Stains used in Histopath Lab

• Staining


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


  A colored compound used to produce a contrast between different tissues and cellular components based on their varying affinities for most dyes and stains
• Why we stain


  • Most cells are colorless and transparent, so histological sections have to be stained in some way to make the cells visible
  • The main reason is to enhance contrast and visualization of the cell or certain cellular components under a microscope
  • Cells may be stained to highlight metabolic processes, differentiate between live and dead cells, demonstrate the relationship between internal and external structures, and identify different types of cells
• Types of dye


  • Natural dye
  • Synthetic dye
• Natural dye


  Derived from plants and animals
• Synthetic dye


  Usually from hydrocarbons like benzene
• Natural dyes


  • Hematoxylin
  • Cochineal dye
  • Orcein
• Hematoxylin


  Source: "Hematoxylin campechianum", Coloring agent: Hematin, Used for routine histologic studies, Has a powerful nuclear and chromatin staining capacity, Can be useful with any fixative, Permanent stain
• Cochineal dye


  Source: "Coccus cacti" (female cochineal bug), Dye: Carmine, Powerful nuclear and chromatin stain for fresh and smear preparations, Used for neuropathological studies and demonstration of glycogen
• Orcein


  Source: Extracted from lichens, Colorless, treated with ammonia and exposed to air, Stains elastic fibers, Not used as a cytological stain, Used mainly as an indicator
• Synthetic dyes


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


  The active coloring substance is found in the acidic component, Have affinity towards the basic cell structure (collagen, eosinophilic granules of leukocyte, etc.), Acidophilic: basic cell structure that have an affinity for the acid dye ions
• Basic dyes


  The active coloring substance is found in the basic component, Have affinity towards acidic cell structure (chromatin, mucus, cartilage matrix, etc.), Basophilic: refers to acidic cell structures that have an affinity for basic dye ions
• Neutral dyes


  Combined acid and basic dyes, Stain cytoplasm and nucleus simultaneously but with different colors
• Common staining solutions


  • Hematoxylin
  • Eosin
  • Romanowsky
• Hematoxylin


  Not in itself a dye, Used for routine histologic studies, Used with a mordant, Categorized depends on the mordant used (Alum, Iron, Copper, Tungsten)
• Aluminum hematoxylin solutions


  • Erlich's Hematoxylin
  • Harris Hematoxylin
  • Mayer's Hematoxylin
• Erlich's Hematoxylin


  Recommended for bone and cartilage, Recommended nuclear stain for immunohistochemistry and cytochemical staining, Takes about 2 months to ripen, Not applicable for frozen sections, Ripened with Sodium iodate, Glycerin slows oxidation and prolongs shelf-life
• Harris Hematoxylin


  Routinely used in nuclear staining, Ripened with Mercuric Oxide (HgO), Nuclear stain in Papanicolaou, Stains sex chromosomes, Addition of glacial acetic acid gives a precise nuclear stain, Employs progressive staining
• Mayer's Hematoxylin


  Ripened with Sodium Iodate, Primarily a regressive stain but can also be used as progressive, Used as a nuclear counterstain to demonstrate the presence of cytoplasmic glycogen by special stain, Used in instances when acid-alcohol differentiation might destroy or decolorize the stained cytoplasmic components like mucopolysaccharides
• Iron hematoxylin solutions


  • Regaud's Hematoxylin for Mitochondria
  • Weighert's Hematoxylin Solution
  • Heidenhan's Hematoxylin Solution
• Regaud's Hematoxylin for Mitochondria


  Modified Iron hematoxylin, Used to demonstrate mitochondria in light microscopy, The most permanent and the simplest
• Weighert's Hematoxylin Solution


  Mordant/oxidizer: Ferric Ammonium Chloride, Standard Iron Hematoxylin Solution, Used in demonstrating muscle fibers and connective tissues
• Heidenhan's Hematoxylin Solution


