School of Anatomy and Human Biology - The University of Western Australia


Blue Histology


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Histological Stains

by Alan Cockson

Alcian Blue (AB)

Designed to show Mucopolysaccharides or Glycosaminoglycans

Alcian blue is one of the most widely used cationic (it has many positive charges on the molecule) dyes for the demonstration of glycosaminoglycans. It is thought to work by forming reversible electrostatic bonds between the cationic dye and the negative (anionic) sites on the polysaccharide. These electrostatic bonds can be readily broken, revealing what appears to be a variation in bonding among different types of glycosominoglycans. When the concentration of electrolyte required to break the bond is increased progressively, then neutral, sulphated and phosphated mucopolysaccharides may be identified in tissue sections. It may be combined with the H&E and VG staining methods.

Structure Colour
nuclei   black or grey
polysaccharides blue to bluish green
other tissue elements as the counterstain

Elastic Stain (EL)

Designed to show elastic fibres in stark contrast to other tissue elements.

Why elastica stains with certain dyes is unclear. It seems that aldehyde residues are involved when the staining solution is pH 9-10, since blocking these groups impairs the staining. When orcein is used as the stain then dye-amine reactions may be involved. More recently van der Wall's forces have been suggested. Whatever the reaction involved, it's known that certain dyes used under very specific conditions colour elastica.
Can be applied to H&E and VG stained sections.

Structure Colour
elastin very deep purple (depending on stain used)

Haematoxylin and Eosin (H&E)

Designed to show basophilic structures blue, black, purple or grey and acidophilic structures in shades of pink and red.

The stain haematoxylin is not a dye, but develops colouring properties on oxidation to haematin. This substance haematin has little or no affinity for tissue elements and requires an inorganic ion to act as a "go between" (called a mordant) between the dye and the tissue. The mordant may be incorporated into the haematoxylin dye bath, the most common method. Or one may pre-treat the tissue with the metal salt and then stain by the haematoxylin.
Eosin is an acid dye, which requires an acidic environment to work. In solution the dye molecule is negatively charged and thus attaches to positive site in the tissue by salt bridges.

Structure Colour
nuclei black
cytoplasm pink or red shades

The most widely used staining technique is that of Haematoxylin and Eosin, commonly called H&E. In this method the nuclei of cells is stained by the haematoxylin whilst the cytoplasm is coloured by the eosin. This technique in various forms has been around for over one hundred years. Waldeyer was the first person to try staining histological section with an extract of Logwood in 1863 ( Henle Pfeifer. Z. Rat. Med. (3 Reihe) 20 (1863) 193-256). Logwood (Haematoxylon campechianum) is a small leguminous tree found in Central America. This contains a non-quinonoid substance, haematoxylin, which is not a dye and is indeed colourless. This chemical is readily oxidised to haematein and it is this oxidised form of haematoxylin, which is the colouring agent in the above technique.
Two years after Waldeyer’s publication Bohmer showed that the inclusion of alum (aluminium potassium sulphate) to the dye bath greatly enhanced the staining of nuclei. He made further experiments, which were published in 1868 ( Aerztl. Intelligenzbl. (Munich) 12 (1868) 539-550). In the years following these discoveries many improvements were made to the staining method, notably by Ehrlich who devised a stable solution of haematoxylin with a long shelf life (Z. wiss. Mikr. 3 (1886) 150). In 1886 Benda used a ferric salt instead of alum and thus opened the way for the Iron-Haematoxylin techniques (Arch. Anat. Physiol. - Anat. Abt. [Physiol. Abt.]: 562-564).
In the 1970’s there was a shortage of logwood and hence haematoxylin. This was due to the clear felling of forests in Central America and Brazil. The cost of the dye rose to an unprecedented hight, which increased the cost of diagnostic histopathology. Much research has been done to develop alternative nuclear stains, but to this day, none have replaced the traditional H&E sequence.

Leishman / Giemsa Stain

Designed to differentiate blood cells and to stain intracellular parasites in red blood cells and plasma, e.g. Plasmodium falciparum (malaria parasite).

When solutions of the dyes methylene blue and eosin are mixed a precipitate is formed. This precipitate is soluble in ethanol and methanol. The dried precipitate known as "methylene azure" is sold as Leishman's Stain, variations are known as Wrights Stain (America) and Giemsa and May-Grünwald stains in Germany and Europe. When the stain is applied to a blood smear and diluted with a buffer of correct pH (usually about pH 6.8) it is possible to type the different kinds of lymphocytes and leucocytes.

