With the use of stains and dyes, histology allows researchers to visualize particular tissue structures, chemical elements within cells, tissues, and even microorganisms. The advent and evolution of histology follows that of microscopy as outlined in ‘A (very) Short History of Histology’. Histology, which means ‘tissue science’ became an academic discipline in its own right in the 19th century, after the French anatomist, Bichat, introduced the concept of tissue in 1801. Karl Meyer, a German anatomist, however, was the first to coin the term “histology” in 1819.
Historically, histologists relied on readily available chemicals. Although some older staining methods have since been abandoned because the chemicals proved to be toxic (YIKES!), many, which are still in use today, have stood the test of time. They have proven to be efficient, accurate, and less complex.
BitesizeBio has already covered some of these staining procedures in depth. The aim of this article is to provide you with a brief overview of many of the available histological stains. Although by no means exhaustive, the table below gives a rundown of the top dyes and stains dominating today’s world of medical/diagnostic histology.
Used to stain
|1||4′, 6-diamidino-2-phenylindole (DAPI)||Nuclei: blue||First synthesizsed in 1971 by Otto Dann’s lab as a drug to treat trypanosomiasis1.
Selectively binds to double-stranded DNA, with no (or very little) cytoplasmic staining.
Can be easily used as a counterstain for green or red fluorescent labels.
Can be used to stain DNA in mammalian, bacterial, and metazoan cells.
|2||Acid Fast||Rod-shaped bacteria: red-pink||The Ziehl-Neelsen approach for Acid Fast Red staining, which was first described in 1800s, is the most widely used.
A differential stain used to identify acid-fast bacterial organisms, such as the members of the generaus Mycobacterium and Nocardia.
Particularly important for in the diagnosis of tuberculosis.
|3||Alkaline phosphatase||Pluripotent cells: red/purple||Universal stem cell membrane marker.
Commonly used to screen colonies during early stages of the reprogramming workflow (the stain maintains stem cell viability).
Can also be used as a negative selection tool at later stages to identify undifferentiated cells.
|4||Bielschowsky Stain||Axons, plaque neurites, and tangles: black|
Plaque and vascular amyloid: generally brown to dark brown
Background: yellow to brown
|A silver staining method introduced by Max Bielschowsky2, who improved the approach developed by Ramon y Cajal.
It can be used to visualize nerve fibers.
Routinely used to study Alzheimer’s disease.
|5||Congo Red||Amyloid fibrils: pale orange-red (apple green birefringence under polarized light)||Although it had been around for a while, German physician, Hermann Bennhold, was the first to discover that Congo red binds to amyloid , in 19233.
Remains the ‘gold standard’ test by amongst diagnosticians to identify amyloid in in the tissues (most often in patients with Alzheimer’s disease).
|6||Gram Stain||Gram-positive bacteria: blue-black|
Gram-negative bacteria: red-pink
|Developed by Hans Christian Gram in 18844.
This stain classifies bacteria as either, gram-positive cells (e.g. Staphylococcus spp.), which usually has have a thicker peptidoglycan mesh, or gram-negative cells (e.g. Escherichia coli, Salmonella spp.), which usually haves a lipid-polysaccharide layer external to the peptidoglycans.
|7||Grocott-Gomori's (or Gömöri) Methenamine Silver||Fungal elements: black (with sharp margins and cleared center)|
Background: light green
|A ‘broad spectrum’ fungal stain, which is better than the PAS stain (see below) at detecting even degenerated and dead fungi.
Particularly useful for staining carbohydrates.
Used for general screening for fungal infections.
|8, 9||Hematoxylin and Eosin||Nuclei: blue (hematoxylin)|
Endoplasmic reticulum: blue (hematoxylin)
Elastic fibers: pink (eosin)
Collagen fibers: pink (eosin)
Reticular fibers: pink (eosin)
Red blood cells: orange/red
|Most popular ‘general purpose stain’ used for routine tissue preparation.
