water

  7. Hematoxylin

  8. Tap Water

  9. Acid Alcohol

  10. Tap Water

  11. Ammonia Water

  12. Tap Water

  13. Eosin

  14. Ethanol

  15. Methanol

  16. Drain

  17. Xylene 1

  18. Xylene 2
• FITE FARACO Staining


  1. Xylene- Mineral Oil Mixture

  2. Drain and Blot

  3. Carbol Fuchsin

  4. Tap Water

  5. Acid Alcohol

  6. Drain and Blot

  7. Methylene Blue

  8. Tap Water

  9. Blot, Air dry and Mount
• Pap Smear Stain


  1. Distilled Water

  2. Reagent 1 (Modified Hematoxylin Stain)

  3. Running Tap Water

  4. Reagent 3 (2-propanol)

  5. Reagent 2 (Modified Plychromatic Stain)

  6. Reagent 3 (2-propanol)

  7. Reagent 4 (Xylene)

  8. Reagent 4 (Xylene)
• Drain and Blot every after staining solution
• Keratinized Cytoplasm

  Pink to Intensity pink
• Cell Nuclei

  Blue, Dark Violet, Black
• Microorganisms

  Blue Violet
• H & E and Pap's Staining


  Staining techniques used in histopathology laboratories to render different tissue constituents more visible through variation in colors, enabling differentiation and identification of cell and tissue components

  
• Hematoxylin & Eosin Staining Technique

  1. Specimen preparation

  2. Fixation

  3. Decalcification

  4. Dehydration

  5. Clearing

  6. Infiltration

  7. Embedding

  8. Blocking/Casting

  9. Trimming

  10. Section cutting

  11. Staining

  
• Staining


  • Renders different tissue constituents more visible through variation in colors
  • Enables differentiation and identification of cell and tissue components

  
• Progressive Staining


  Tissue elements are stained in a definite sequence, the staining solution is applied for specific periods of time until the desired intensity of coloring is attained, and the dye is not washed or decolorized
• Regressive Staining

  Tissue is first overstained to obliterate cellular details, then 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 surrounding tissues
• Hematoxylin


  A natural dye derived from the heartwood of a Mexican tree, the active coloring agent is known as hematin which is formed by the oxidation of hematoxylin
• Mordants


  Substances that combine with the tissue and the staining solution, forming a bridge that allows the staining reaction to take place
• Aluminum Hematoxylin Solutions (Alum Hematoxylin Stains)


  Recommended for both progressive and regressive staining of tissues, using aluminum salts as mordants to increase selectivity for nuclei
• Bluing of Alum Hematoxylin Stained Sections


  The process of neutralizing the acid and freeing the hydroxide group to form an insoluble blue aluminum hematin-tissue lake, using alkaline solutions like tap water, lithium carbonate, or Scott's Tap Water Substitute
• Ehrlich's Hematoxylin


  An aluminum hematoxylin solution used for regressive staining, with rapid ripening achieved by the addition of sodium iodate
• The most critical step of gram staining is the decolorization step, as the crystal violet stain will be removed from both Gram positive and Gram negative cells if the decolorizing agent (e.g. alcohol) is left on too long

  
• Natural ripening


  1. Place in lightly plugged flask

  2. Shake daily
• Transfer to well-stoppered bottle


  Store in warm place
• Natural ripening of aluminum hematoxylin


  Takes about two months but staining property lasts for months to years
• Hasten ripening of hematoxylin


  1. Partially oxidize with sodium iodide

  2. Add 0.3 grams sodium

  
• Hematoxylin solution oxidizes


  • Color changes from purplish to deep red
  • Pungent odor of acetic acid replaced by acetic aroma
• Glycerin


  Acts as a stabilizer, retards evaporation and slows down ripening
• Hematoxylin


  Generally used for regressive staining and differentiated with 1% Hydrochloric acid in 70% alcohol until the nucleus is selectively stained

  
• Hematoxylin


  • Stains mucopolysaccharides substances such as cartilage and cement lines of bones intensely blue
  • Suitable for tissues that have been subjected to acid decalcification and is especially useful for tissues that have become acidic during prolonged storage in formalin
  • Not ideal for frozen sections

  
• Staining time for Harris Hematoxylin

  15-40 minutes
• Harris Hematoxylin


  Used for regressive staining

  
• Harris Hematoxylin formula


  • Hematoxylin
  • Absolute ethyl alcohol
  • Ammonium/potassium alum
  • Distilled water
  • Mercuric oxide (oxidizing agent)
  • Glacial acetic acid
• Making Harris Hematoxylin


  1. Dissolve hematoxylin in absolute alcohol with gentle heating

  2. Dissolve ammonium or potassium alum in distilled water, boil

  3. Add hematoxylin solution, boil

  4. Add mercuric oxide

  5. Plunge into cold water for rapid cooling

  
• A large beaker should be used because the violent liberation of oxygen will cause the solution to explode from a narrow mounted flask
• The solution should assume a dark purple color when ripened by mercuric oxide
• The addition of 4% glacial acetic acid will give a more precise nuclear staining
• Finish making Harris Hematoxylin

  Filter and transfer into well-stoppered bottle
• Harris Hematoxylin


  • Good regressive stain that may either be used immediately or stored for future use since it remains stable for a long time about 6 months
  • Since most of the alcohol is evaporated in the process of boiling, 10mL of ethyl alcohol may be added to the final solution to help prevent the growth of molds
  • Widely used for routine nuclear staining
  • Also used in exfoliative cytology and for staining of sex chromosomes
  • Usual staining time is 5-20 minutes
  • Best results are obtained when the solution is made every 2 to 3 months
  • Deformation of precipitate in the stored staining solution indicates deterioration in nuclear staining property

  
• Cole's Hematoxylin


  • Another alum hematoxylin solution recommended for routine purposes especially used in sequence with celestine blue
  • It is artificially ripened with an alcoholic iodine solution
  • Ready for immediate use but may need filtering after storage as with Harris hematoxylin

  
• Making Cole's Hematoxylin


  1. Dissolve hematoxylin in warm distilled water and mix with iodine

  2. Add alum solution and boil

  3. Cool and filter before use
• Staining time using Cole's hematoxylin

  Around 10 minutes
• Mayer's Hematoxylin


  • Widely used hematoxylin stain
  • Both regressive and progressive stain
  • An alum hematoxylin that is chemically ripened with sodium iodate
  • Used as a nuclear counterstain to demonstrate the presence of cytoplasmic glycogen by special stain
  • Also used in instances when acid alcohol differentiation might destroy or decolorize the stained cytoplasmic components like mucopolysaccharides
  • Stain applied for a short period 5 to 10 mins until nuclei are stained and then blued without any differentiation
  • Used in celestine blue hemalum method of nuclear staining