BIOLOGICAL  STAIN COMMISSION

Glossary of Staining Methods, Reagents, Immunostaining, Terminology and Eponyms

Version 1.0.  Compiled for the Biological Stain Commission by Richard W. Dapson, Richard W. Horobin and John A. Kiernan.

Copyright © December 2018  Biological  Stain Commission, Inc.  

The headwords are in black bold, with cross references to other items in the glossary in underlined bold italic.  Click on an underlined term
to move to its place; it will appear at the top of your window or screen.
Any whole word can be located by using the Find in This Page function (Ctrl+F) of your web browsing program.
 
Microtechnique now includes sub-disciplines whose practitioners may not be familiar with related fields. For example, a researcher or technologist familiar with immunohistochemistry may not understand the rationale and terminology of staining with dyes, or of enzyme activity histochemistry. Likewise, a user of fluorescent probes may have little knowledge of fluorescent labels, and vice versa. This glossary is for the curious, such as those who have used a procedure and wonder, for example, who Mallory or Papanicolaou was, or what an instruction to differentiate means. Many terms in the glossary provide insight into the mechanisms of techniques. Similar words with different meanings are explained (e.g., albumen/albumin, selectivity/sensitivity). The glossary summarizes the properties of many dyes and other reagents used to color tissue components, and the actions of fixatives and other specimen preparative techniques. Entries are cross-referenced through numerous links.
The authors recognize that many relevant terms are not yet included in the glossary, and they intend to expand and update it from time to time.

Absorption. (1) Neutralization of specific antibodies in an antiserum, by adding an excess of the appropriate antigen; a negative control for immunostaining. (2) Removal of unreacted fluorochrome from a solution containing fluorescently labeled  proteins, by treatment with activated charcoal. (3) Treatment of a labeled antiserum with powdered acetone-fixed tissue, such as liver or kidney, to remove unwanted labeled proteins that can bind to tissues by non-immune mechanisms. (The tissue powder must not contain the antigen to which the antiserum was raised.)  See also Affinity-purified. (4) The wavelength of light from a microscope’s lamp that is absorbed by a dye or pigment, or that stimulates a fluorochrome to emit light of a different color.

Acetal. A compound formed by condensation of one molecule of an aldehyde with two molecules of an alcohol to give the structure Rʹ–O–CH(R)–O–Rʹ.  (R and Rʹ are alkyl or aryl groups).

Acid dye. A dye in which the colored component is an anion, e.g. Congo red and eosin Y. Such dyes are not acids, the term derives from 19th century textile dyeing, when they are used to color silk and wool from acid dyebaths.

Acid dyeing. The uptake of the anion of an acid dye into those cell and tissue structures where biopolymers that are cationic predominate, typically proteins. These are cationic if acid dyeing is carried out at low pH, when amines are present as –NH3+ and carboxylic acids as non-ionized free acids (i.e. –COOH). The affinity of an acid dye for such proteins is only partly due to coulombic forces between dye anions and tissue cations, and substantially due to the various short range dye–biopolymer van der Waals forces. However the coulombic forces do control the selectivity of the staining.

Acid-fast stain. A stain for bacteria possessing a waxy cell wall (acid-fast bacteria, Mycobacteria). Basic fuchsine dissolved in an alcoholic phenol solution (carbol fuchsine) penetrates all bacterial cell walls but is retained during an acidic differentiation step only in organisms with waxy cell walls. Several variants are popular: Fite's, Kinyoun's and Ziehl-Neelsen's methods, differing in reagent concentration, times, temperature and type of acid used in differentiatio

Acid fuchsin. See Acid fuchsine.

Acid fuchsine. An acid dye of moderate size, of the aminotriarylmethane class, made by sulfonation of basic fuchsine. Acid fuchsine is used in mixtures with other acid dyes that have bigger or smaller colored anions. A solution in saturated aqueous picric acid (smaller) is Van Gieson’s stain, which colors collagen fibers red and cytoplasm yellow. Acid fuchsine is also used in some trichrome methods, including Mallory’s stain and some variants of Masson’s trichrome. The dye is very soluble in water and slightly so in ethanol. Synonyms: CI 42685, Acid violet 19, acid fuchsin, acid magenta.  Commercial lots are available certified by the Biological Stain Commission.


Acidophilic. Literally, acid [dye] loving. Usually proteins, which take up acid dyes. Examples of acidophilic tissue components are collagen fibres, nuclear histones and erythrocytes.

Acridine orange. A basic dye in the acridine family, with small cations, used as a fluorochrome. It can bind to nucleic acids, with different fluorescent emission colors for DNA and RNA. Soluble in water; less soluble in ethanol. Synonyms: CI 46005, Basic orange 14.

Addition reaction
. See adduct, contrast with condensation reaction.

Adduct
. (1) Compound formed by combination of two molecules without the loss of any atoms. Often used when the exact structure of the addition compound is uncertain.  (2) In fixation, the addition of a fixative molecule to its substrate. The adduct retains chemical reactivity that could lead to crosslinking. Contrast with condensation reaction.

Affinity. The degree to which a staining reagent (e.g. dye or enzyme substrate or labeled antibody or silver ion) transfers from the staining solution into the tissue. The greater the transfer, the higher the affinity. Contribu­tions to affinity are factors which favour this. For instance coulombic forces and other reagent-tissue attractions, and positive entropy changes such as occur during hydropho­bic bonding. An alternative histochemical usage regards affinity as the attractive forces binding stains to tissue – however this latter is better seen as just one contribution to affinity. Synonym: substantivity.