Hematoxylin is extracted from the Haematoxylum campechianum tree; it was first used by CG Reichel in 1758, but only produced good results as a histological stain in 1865, when it was used in combination with alum by Böhmer. It binds and stains acidic structures5.
Eosin, a yellow-red dye, was synthesizsed by a Polish chemist, Heinrich Caro (who named it after the nickname of a girl he liked!). Emil Fischer, a German chemist, then worked on the novel compound first publishing a paper on Eosin Y (for ‘yellowish’) in 18756. Eosin binds and stains basic structures.
Hematoxylin and eosin were used in combination by a chemist, Wissowzky, in 18767.
|10||Hoechst stain||Nuclei: blue||Part of a family of blue fluorescent dyes developed by a German life sciences company, Hoechst AG.
Hoechst dyes specificallySpecifically binds to A/T-rich regions of double stranded DNA, with no (or very little) cytoplasmic staining.
It isThese dyes are less toxic and more cell-permeable than DAPI (see above).
|11||Luxol Fast Blue||Myelin fibres: blue to blue/green|
Neurons: violet (when counterstained)
Red blood cells: blue
|Created by Heinrich Klüver and Elizabeth Barrera in 19538.
AIt is a copper phthalocyanine dye that is soluble in alcohol and attracted to the bases found in the lipoproteins of the myelin sheath.
Commonly used to detect (de)myelination in the central nervous system.
Often accompanied by a counterstain (e.g. hematoxylin and eosin).
|12||Methylene Blue||Nucleus and cytoplasm: blue||Cationic dye that binds to anions in the tissue, such as carboxylic acid, sulfuric acid, and phosphoric acid groups.|
|13||Oil Red O||Neutral triglycerides and lipids (frozen sections), lipoproteins (paraffin sections), and lipofuscin: Bright red||Introduced in 1926 by RW French9
Closely related to the Sudan dyes
Used to demonstrate the presence of fats or lipids in fresh, frozen tissue sections.
Also used in forensic pathology to enhance latent prints produced by oily fingers.
|14||Periodic-acid Schiff||Glycogen and other carbohydrates: magenta|
Collagen fibers: pink
|Useful for researching/diagnossing glycogen storage diseases or diseases of the basement membrane.
Hematoxylin is typically used as a counter stain to visualizse other tissue elements.
A light green counter stain is preferred when PAS is used to demonstrate fungal organisms.
|15||Perl’s Prussian Blue||Iron deposits: Blue or purple|
Other tissue components: red (when counterstained with neutral red)
|Prussian blue was accidently developed in 1704 by a chemist, but it was first introduced as a histological stain by the German pathologist, Max Perls, in 186710.
Important stain to identify patients with hemosiderin (type of iron-storage complex found inside cells) deposits, for instance in some liver diseases or hemolytic anemia.
|16||Sudan Black B||Neutral triglycerides and lipids (frozen sections); some lipoproteins (paraffin-embedded sections): blue-black|
|Similar to other synthetic Sudan stains (including Oil Red O).
Usually tissue sections are counterstained (with hematoxylin/nuclear fast red).
Can also be used to stain myeloblasts, but not lymphoblasts.
|17||Toluidine Blue||Mast cell granules and polysaccharides: violet|
Red blood cells: blue
Collagen fibres: blue
|Developed by William Henry Perkin in 185611.
Basic dye that selectively stains acidic tissue components.
It is also useful for staining thin sections of resin-embedded tissues for electron microscopy, in order to help with the orientation and visualizsation of samples.
|18||Trichrome||Collagen, bone: green-blue|
Muscle, fibrin, cytoplasm: red
Red blood cells: yellow or red
Nuclei: dark red-black
|Original trichrome recipe was formulated by pathologist, Claude L. Pierre Masson, in the early 1900s12.
Technique using three (acidic) dyes to produce different colouration of (basic) tissue elements.
This stain is routinely used in diagnostic labs to evaluate liver diseases, such as cirrhosis.