  Mordant/Oxidizer: Iron Alum, Cytological Stain, For the study of mitosis and for nuclear and cytoplasmic inclusions, Result: Gray-Black
• Tungsten Hematoxylin Solution


  Phosphotungstic Hematoxylin: Mordant: 1% aqueous phosphotungstic acid, Oxidizer: Potassium permanganate, Naturally ripened, Progressive stain, For CNS and general staining of tissues
• Eosin


  One of the most valuable stains used for differentially staining connective tissues and cytoplasm, Stains its specimen orange, An acid dye which can be termed as acidophilic, oxyphilic and eosinophilic, A counterstain for hematoxylin
• Eosin variants


  • Yellowish (Eosin Y)
  • Eosin B (Eosin bluish or Imperial red)
• Yellowish (Eosin Y)


  The most commonly used, Readily soluble in water, less in alcohol, Available in both aqueous and alcoholic solutions, Shows a green yellow fluorescence especially in alcoholic medium, The aqueous stain is generally used as a 1% solution for 15 seconds to 3 minutes, Slightly longer staining time is required after formalin than after Zenker's solution
• Eosin B (Eosin bluish or Imperial red)


  Has a very faint bluish cast, The two dyes are interchangeable, and the use of one or the other is more a matter of preference and tradition
• Romanowsky stain


  Common variants: Wright's Stain, Jenner's Stain, Leishman Stain, Giemsa Stain
• Other stains


  • Acid Fuchsin-Picric Acid (Von Gieson's Stain)
  • Acid Fuchsin (Masson Stain)
  • Picro-Fuchsin Solution
  • Acridine Orange
  • Acridine Red 3B
  • Alcian Blue
  • Alizarin Red
  • Aniline Blue
  • Azocarmine
  • Basic-Fuchsin
  • Benzidine
• Hydrochloric acid treatment


  1. Cool to 25C

  2. Add sodium metabisulphite

  3. Let stand for 24 hours until the solution becomes pale-straw in color

  4. Filter through activated charcoal to form a colorless filtrate

  5. Store in a refrigerator
• D. Mallory's Fuchsin Stain


  Formula: Basic fuchsin 0.5 gm, 95% ethyl alcohol 50 ml, Distilled water 50 ml. Dissolve fuchsin in alcohol by gentle heating. Add water, cool, and filter.
• E. Aldehyde Fuchsin (Gamori's Stain)


  Formula: Concentrated hydrochloric acid 1 ml, Paraldehyde 1 ml, 0.5% basic fuchsin in 70% alcohol 100 ml. Stand at room temperature for 24 hours until the mixture becomes deep purple in color. Store in the refrigerator.
• Benzidine


  Used for staining hemoglobin. Solution A: Benzidine 0.5 gm, Absolute alcohol 50 ml, Sodium nitroprusside 0.1 gm, Distilled water up to 100 ml. Solution B: Absolute alcohol 50 ml, Glacial acetic acid 2 ml, Hydrogen peroxide 30% 0.5 ml, Sodium nitroprusside 0.1 gm, Distilled water up to 100 ml.
• Bismarck Brown


  Used as contrast stain for Gram's technique, in acid fast and Papanicolaou method, and for staining diphtheria organisms.
• Carmine


  Used as a chromatin stain for fresh materials in smear preparations.
• Best Carmine Stain (Stock Solution)


  Formula: Carmine 2 gm, Potassium carbonate 1 gm, Potassium chloride 5 gm, Distilled water 60 ml, Concentrated ammonia 20 ml. Grind carmine in a mortar and add potassium carbonate and potassium chloride to water. Boil gently in a large flask (to avoid frothing) for 5 minutes. Cool and add ammonia. Store in a dark bottle inside the refrigerator.
• Best Carmine Working Solution

  Formula: Best carmine (stock solution) 2 parts, Ammonia concentrated 2 parts, Absolute methyl alcohol 3 parts.
• Best's Differentiator


  Formula: Absolute methyl alcohol 40 ml, Absolute ethyl alcohol 80 ml, Distilled water 100 ml.