ch 17 histo

• Biological stains


  Coloring substances prepared from dyes which may generally be divided into natural dyes and synthetic (artificial) dyes
• Natural dyes


  • Cochineal dyes
  • Logwood dyes
  • Vegetable extracts
• Synthetic (artificial) dyes


  • Aniline dyes
  • Coal tar dyes
• Hematoxylin


  A natural dye derived by extraction from the core or the heartwood of a Mexican tree known as "Hematoxylin Campechianum"
• Hematoxylin


  • It has powerful nuclear and chromatin staining capacity, and its striking polychrome properties which may be produced with proper differentiation
  • It may be used after almost any fixative and is a permanent stain
  • It is not a true basic dye, the active coloring agent is hematin, which is formed by the oxidation of hematoxylin, a process known as "ripening"
• Mordants


  Substances that combine with the tissue and the staining solution, forming a "bridge" that allows staining reaction to take place
• Hematoxylin compounds


  • Alum hematoxylin
  • Iron hematoxylin
  • Copper hematoxylin
• Hematoxylin and eosin (H&E) staining


  Hematoxylin stains cell nuclei blue, while eosin stains cytoplasm, connective tissue and other extracellular substances pink or red
• Cochineal dye


  An old histologic dye extracted from the female cochineal bug (Coccus Cacti), which is treated with alum to produce the dye, carmine
• Orcein


  A vegetable dye extracted from certain lichens which are normally colorless, but which, when treated with ammonia and exposed to air, produce blue or violet colors
• Litmus


  Obtained from lichens, treated with lime and soda, and exposed to ammonia and air, used mainly as an indicator rather than a cytological stain
• Synthetic dyes


  Also known as "Coal Tar Dyes", derived from the hydrocarbon benzene (C6H6), and are collectively known as Aniline Dyes
• Chromophores


  Substances with definite atomic groupings and are capable of producing visible colors
• Chromogens


  Simple benzene compounds which contain chromophores, but any color they impart to the tissue is not permanent and can be easily removed
• Auxochrome


  An auxiliary radical or substance which imparts to the compound the property of electrolytic dissociation, thereby altering the shade of the dye, enabling it to form salts with another compound, and ultimately retaining its color
• Types of dyes


  • Acid dyes
  • Basic dyes
  • Neutral dyes
• Acid dyes


  The active coloring substance is found in the acid component, and the inactive base is usually the sodium salt of a sulfonate of rosaniline
• Basic dyes


  The active coloring substance is found in a basic component that combines with the acid radical (usually taken from sulfuric, acetic or hydrochloric acid)
• Neutral dyes


  Formed by combining aqueous solutions of acid and basic dyes, capable of staining cytoplasm and nucleus simultaneously and differentially
• Hematoxylin


  The staining solution most commonly used for routine histologic studies, using alum and iron as mordants to form colored complexes
• Hematoxylin (in H&E staining)


  A basic dye that stains acidic (or basophilic) structures a purplish blue, using aluminum ions as a mordant
• Eosin (in H&E staining)

  An acidic dye that stains basic (or acidophilic) structures red or pink
• Alum hematoxylin stains


  Recommended for progressive staining of tissues, to be later counterstained with eosin, Congo red or safranin
• Ehrlich's hematoxylin and Harris hematoxylin

  The two main alum hematoxylin solutions employed, with rapid ripening brought about by sodium iodate and mercuric chloride respectively
• Blueing of alum-hematoxylin stained sections

  Passing the stained sections to an alkaline solution (e.g. 1% hydroxide) to neutralize the acid and form an insoluble blue aluminum hematin-tissue-lake
• Alum hematoxylin


  Stain that becomes red in acid solutions due to lack of -OH ions
• Blueing of alum-hematoxylin-stained sections

  Pass sections to alkaline solution (e.g. 1% hydroxide) to neutralize acid and free OH group, forming insoluble blue aluminum hematin-tissue-lake
• Blueing with warm tap water

  Commonly used as it is generally sufficiently alkaline
• Blueing with other alkaline solutions

  Lithium carbonate (1% w/v), bicarbonate (0.2-0.5% w/v), potassium or sodium acetate, Scott's Tap Water Substitute
• Blueing with ammonia, lithium carbonate or Scott's Tap Water Substitute

  Has more rapid action (15, 30 and 60 seconds respectively) compared to 5-15 minutes for warm tap water
• Ammonia water for blueing

  Prepared by mixing 2 cc strong ammonium hydroxide with 98 cc tap water
• Lithium carbonate has a tendency to form crystalline deposits unless slides are agitated and washed well afterwards
• Use of very cold water for blueing

  Slows down the process, may produce pink artifact discolorations
• Warming the blueing solution

  Accelerates the process
• Ehrlich's hematoxylin


  Alum hematoxylin stain used for regressive staining, differentiated with 1% hydrochloric acid in 70% alcohol
• Ehrlich's hematoxylin


  • Stains mucopolysaccharide substances like cartilage and cement lines of bones intensely blue
  • Suitable for tissues subjected to acid decalcification or stored in formalin
  • Not ideal for frozen sections
• Staining time for Ehrlich's hematoxylin

  15-40 minutes
• Harris hematoxylin


  Alum hematoxylin stain that is a good regressive stain, may be used immediately or stored for up to 6 months
• Harris hematoxylin


  • Widely used for routine nuclear staining, exfoliative cytology, staining of sex chromosomes
  • Staining time is 5-20 minutes
  • Best results when solution is made every 2-3 months
  • Precipitate formation indicates deterioration in nuclear staining property
• Cole's hematoxylin


  Alum hematoxylin solution artificially ripened with alcoholic iodine, ready for immediate use