staining solutions

• Categories of Staining


  • Natural Dyes
  • Synthetic Dyes
• Natural Dyes


  Sources: plants and animals
• Most common natural dyes


  • Hematoxylin
  • Cochineal dyes
  • Orcein
• Hematoxylin


  • Most valuable for cytologist
  • Is NOT a stain itself
  • Is natural dye derived by the extraction from the core of the heartwood of a Mexican tree known as "Hematoxylin Campechianum"
• Oxidizing agents for ripening Hematoxylin

  • Hydrogen peroxide
  • Mercuric oxide
  • Potassium permanganate
  • Sodium perborate
  • Sodium iodate
• Cochineal dyes


  • Extracted from the female cochineal bug (Coccus Cacti), which is treated with alum to produce the dye, carmine
  • Carmine is a powerful chromatin and nuclear stain for fresh material and smear preparations
  • Picric acid: used extensively in neuropathological studies
  • Aluminum chloride: used for glycogen demonstration
• Orcein


  • Vegetable dye
  • Derived from certain lichens
  • A weak acid, and is soluble in alkali
  • Mainly used for staining elastic fibers
• Synthetic Dyes


  • Known as Coal Tar Dyes
  • Derive from the hydrocarbon benzene (C6H6) and are collectively known as Aniline dyes
• Components of Synthetic Dyes


  • Chromophores
  • Auxochromes
• Chromophores


  • Has definite atomic groupings
  • Produce visible colors
• Auxochromes


  • An auxiliary radical
  • Imparts electrolytic dissociation
  • Alters the shade of the dye
  • Dyeing property
• Dye modifiers


  • Ethyl groups
  • Methyl groups
  • Sulphonic groups
• Types of Chromophores


  • Quinoid ring
  • Azo groups
  • Xanthene
  • Quinone-imine group
• Types of Auxochromes


  • Cationic: amino group
  • Anionic: Hydroxyl and Carboxyl
• Classification of Dyes


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


  • Active coloring component: ACID
  • Inactive coloring component: BASE
  • Example: Picric acid
  • Fixes, Differentiates, and Stains tissues
  • For Van Gieson's stain for connective tissue
  • For microscopic study of fungi
• Basic dyes


  • Active component: Base
  • Example: Methylene blue
  • Used as an indicator and a dye
  • Mercuric chloride and formaldehyde fixed tissues: favored
• Neutral dyes


  • Combination of aqueous solution of acid and basic dyes
  • Stains cytoplasm and nucleus simultaneously and differentially
  • Insoluble/barely soluble in water
  • Examples: Romanowsky dye, Giemsa stain, Irishman's stain
• Common Staining Solutions Used


  • Hematoxylin
  • Eosin
  • Acid Fuchsin-Picric Acid
  • Acridine Orange
  • Acridine Red 3B
  • Alcian Blue
  • Aniline Blue
  • Basic Fuchsin
  • Benzidine
  • Bismarck Brown
• Aluminum Hematoxylin Solutions


  • Ehrlich's Hematoxylin
  • Harris Hematoxylin
  • Cole's Hematoxylin
  • Mayer's Hematoxylin
• Aluminum Hematoxylin Solutions


  • Give a "blue-lake"
  • Ripening agents: Sodium iodate, Mercuric chloride
  • During staining, these are passed on to an alkaline solution to neutralize the acid and free OH group, to form an insoluble blue aluminum hematin-tissue lake
• Blueing examples


  • Lithium carbonate
  • Bicarbonate
  • Potassium or sodium acetate
  • Scott's Tap Water Substitute
  • Ammonia water
• Ehrlich's Hematoxylin


  • Naturally ripened
  • Blue: cartilage and cement lines of bones
  • Glycerin: stabilizer, retards evaporation of the solution, slows down ripening
  • Recommended for tissues that have become acidic
• Harris Hematoxylin


  • Widely used for routine nuclear staining, exfoliative cytology, staining sex chromosome
  • Should assume a dark purple color when ripened with mercuric oxide
  • 4% glacial acetic acid: give more precise nuclear staining
  • 10mL of ethanol: prevents mold growth
• Cole's Hematoxylin


  • Recommended for routine purposes
  • Artificially ripened with an alcoholic iodine solution
  • Ready for immediate use
  • Staining time: 10 minutes
• Mayer's Hematoxylin


  • Chemically ripened with sodium iodate
  • Is a nuclear counterstain
  • To demonstrate cytoplasmic glycogen
  • Used in acid-alcohol differentiation
  • Used in Celestine Blue hemalum method
• Iron Hematoxylin


  • Weigert's Hematoxylin Solution
  • Heidenhain's Hematoxylin
• Weigert's Hematoxylin Solution


  • Color: deep purple blue-black-violet, through violet, purple, brown and yellowish brown within 2 to 3 weeks
  • Used for demonstrating muscle fibers and connective tissues
• Heidenhain's Hematoxylin


  • Oxidant/mordant: ferric amonium sulfate
  • Is a cytological stain recommended for regressive staining of thin sections
  • Color: black or dark grey
  • Used to demonstrate: chromatin, chromosomes, nucleoli, centrisome, and mitochondria
• Phosphotungstic Acid Hematoxylin (PTAH)


  • Mordant: 1% aqueous phoshotungstic acid
  • Immediate ripening: adding 50mL of 0.25% K permanganate
  • Color: reddish brown to purple
  • Blue: nuclei, fibrin, muscle striations, myofibrils and fibroglia
  • Orange-red/brownish red to brick red stain: collagen, bone and cartilage
  • Demonstrates in paraffin, celloidin and frozen sections
• Eosin


  • Used for staining connective tissues and cytoplasm
  • Used as a counterstain after hematoxylin and before methylene blue
  • Background stain: pleasing and colorful contrast
• Forms of Eosin


  • Yellowish (Eosin Y)
  • Bluish
  • Ethyl eosin
• Yellowish Eosin (Eosin Y)


  • Most commonly used
  • Gives green yellow fluorescence in alcoholic medium
• Other Stains Used


  • Acid Fuchsin-Picric Acid (Van Gieson's stain)
  • Acridine Orange
  • Acridine Red 3B
  • Alcian Blue
  • Aniline Blue
  • Basic Fuchsin
  • Benzidine
  • Bismarck Brown
  • Congo Red
  • Giemsa stain
  • Gold sublimate
  • Iodine
  • Janus Green B
  • Malachite Green
  • Night Blue
  • Orcein
  • Silver Nitrate
  • Victoria Blue
• Oil Soluble Dyes (Lysochromes)


  • Sudan Black
  • Sudan IV / Scharlach R
  • Sudan III
• Sudan Black


  • Most sensitive
  • Has much greater affinity to phospholipids
  • Imparts black color on lipids
  • Recommended for formol calcium fixed tissues
• Sudan IV


  • Does not color phospholipids or the fine lipid droplets
  • 1 part 2% benzoic acid and one part acetone
  • Recommended for triglycerides and neutral lipids
• Sudan III


  • Was the first sudan dye
  • Fat soluble
  • Good fat stain for CNS
• Chief Solvents Used for Stains


  • Water
  • Alcohol
  • Aniline water
  • Phenol
• Dye Tissue Mechanisms


  • Electrostatic
  • Hydrogen bonding
  • Van der Waals forces
  • Natural affinity
  • Physical