Appendix A: Staining and Commonly Used Stains

To enhance contrast in the normally colorless tissue sections, tissue sections are commonly stained. For light microscopic examinations, colored agents (chromophores) are used. For the electron microscope, electron-dense heavy metal salts are employed (e.g., lead citrate, uranyl acetate).

The earliest histologists used naturally occurring dyes to stain their sections. With the invention of chemical dyes a greater array of stains became available. Although there is some understanding of staining mechanisms, even today the precise mechanism of many stains is not known.

Students frequently hear or read that certain dyes are either basic or acidic. This is because synthetic dyes are prepared so that the coloring part of the dye is either acid (anionic) or basic (cationic) in its chemical behavior. The cationic or basic dye has an affinity for nuclei and ribosomes that exist in the tissue with a net negative charge and are termed basophilic; the anionic or acid dye has an affinity for positively charged cytoplasm and other components like mitochondria and cilia, which are then termed acidophilic structures. Thus:

Basic stain:   [dye]+ OH-
stains basophilic structures, e.g., nuclei, ribosomes, GAGs

Acid stain:   [dye]- H+
stains acidophilic structures, e.g., mitochondria, collagen

A tissue section contains many proteins that differ in their isoelectric points. At an ideal pH, certain tissue components will show a relative acidophilia whereas others display a relative basophilia.

Frequently, basic dyes (methylene blue, toluidine blue, thionine) will react with a specific tissue component and impart to it a color different from that of the dye itself. This phenomenon is called metachromasia and the cell or tissue components that exhibit it are said to be metachromatic.

Following is an alphabetical list of stains commonly used in light microscopic histology. Some are general purpose stains and some are used to specifically demonstrate certain materials in sections. Examples of microscope slides that illustrate the stain described are provided in brackets [#] at the end of the description.

1. H&E Stain (Hematoxylin and Eosin)
Most widely used and important general purpose stain combination. May be used after any fixation except fixation with osmium tetroxide. Hematoxylin, a natural dye product, acts as a basic dye that stains blue or black. Nuclear heterochromatin stains blue and the cytoplasm of cells rich in ribonucleoprotein also stains blue. The cytoplasm of cells with minimal amounts of ribonucleoprotein tends to be lavender in color. The aniline dye, eosin, is an acid dye that stains cytoplasm, muscle, and connective tissues various shades of pink and orange. This difference in staining intensity is useful in differentiating one tissue from another. Although it is an esthetically pleasing combination and widely used, it is limited in its ability to differentiate cytoplasmic organelles. Common basic dyes often substituted for hematoxylin include methylene blue, toluidine blue, thionine, carmine, basic fuchsin, and azure II. Commonly substituted acid dyes include orange G or phyloxine.

Many of the microscope slides are prepared using material embedded in plastic rather than in paraffin. The plastic used is glycol methacrylate and it is commonly used in histology and pathology because some of the artifacts (shrinkage and distortion) caused by hot paraffin can be largely avoided. Furthermore, plastic embedded sections can be cut at 1 or 2 micrometers thick, allowing for improved visualization of the tissue. Because H∓E stains can be problematic with methacrylate, some of the plastic embedded material is stained using a combination of substitute dyes that look similar to H&E but without the problems. The basic dye used is often celestine blue and stains nucleic acids blue; the acid dye is a solution of ponceau de xylinine and acid fuchsin that stains collagen and muscle fibers an orange-red, and eosinophil granules red. [Slide #125]

2. Lee's Stain (Methylene Blue and Basic Fuchsin)
This stain is often used for general purpose tissue staining, being better for muscle tissue than H&E. Nuclei stain blue; cytoplasm mitochondria, cilia and certain cell granules stain shades of red and pink; cartilage and most cell granules stain blue to purple. [Slide #109, "blue" section]

3. Mallory's Connective Tissue Stain (Mallory Trichrome)
One of the most beautiful of all stains. Several modifications of the original method have been developed. It is often used to differentiate acidophilic extracellular fibers from acidophilic cytoplasm. Basic ingredients are acid fuchsin, aniline blue, orange G and phosphotungstic acid. Collagen and reticular fibers stain blue; nuclei and smooth muscle stain red; elastic fibers stain pink; red blood cells and myelin stain orange. [Slide #53]

4. Periodic Acid-Schiff Method (PAS)
Principally used to demonstrate structures rich in carbohydrate macromolecules such as glycogen, glycoprotein, and proteoglycans found in ground substance of connective tissues, basement membranes and mucus. This method depends on the selective oxidation by periodic acid of free hydroxyl groups on two adjacent hydroxyl groups converting the alcohols to aldehydes. The aldehydes are then detected by the Schiff reagent, which stains them reddish purple. Other tissue components stain according to the counterstain used. Lead-hematoxylin or another basic stain is often the counterstain. [Slide #122]

5. Phosphotungstic Acid Hematoxylin (PTAH)
This is an ideal stain for the demonstration of striated muscle fibers and mitochondria, which stain blue. A counterstain is often not used. [Slide #38, no counterstain]

6. Silver Stains
Certain tissue components called argyrphilic have a natural affinity for silver salts. The glycoproteins in these materials reduce silver salts to silver metal, depositing a black stain around the argyrophilic materials. Reticular fibers and the granules in diffuse endocrine cells are argyrophilic. Usually a counterstain is not used and the unstained cellular elements are seen as colorless shadows. [Slide #70, no counterstain]

7. Sudan Stains
Sudan dyes are used to stain lipids. The Sudan dyes, e.g., sudan IV, dissolve in droplets containing triglycerides and color them intensely. In order to apply this dye, care must be taken during tissue preparation to retain the lipid, which is often washed out by standard tissue preparation procedures. [Slide #9]

8. Wright's Stain
Wright's stain is a neutral stain produced by the interaction of an acidic and a basic dye, producing a large salt molecule with a colored dye in both its parts. The Romanovsky-type mixtures, (including Wright's and Giemsa stains) are the best known of these neutral stains and they are formed by the interaction of methylene blue and eosin.

With the Wright's stain, blood cells exhibit four major staining properties that allow the cell types to be distinguished. Basophilia (affinity for methylene blue), azurophilia (affinity for the oxidation products of methylene blue called azures, which are reddish purple), acidophilia (affinity for eosin), and neutrophilia (affinity for a complex of dyes in the mixture, which are pale lilac). In a stained blood smear, erythrocytes bind eosin and appear orange to pink, nuclei purplish blue, basophilic granules very dark bluish purple, eosinophilic granules red to red-orange, neutrophilic granules reddish-brown to lilac, platelets violet to purple, and lymphocyte cytoplasm stains pale blue. [Slide #18]

9. Verhoeff Stain
A good stain for connective tissue, especially the elastin. Elastin stains a dark brown-black. A counter stain may or may not be used with the Verhoeff's stain. [Slide #29, no counterstain]

Stains Used in Electron Microscopy
Heavy metal salts are used to stain biological materials for examination in the transmission electron microscope. The majority of the popular electron stains are general purpose stains and they are not very specific. Uranyl acetate stains membranous structures and structures containing nucleic acids. Lead citrate is the other commonly used stain. The lead binds to RNA-containing structures and hydroxyl groups of carbohydrates. [Slide EM#01]

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