Stains and Staining Solutions

Staining and Dyeing

  • Allows observation and examination of tissue architecture.

  • Reveals physical features and structural relationships between tissues and cells.

Principle of Staining

  • Different tissues and cells have varying affinities for dyes or stains.

  • Alkaline structures are stained by acidic stains.

  • Acidic structures are stained by alkaline/basic stains.

Classification of Dyes

Natural Dyes

  • Obtained from plants and animals.

  • Previously used for dyeing wool and cotton.

  • Oldest dyeing source used by Leeuwenhoek with Saffron.

  • Examples:

    • Cochineal dyes and derivatives

    • Orcein

    • Saffron

    • Hematoxylin

Synthetic (Artificial) Dyes

  • Derived from hydrocarbon benzene; aniline dyes.

  • Sometimes known as “coal tar dyes”.

  • William H. Perkin discovered the first aniline dye, Mauve (Mauve-6,6’-dibromoindigo, aniline purple).

Composition of a Dye

  • A dye should consist of two substances:

    • Chromophores

    • Auxochromes

Natural Dyes in Detail

Cochineal Dyes

  • Extracted from the female cochineal bug (Coccus cacti).

  • Treated with alum to produce carmine.

  • Used as a chromatin and nuclear stain for fresh material and smear preparations.

    • Carmine + Picric Acid = Picrocarmine (for neuropathologic studies)

    • Carmine + Aluminum Chloride = Best’s Carmine Stain (for glycogen demonstration)

Orcein

  • Plant-based dye from specific lichens.

  • Naturally colorless but develops blue or violet hues when exposed to air after ammonia treatment.

  • Weak acid, soluble in alkali, mainly for staining elastic fibers.

    • Lichens + treated with lime and soda + exposed to ammonia and air = LITMUS
      *Note: Litmus is mainly an indicator with poor staining properties.

Saffron

  • Spice from the stigmas of the saffron crocus flower, Crocus sativus.

  • Used to season and color food.

  • Recognized for its bright yellow-orange hue, distinct aroma, and unique taste.

Hematoxylin

  • Derived by extraction from the core/heartwood of Hematoxylin campechianum (Mexican tree).

  • Most valuable staining reagent.

  • Can be used after almost any fixative and is a permanent stain.

  • Not a true basic dye.

  • Active coloring agent is hematin, formed by hematoxylin oxidation (“ripening”).

Hematoxylin Mordants

  • Ripened hematoxylin is seldom used alone; often used with alum, iron, chromium, and copper salts, which act as mordants.

  • Alum (Potassium Aluminum Sulfate) / Aluminum Hematoxylin Stain

    • Progressive or Regressive Staining

    • Counterstained with Eosin, Congo Red, and Safranin

    • Examples: Ehrlich’s solution, Harris’ Solution

    • Most commonly used mordant

    • Gives a “blue lake”

    • Increases nuclei selectivity, especially with acid addition or as a differentiating agent.

    • BLUEING - process of converting the reddish-purple color of hematoxylin to a blue hue, enhancing contrast and improving visualization of tissue structures under the microscope (e.g., 1% alcoholic ammonia or ammonia water, 1% aqueous lithium carbonate, Scott’s tap water/Scott’s tap water substitute)

  • Iron Hematoxylin Compounds

    • Differential or Regressive Staining

    • Using Acid-Alcohol as a differentiating agent

    • Example: Weigert’s Stain

  • Copper Hematoxylin Solutions

    • For the study of spermatogenesis

    • Ferric Salt - Causes an intense blue-black when used as a mordant

Types of Hematoxylins based on Mordants

  • ALUM HEMATOXYLINS: Mayer’s, Ehrlich’s, Delafield’s, Gill’s, Harris’, Cole’s

  • IRON HEMATOXYLINS: Weigert’s, Heidenhain’s

  • TUNGSTEN HEMATOXYLIN: Mallory’s Phosphotungstic Acid Hematoxylin (MPTAH)

  • COPPER HEMATOXYLINS: Used for the demonstration of spermatogenesis

Synthetic Dyes

Dye Components

  • A dye consists of a chromophore and an auxochrome.

