Stains and staining solutions

• Natural dyes


  hose obtained from plants and animals, previously utilized for dyeing of wool and cotton
• Hematoxylin


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


  It is by far the most valuable staining reagent used by the cytologist due to its 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.
• hematin


  active coloring agent
• ripening


  formed by the oxidation of hematoxylin
• Mordants


  ubstances that combine with the tissue and the staining solution, forming a "bridge" that allows staining reaction to take place
• Alum hematoxylin stains


  ecommended for progressive staining of tissues, and are usually counterstained with Eosin, Congo Red and Safranin. Both the Ehrlich’s solution and the Harris’solution contain Alum Hematoxylin. Rapid ripening of Ehrlich’s reagent, however, is brought about by the addition of Sodium Iodate; while Harris solution is ripened with Mercuric Chloride.
• Iron hematoxylin compounds


  used only for differential or regressive staining, using Acid-Alcohol as a differentiating agent. An example of an Iron Hematoxylin compound is Weigert’s Stain using Iron (Ferric) Chloride.
• Copper hematoxylin solutions


  utilized for the study of spermatogenesis.
• Cochineal dye


  old histologic dye extracted from the female cochineal bug (Coccus Cacti), which is treated with alum to produce the dye, carmine. It is widely used as a powerful chromatin and nuclear stain for fresh material and smear preparations.
• ORCEIN


  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. It is a weak acid, is soluble in alkali, and is mainly used for staining elastic fibers.
• Synthetic dyes


  sometimes known as "Coal Tar Dyes" since they were originally manufactured from substances that have been taken from coal tar. They are derived from the hydro-carbon benzene (C6H6 ), and are collectively known as Aniline Dyes.
• Chromophores


  substances with definite atomic groupings and are capable of producing visible colors. Simple benzene compounds which contain such substances are known as chromogens.
• Acid Dyes


  active coloring substance is found in the acid component, and the inactive base, e.g. acid fuchsin, is usually the sodium salt of a sulfonate of rosaniline. One example of such a dye is picric acid, which has the ability to form salt with an alkali.
• Basic Dyes


  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. Because they are made up of large molecular complexes, neutral dyes are insoluble or barely soluble in water, but they are usually soluble in alcohol. Ethyl alcohol or acetic acid-fixed tissues, on the other hand, readily take in both basic and acidic dyes.
• Hematoxylin


  staining solution most commonly used for routine histologic studies. The mordants used to demonstrate nuclear end cytoplasmic structures are alum and iron, forming lakes or colored complexes (dyemordant-tissue complexes), the color of which will depend on the salt used. Aluminum salt lakes are usually colored blue while ferric salt lakes are colored blue-black
• Aluminum Hematoxylin Solutions


  recommended for progressive staining of tissues
• The alum hematoxylins can also be used for regressive staining, meaning that the section is overstained, and then differentiated in acid alcohol followed by "blueing".
• Rapid ripening of Ehrlich's reagent is brought about by the addition of sodium iodate
• Harris solution is ripened with mercuric chloride.
• During staining, alum hematoxylin stained sections are usually passed on to an alkaline solution (e.g. 1% hydroxide) in order to neutralize the acid and free the OH group, to form an insoluble blue aluminum hematin-tissue-lake. Such procedure is known as blueing
• Ehrlich's Hematoxylin

  FORMULA:

  Hematoxylin 2 gm

  Absolute ethyl alcohol 100 ml

  Aluminum potassium Sulfate 15 gm approximately

  Glycerin 100 ml Distilled water 100 ml

  Glacial acetic acid 10 ml
• Harris Hematoxylin


  FORMULA:

  Hematoxylin 1 gm

  Absolute ethyl alcohol 10 ml

  Ammonium/Potassium alum 20 gm

  Distilled water 190 ml

  Mercuric oxide (red) 0.5 gm Glacial acetic acid 10 ml
• Harris hematoxylin is a 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).
• Cole's Hematoxylin


  another alum hematoxylin solution recommended for routine purposes, especially used in sequence with Celestine blue
• Cole's Hematoxylin

  This alum hematoxylin is artificially ripened with an alcoholic iodine solution
• Cole's Hematoxylin

  FORMULA:

  Hematoxylin 1.5 gm

  1% Iodine in 95% Alcohol 50 ml Sat. Aq. Ammonium Alum 700 ml Distilled Water 250 ml
• Mayer's Hematoxylin


  chemically ripened with sodium iodate. Like any alum hematoxylin, it can be used as a regressive stain, but it is also useful as a progressive stain.
• Mayer's Hematoxylin


  used as a n uclear counterstain to demonstrate the presence of cytoplasmic glycogen by special stain. It is also used in instances when acid-alcohol differentiation might destroy or decolorize the stained cytoplasmic components like mucopolysaccharides. It is used in Celestine Blue hemalum method of nuclear staining
• Mayer's Hematoxylin

  FORMULA:

  Hematoxylin 1 gm

  Sodium iodate 0.2 gm

  Potassium alum 50 gm

  Citric acid 1 gm

  Chloral hydrate 50 gm

  Distilled water 1000 ml
• Iron Hematoxylin Solutions


  used only for differential or regressive staining, using acid-alcohol as a differentiating agent. Two main iron hematoxylin solutions are employed for routine work in the laboratory: Weigert's Solution, using ferric ammonium chloride, and Heidenhain's solution, using ferric ammonium sulfate (iron alum) as mordants. The dye lake obtained when ferric salts are used as mordants is an intense blue-black one.
• Regaud's Hematoxylin for Mitochondria


  used to demonstrate mitochondria by light microscopy
• Weigert's Hematoxylin Solution

  FORMULA:

  SOLUTION A:

  Hematoxylin 1 gm

  Absolute ethyl alcohol ml

  SOLUTION B:

  30% anhydrous ferric chloride 4 ml

  Concentrated hydrochloric acid 1 ml

  Distilled water 100 ml
• Heidenhain's Hematoxylin


  popular cytological stain, especially for the study of mitosis. It can be used after almost any fixative. Chromatin material (nuclear network and chromosomes) blue black.
• Heidenhain's Hematoxylin

  FORMULA:

  MORDANT DIFFERENTIATOR:

  Ferric ammonium sulfate 2.5 gm

  Distilled water 100 ml

  HEMATOXYLIN STAIN:

  Hematoxylin 1.5 gm 95% ethyl alcohol 10 ml

  Distilled water 90 ml
• Phosphotongstic Acid Hematoxylin (PTAH)


  Natural ripening of the tungsten hematoxylin solution is achieved with light and air, but will take some months to ripen.
• Phosphotongstic Acid Hematoxylin (PTAH)

  FORMULA:

  Hematoxylin 1 gm

  Phosphotungstic acid 20 gm

  Distilled Water 1000 ml
• Eosin


  one of the most valuable stains used for differentially staining connective tissues and cytoplasm
• Yellowish (Eosin Y)

  the most commonly used. It is readily soluble in water, less in alcohol, available in both aqueous and alcoholic solutions, showing a green yellow fluorescence especially in alcoholic medium.