Various staining approaches exist, of which Masson’s Trichrome and Gömöri’s Trichrome are the most commonly used today.
|19||Verhoeff-van Gieson Stain||Elastic fibers and cell nuclei: black (Verhoeff component)|
Collagen and muscle: Red (van Gieson component)
Cell cytoplasm and other components: yellow
|Ira van Gieson first described this staining procedure in 188913, which was modified by Frederick Herman Verhoeff in 190814.
The Verhoeff component is an iron-hematoxylin stain, while the van Gieson component is a collagen-specific counterstain, which is comprised of picric acid and acid fuchsin.
Used to validate the presence or absence of elastic fibers in tissues.
|20||Warthin-Starry||Microorganism: dark brown to black|
Background: golden brown (due to lower concentrations of silver deposits)
|First developed by American pathologists, Aldred Scott Warthin and Allen Chronister Starry, in 192015.
Silver nitrate-based staining method
Considered the best approach to detect Gram-negative organisms, such as small bacilli and spirochaetes.
Did we miss any histological stains that you commonly use in your lab? Comment below.
Want to know more about histology? Visit the Bitesize Bio Histology Hub for tips and trick for all your histology experiments.
- Dann, O. et al. (1971). Trypanocide Diamidine des 2?Phenyl?benzofurans, 2?Phenyl?indens und 2?Phenyl?indols. Justus Liebigs Ann. Chem., 749: 68–89.
- Bielschowsky, M. (1908) Eine Modifikation meines Silverimprägnationsverfahrenszur Darstellung der Neurofibrillen. J für Psychologie Neurologie., 12:135–137
- Bennhold, H. (1923) Über die Ausscheidung intravenös einverleibten Kongorotes bei den verschiedensten Erkrankungen insbesondere bei Amyloidosis. Arch Klin Med., 142: 32–46
- Gram, H. C. J., and Friedlaender, C. (1884) Ueber die isolirte Färbung der Schizomyceten: in Schnitt-und Trockenpräparaten. Theodor Fischer’s medicinischer Buchhandlung.
- Titford, M. (2005) The long history of hematoxylin. Biotechnic & histochemistry 80.2 (2005): 73-78.
- Fischer, E. (1876). Eosin als Tinctionsmittel für mikroskopische Präparate. Archiv für mikroskopische Anatomie, 12(1), 349-352.
- Wissowsky, A. (1877) Ueber das Eosin als Reagens auf Hämoglobin und die Bildung von Blutgefässen und Blutkörperchen bei Säugethier- und Hühnerembryonen. Archiv für mikroskopische Anatomie., 13: 479–496.
- Klüver, H., and Barrera, E. (1953) A Method for the Combined Staining of Cells and Fibers in the Nervous System. J Neuropath Exp Neurol., 12: 400–403.
- French, R. W. (1926). Fat stains. Stain Tech., 1: 79.
- Perls, M. (1867) Nachweis von Eisenoxyd in gewissen Pigmenten. Virchows Arch., 39: 42–48.
- Perkin, W. H., and Church, A. H. (1856) Jahresbericht ueber die Fortschritte der Chemie und Verwandter Theile Anderer Wissenschaften. J Chem Soc., 8: 48.
- Masson, P. (1929) Some histological methods: trichrome staining and their preliminary technique. J Tech Methods., 12:75–90.
- Van Gieson, I. (1889) Laboratory notes of technical methods for the nervous system. (Haematoxylin, acid fuchsin and picric acid as nervous tissue stain). N Y Med J., 50:57–60.
- Verhoeff F.H. (1908) Some New Staining Methods of Wide Applicability. JAMA., 50:876–877.
- Warthin, A.S., and Chronister, A.C. (1920). A more rapid and improved method of demonstrating spirochetes in tissues (Warthin and Starry’s cover-glass method). Am J Syphilis., 4: 97–103.
For Additional Reading:
Suvarna SK, Layton C, Bancroft JD (2013) Bancrofts’s theory and practice of histological techniques. London: Churchill Livingstone Elsevier.