Chromophores

  • “Color Bearer”

  • Capable of producing visible colors

  • Benzene+Chromophores=ChromogensBenzene + Chromophores = Chromogens

  • The color they impart is not permanent

Auxochrome

  • “Color Increaser”

  • Auxiliary radicals impart electrolytic dissociation, altering the dye's shade, giving it the property of forming salts with another compound and retaining its color.

Coloring and Dyeing Properties

  • The coloring property is attributed to the chromophore, and the dyeing property to the salt-forming auxochrome.

Types of Synthetic Dyes

  • Based on active coloring substance:

    • Acid Dyes

      • The active coloring substance is in the acid component.

      • Inactive base, e.g., acid fuchsin, is usually the sodium salt of a sulfonate of ronailine.

      • Example: Picric Acid

    • Basic Dyes

      • The active coloring substance is in a basic component.

      • Example: Methylene Blue

    • Neutral Dyes

      • Formed by combining aqueous solutions of acid and basic dye.

      • Example: Romanowsky Dye, Giemsa Stain, Irishman’s Stain

Common Staining Solutions

Hematoxylin

  • Commonly used for routine histologic studies (Hematoxylin and Eosin/H&E).

  • Considered a basic dye - used to stain acidic (or basophilic) structures a purplish-blue.

Eosin

  • Used for differentially staining connective tissues and cytoplasm.

  • A red general cytoplasmic stain that combines with hemoglobin to give an orange color.

  • An acid dye - counterstain to hematoxylin, imparting a pink or red color to cytoplasmic material, cell membranes, and some extracellular structures

  • Commonly used as a background stain

Types of Eosin

  • EOSIN YELLOWISH (EOSIN Y)

    • Most commonly used

    • Readily soluble in water, less in alcohol; available in both aqueous and alcoholic solutions, showing a green-yellow fluorescence, especially in alcoholic medium

  • EOSIN B

    • Eosin Bluish or Imperial Red

    • Very faint bluish cast

Eosin Solutions

  • 5% AQUEOUS EOSIN Y

    • Contains Eosin Y (5gm), distilled water (100 mL), Thymol crystals (prevent molds)

  • EOSIN, STOCK ALCOHOLIC SOLUTION

    • Contains Eosin Y (1gm), distilled water (20 mL), 95% alcohol (80 mL)

  • EOSIN-PHLOXINE B SOLUTION

    • Combining Eosin Y and Phloxin B produces cytoplasmic stain that demonstrates various components more dramatically

    • Contains Glacial Acetic Acid

H&E Staining Results

  • Cytoplasmic & Proteins in Edema Fluid: Pale Pink

  • Decalcified Bone, Osteoid, and Collagen: Pink

  • Muscle Fiber: Deep Pink

  • RBC, Eosinophil Granules, and Keratin: Bright Orange to Red

  • Calcium and Calcified Bone: Purplish Blue

  • Plasma Cells, Osteoblast, and Basophilic Cytoplasm: Purplish Pink

  • Karyosome: Dark Blue

  • Nucleus: Blue to Blue-Black

  • Cartilage: Light Blue to Dark Blue

Romanowsky Stains

  • Combination of eosinate (chemically reduced eosin) and methylene blue (sometimes with its oxidation products azure A and azure B)

  • Examples: Wright’s Stain, Jenner’s Stain, Leishman Stain, Giemsa Stain

  • Used to examine blood or bone marrow samples.

  • Preferred over H&E for blood cell inspection because different types of WBCs can be distinguished.

  • Suited to detect blood-borne parasites (e.g., malaria).