Structure Colour
red blood cells red to yellowish red
neutrophils dark purple multi-lobed nucleus, pale pink cytoplasm with reddish-lilac small granules
eosinophils blue nuclei, pale pink cytoplasm, red to orange-red large granules
basophils Purple to dark blue nucleus, dark purple, almost black large granules
lymphocytes dark purple to deep bluish purple nuclei, sky blue cytoplasm
platelets violet to purple granules

Luxol Fast Blue and Cresyl Violet

Designed to show myelin sheath and Nissl substance

The Luxol dyes are insoluble in water and are used as solutions in alcohol or other moderately polar liquids. These dyes stain phospholipids, they probably also enter hydrophobic domains of protein molecules. If the staining solution is applied for long enough almost everything in the section will be coloured. Most of this "background" colouring can be removed by washing the section in 70% ethanol to which a little lithium carbonate has been added. Thus we may "differentiate" the section to leave the stain only in the myelin sheath. When a suitable counter stain, e.g. Cresyl Violet or other cationic dye is applied it will bind not only to the nuclei and Nissl substance but will combine with the luxol fast blue anions present in the myelin.

Structure Colour
Nissl substance red
nuclei red
myelin blue to bluish purple

Osmium Tetroxide

Designed to show fats.

Osmium Tetroxide has a great affinity for lipids. The osmium is chemically bound to the fat and as such acts as a fixative. Thus in electron microscopy it is usual to fix firstly in glutaraldehyde then wash the tissue and post-fix in osmium. When using osmium as a 'stain' we make use of the fact that osmium oxidises tissue fats and forms a black substance which is easily seen in the light microscope.

Phosphotungstic Acid Haematoxylin (PTAH)

Designed to show muscle striations and certain pathological processes in the CNS.

When phosphotungstic acid is added to a solution of haematoxylin the resulting mixture stains certain tissues in contrasting colours. The staining is similar to H&E in that basophilic elements stain dark blue or brownish blue whilst other elements stain brownish red. In diagnostic work it is useful in demonstrating gliosis in the CNS, tumours in skeletal muscle and fibrin deposits in a wide variety of lesions.

Structure Colour
muscle blue-black or dark brown with striations
connective tissue pale orange-pink or brownish red
fibrin, neuroglia deep blue
coarse elastic fibres purple
cartilage and bone yellowish to brownish red

Schmorl's Stain

Designed to show canaliculi and lamellae in bone sections.

This is not a staining reaction in the classical sense of the word. The stain is made up of two colouring agents, ammoniacal thionin and aqueous saturated picric acid. The thionin precipitates within the lacunae and canaliculi whilst the picric acid forms picrates in the bone matrix. The result being that lacunae and canaliculi are dark brown to black whilst bone matrix is yellow or brownish-yellow.

Silver Stain for Reticulin (RE)

Designed to show reticulin fibres in connective tissue and various organs.

The use of silver for the demonstration of neuronal processes in the nervous system and reticulin in the connective tissues is not a staining procedure like the ones described above. For reasons not yet fully understood certain tissue elements become preferentially impregnated with silver salts. These silver salts are made visible by converting them into metallic silver by the same process as used in black and white photography.

Structure Colour
reticulin fibres black
other tissue elements colour of counterstain (green, pink, blue etc.)

Trichrome Stain

Designed for the selective demonstration of muscle, collagen fibres, fibrin and red blood cells.

As the name implies the tissue is coloured in three colours, each colour being selective for the tissue element demonstrated. Basically the nucleus is coloured by a nuclear stain, often by an iron haematoxylin. Connective tissue and muscle are coloured differently because the staining affinity of the tissue may be affected by the dye molecular size, density of the tissue, pH of the dye bath and/ or the use of colourless "dyes". These colourless "dyes" are either phosphomolybdic acid (PMA) or phosphotungstic acid (PTA). These two compound play a rather complex role in the dying of connective tissues, but basically can be thought of as blocking the reaction of one of the other dye molecules from certain tissue elements.

To give you some idea of the number of techniques involved in "trichrome" staining I have made a Table of several of the more common methods, see below.

Method Nucleus Collagen Muscle RBC's
Masson blue-black blue or green red red
MSB blue blue red yellow
Gomori Rapid grey-blue green red red
Lissamine red blue-black yellow or red red
Azan red blue orange-red red

van Gieson (VG)

Designed to differentiate between muscle and connective tissue.

This is an example of the Big Dye/Little Dye staining model. The little dye is picric acid with a molecular weight of 229 whilst the big dye is acid fuchsin, molecular weight 585. Both dyes are used in the same dye bath and sufficient time given for all tissue elements to be coloured to saturation. However the large molecular weight dye has difficulty penetrating some structures and sits more or less on the surface, and hence washes off easily. Whilst the picric acid penetrates into the tissue and is held by hydrogen bonds. The staining mechanism is a combination of physical properties of the tissue and chemical bonding. Any large molecular weight acid dye will work with picric acid.
The nuclear counter-stain is usually Weigert Haematoxylin.

Structure Colour
nuclei black or grey
muscle yellow
connective tissue red

page content: Alan Cockson
page construction: Lutz Slomianka
last updated: 10/04/00