Histology Stains



For the most part, living or fixed cells have very little inherent color and only very small differences in the refractive indices of their component parts. The phase difference and interference microscopes have greatly increased what we can observe in such material, but these instruments are used primarily in research laboratories.


In order to enhance contrast for routine observation and study of details of cells and tissues we must resort to the process of staining.   By using special selective staining methods it is possible to differentiate many structural elements of tissues by their color or intensity of staining.   There are many different methods for staining, and their principles are (numbered references below):  (a) affinity between the dye and substance that constitutes the structure (1, 2, 3, 6, 8, 10, 11), (b) solubility of the dye in the substance that constitutes the structure (4), (c) chemical reaction between the dye and the substances that constitute the structure (7, 9) (d) utilization of a biological phenomenon of cells (e.g. phagocytosis) (5), (e) visualization of the products of the enzyme reaction, (f) visualization of the products of immunoreaction, etc.   Below is a brief description of the stains used for the slides in your histology set.   A discussion of stains, dyes and histochemistry is also found in your textbook.

1)  Hematoxylin and Eosin (H&E:  most slides; [Example; Example])

This is a good general stain and is widely used.   Most of your slides, when it is not otherwise stated are stained with H&E.  Hematoxylin acts as a basic stain via a substance called hematein which is formed in solutions of hematoxylin.   It stains nucleic acids in the nucleus (chromatin and nucleolus) and cytoplasm (ribosomes) blue.   Eosin is an acid aniline dye which stains the more basic proteins and other materials pink or red.   It is thus mainly a cytoplasmic stain. Collagen and red blood cells stain pinkish or reddish with eosin.   Cartilage matrix stains purple if the proteoglycans were preserved during processing.

2)   Iron Hematoxylin (Slide 34)

In this stain iron acts as a mordant to bind the hematoxylin.   It is a good nuclear stain, rendering the chromatin and nucleolus black.  Centrioles and basal bodies are black.   The terminal web also binds this stain.  Mitochondria, if adequately preserved, are black.  A counterstain (eosin, acid fuchsin, etc.) is used to stain the cytoplasm.

3a)  Mallory's Trichrome Connective Tissue Stain (Slides 20, 30, 31, and 98) [Example]

A stain composed of aniline blue, acid fuchsin, and orange G.   It is a good stain to use for distinguishing cellular from extracellular components.   Collagen fibers stain an intense blue.  Mucus and ground substance  take on varying shades of blue.  Cytoplasm and neuroglia stain red.  Elastic fibrils, red blood cells and nucleoli stain pink or yellow.

Note:  In the case of slides 30 and 98, some are H&E stain and others are Mallory's Trichrome stain.

3b)  Masson stain (Slide 6) [Example]

4)  Osmic Acid or Osmium Tetroxide (Slide 19: [Example])

Used in light microscopy to show fat, myelin and the Golgi complex, all of which stain black.  It is also a fixative used routinely in electron microscopy.

5)  Trypan Blue Vital Stain (Slide 65) and Titanium Dioxide (Slide 111) [Example]

Trypan blue or titanium dioxide (TiO2) injected into an animal intraperitoneally are taken up by phagocytic cells.

6)  Mallory's Phosphotungstic Acid Hematoxylin (PTAH: Slide 46) [Example]

Stains muscle deep blue, showing cross striations of skeletal muscle especially well.  The ground substance of cartilage and bone stains yellowish to brownish-red; coarse elastic fibers stains purple.

7)  Periodic Acid-Schiff (PAS: Slides 109 and 110) [Example; Example; Example]

Structures rich in glycogen and glycoproteins stain red or purple with this technique.  The periodic acid attacks, 1,2 glycols and 1,2 amino alcohols, oxidizing the adjacent groups to aldehydes, which are detected by their reaction with Schiff's reagent to form a reddish color.  If the treatment with periodic acid is carried out for more than 10 minutes, other polysaccharide containing molecules will also be oxidized, e.g. proteoglycans and glycolipids. Many PAS stained tissues are also stained with hematoxylin, hence PAS-H.

8)  Elastic Tissue Stains (Slides 6, 25 and 32) [Example; Example; Example]

Orcein:  Stains elastic fibers brownish red.  Collagen fibers are pale brown or colorless.

Weigert's resorcin-fuchsin:  Stains elastic fibers dark blue-black.

Verhoff's:  Stains elastic tissue black. Neuroglia, myelin and fibrin are pink.

Note:  The stain is designated as "elastic tissue stain" when it is not known which of these methods was used.  Examples of this are slides 6 and 25.

9)  Silver Techniques

Cajal, Bielchowsky and pyridine silver techniques: (Slide 16 [Example]).    These methods involve soaking pieces of tissue in silver containing solutions for long periods of time before embedding, a method called silver impregnation.   In these methods the nerve cells and their processes appear yellow or brown.  The axons of myelinated fibers are light brown and the clear zones around the axon indicate the myelin sheath.   The latter is due to the fact that the myelin has been dissolved out, leaving little stainable material in these zones.  The unmyelinated axons are black or dark brown.  The neurofibrils are stained somewhat more intensely than the rest of the cytoplasm.

Romanes (Slide 17):  A method for staining nerve fibers in paraffin sections with silver.   It involves the use of silver chloride.   Nerve fibers stain purple to black, nuclei red, neurofibrils purple, keratin yellow and bone cells black.   The 'staining' is based on the reduction of silver salts by some substance(s) present in these elements.

Gomori (Slide 38 [Example]):   A silver impregnation of reticular fibers using silver nitrate that gives black reticular fibers.

Foot-Hortega (Slides 40,64, and 69 [Example]):  Another technique of silver impregnation, giving black reticular fibers.

Note:   The stain is designated as "silver technique" when it is not sure which of these methods was used. Example of this are slides 119 and 121.

10)  Nissl (Slide 15, Stained with Gallocyanin Dye [Example])

In this staining procedure a basic dye, such as thionin or gallocyanin, stains basophilic materials, such as RNA (e.g. Nissl bodies of nerve cells or other ribosomal aggregates), bright blue.  Cartilage ground substance and the granules of mast cells stain metachromatically with thionin.


11)  Romanovsky (Slides 11 and 100)

The Romanovsky-type stains are neutral dyes used primarily in the staining of blood smears.  In solution the dye contains ions (eosin-, methylene blue+, and the azures+ which are oxidation products of methylene blue) and neutral salts (eosinates of methylene and the azures).   These stains are not stable in water, so they have to be used immediately after mixing (Giemsa: Slide 100 [Example]).  Alternatively, an alcoholic solution of the dye may be applied, but diluted with water immediately afterwards (Wright: slide 11).   Romanovsky dyes stain the granules of acidophilic and neutrophilic leukocytes red, the granules of basophilic leukocytes blue, red blood cells red, plasma cells and lymphocytes blue.

12)  Toluidine-blue [Example; Example]

This is a stain that is often used by itself on thin (0.5-1μm) plastic-embedded tissues that were fixed and embedded for electron microscopy, but whose thick sections were cut and stained for LM.


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