Other Stains & Lysochromes

Other Stains

  • Acridine Orange: Permits discrimination between dead and living cells, giving GREEN fluorescence for DNA and a RED fluorescence for RNA

  • Acridine Red 3B: For calcium salts and phosphatase activity

  • Alcian Blue: Stain acid mucopolysaccharides; More specific for connective tissue and epithelial mucin

  • Aniline Blue: Cytoplasmic stain used for counterstaining of epithelial sections

  • Basic Fuchsin

    • Carbol fuchsin, Coleman’s fuelgen, Schiff’s, Malory’s fuchsin, Aldehyde Fuchsin (Gomori’s)

    • A plasma stain utilized also for deep staining of acid-fast organisms, mitochondria, and differentiation of smooth muscles with the use of picric acid

  • Benzidine: Used for staining hemoglobin

  • Bismarck Brown: Used for staining diphtheria; Used as a contrast stain for Gram’s technique, acid-fast, and Papanicolaou method

  • Carmine: Used as a chromatin stain for fresh materials in smear preparations

  • Best Carmine: Combined with aluminum chloride to stain glycogen

  • Celestine Blue: Recommended for routine staining of fixed sections

  • Congo Red

    • Best known as indicator; May be utilized as a stain for axis cylinders in embryos; Used for staining elastic tissues, amyloid, and myelin (Krajian’s Method)

  • Crystal Violet

    • Nuclear or chromatin stain used for staining amyloid in fresh sections and platelets; Gentle Violet - mixture of crystal violet, methyl blue, and Dextrin

  • Giemsa

    • Used for staining blood to differentiate leukocytes

  • Gold Sublimate

    • Used for metallic impregnation, made up of gold chloride, and mercuric chloride

  • Iodine

    • Gram’s, Lugol’s

    • Oldest of all stains; Stains amyloid, cellulose, starch, carotenes, and glycogen

  • Janus Green B

    • Used for demonstrating mitochondria (intravital staining)

  • Loeffler’s Polychrome Methylene Blue

    • Used in frozen sections for rapid diagnosis

  • Malachite Green

    • Used as a contrast stain for staining Ascaris lumbricoides eggs and erythrocytes; Used as a bacterial spore stain

  • Mayer’s Carmalum Solution

    • A mordanted dye acting as a basic dye and staining acid substances

  • Methyl Green

    • Stains chromatic green in the presence of an acid

  • Methylene Blue

    • Plasma cells, fresh sputum for malignant cells, evaluation and differentiation of bacterial organisms, Diphtheria diagnosis, and nervous tissue vital staining

  • Methylene Violet

    • Coloring nuclei of leukocytes reddish-purple in the presence of methylene blue

  • Neutral Red

    • For observing cell granules and vacuoles of phagocytic cell

  • Night Blue

    • Substitute for carbon fuchsin in acid-fast staining

  • Orcein

    • An excellent stain for elastic fibers (Taenzer Unna Orcein Method); demonstrates the finest and most delicate fibers in skin (dermatological studies)

  • Osmium Tetroxide

    • Used as fixative; Used to stain fats

  • Prussian Blue

    • Normally utilized for the manufacture of paints; Used as microanatomical contrast stain; For demonstration for the circulatory system by injection (intravital staining)

  • Rhodamine B

    • Used with osmic acid to fix and stain blood and glandular tissues

  • Silver Nitrate

    • Used in identification of Spirochestes, reticulum, nervous tissues, and other fiber stains

  • Toluidine Blue

    • Recommended for staining of Nissl granules or chromophilic bodies; Nuclear stain for fixed tissues; Used as a substitute for thionine in fresh tissue sections

  • Van Gieson’s (Acid Fuchsin Picric Acid)

    • Mixture of picric acid and acid fuchsin for the demonstration of connective tissues, music, and elastic tissue

  • Victoria Blue

    • For demonstration of neuroglia in frozen sections

Lysochromes/Oil Soluble Dyes

  • Sudan Black B (SBB)

    • Greatest affinity for phospholipids or neutral fats (TAG); A more sensitive coloring agent than other lysochromes; Should be discarded if the brownish-black color appeared; Demonstrates lipids that are resistant to paraffin embedding

  • Sudan IV or Sharlach R

    • Recommended for neutral fats (TAG); Does not color phospholipids and fine lipid droplets

  • Sudan III

    • First Sudan Dye introduced into Histochemistry; Fat soluble; A good stain for the CNS