Affinity-purified. This phrase describes a reagent made from an antiserum by capturing antibodies to a specific antigen, using beads of an insoluble polymer with the antigen covalently bound to their surfaces. After washing away unwanted components of the serum, the antibodies are released from the beads by acidification, which weakens antigen-antibody bonds.  Suitably neutralized solutions of affinity-purified antibodies are polyclonal antibodies; they have many uses as primary and secondary reagents in immunostaining.

Aggregation of dyes. Dye molecules bind to many things, including other dye molecules. Resulting dye aggregates can contain two molecules (e.g. methylene blue) or a hundred (e.g. Congo red). Aggregation may occur either in solution or in stained tissues, see metachromasia. Aggregation is favored in dyes with large planar conju­gated systems, facilitating dye–dye van der Waals forces. When aqueous staining solutions are used, a hydrophobic character of the dye also favors aggregation, facilitating dye-dye hydrophobic bonding.

Albumen. The principal protein of egg white. The letter e distinguishes it from the term albumin.

Albumin. A protein that is soluble in water and precipitated by high concentrations of salts (e.g. saturation with ammonium sulfate). Examples are serum albumin and egg albumen. Compare with globulin.

Alcian blue. Any of several basic dyes with a large phthalocyanine chromophore bearing pendent solubilizing cationic side-chains. Acidified aqueous solutions of alcian blue stain anionic carbohydrate macromolecules but not nucleic acids (DNA and RNA). Solubility in water or ethanol varies from batch to batch on account of additives, notably boric acid, included to increase stability of the dye powder. Synonyms: CI 74240, Ingrain blue 1, alcec blue, alcian blue, alcian blue pyridine variant. The Biological Stain Commission recognizes and certifies two categories of alcian blue. Alcian blue 8G is converted to a permanently insoluble pigment by a mildly alkaline rinse after staining; the side-chains probably are not the same as in the original dye with this name. Alcian blue variant dye lots have similar staining specificity but can be extracted from sections by acids.

Alcian blue for acid mucopolysaccharides. Due to basic dyeing, this dye stains sulfate and carboxyl groups at pH 2.5, but only sulfate groups at pH 1.0. The large size of alcian blue results in failure to stain DNA or RNA because of steric hindrance. The large conjugated system and very hydrophilic characterof alcian blue prevent extraction by dehydration alcohols. Pretreating sections with hyaluronidase removes connective tissue mucins containing hyaluronic acid, chondroitin-4- and 6-sulfate, leaving epithelial mucins available for staining with alcian blue.

Alcian blue plus PAS. Detects both acidic and neutral mucopolysaccharides. Neutral mucins stain magenta, sulfated mucins stain blue, and those containing carboxyl groups only may stain purple. See alcian blue for acid mucopolysaccharides and periodic acid Schiff for mechanisms.

Alcoholic formalin. Usually 10% unbuffered aqueous formalin in 70% ethanol (i.e., 1 volume of 40% w/v aqueous formaldehyde diluted with 9 volumes of 70% v/v aqueous ethanol), although it can be buffered to neutrality. This fixative penetrates more quickly than aqueous formalin, especially in fatty tissues.

Aldehyde fuchsine. See Gomori's aldehyde fuchsine.

Aliphatic hydrocarbon. Any of several organic molecules of low toxicity consisting only of carbon and hydrogen atoms. Low molecular weight hydrocarbons, which are used as solvents and clearing agents, may be unbranched (e.g. hexane, octane, decane) or branched (e.g. iso-decane and other isoparaffinic compounds). Mixtures of higher molecular weight hydrocarbons are present in paraffin wax which is used as an embedding medium. Synonym: alkane. Contrast with aromatic hydrocarbon.

Alizarin red S. A small anthraquinone acid dye that can chelate metal ions. Its principal application is histochemical localization of calcified deposits (carbonate or phosphate) in tissues. It can also be used for vital staining of calcium, as in developing bones and teeth, and, often in conjunction with alcian blue, for staining bone and cartilage in cleared preparations of whole small animals. Alizarin red S is soluble in water; slightly soluble in ethanol. Synonyms: CI 58005; CI Mordant red 3. Commercial lots are available certified by the Biological Stain Commission.

Alizarin red S for calcium. This stain demonstrates calcium by chelation of the calcium ions in the mineral deposits with the alizarin red S.

Ammine
. A metal coordination complex with ammonia, such as the silver ammine ion Ag(NH3)2+, which is present in many solutions used in silver staining methods
.  Do not confuse ammines with amines, which are organic compounds in which one or more hydrocarbon radicals replace hydrogen atoms of ammonia.

Amplification
. The generation of several molecules of a visible substance at the site of detection of each single molecule of interest in a specimen. For example, one antibody molecule might carry several fluorescent labeling molecules. Enzymatic labels can use indefinite amounts of substrate to generate insoluble, colored products. A physical developer can deposit black metallic silver on submicroscopic colloidal gold particles, increasing their diameters hundreds of times.

Amplification of staining. Using a secondary staining step to increase the coloration intensity of an initial stain, without changing the initial pattern of selectivity. In this way, physical development and gold toning can amplify a silver stain; and a DAB stain derived from a peroxidase labeled antibody is amplified using a silver stain. Amplification occurs in all techniques of enzyme histochemistry.


Amyloid.
A group of unrelated peptides or proteins that share several characteristics. Each one is derived from a naturally occurring, soluble molecule of identical
primary structure that becomes misfolded and insoluble. Each then self-replicates and self-aggregates into larger (sometimes massive) molecules. Amyloids are usually pathological, arising either from unknown etiology (e.g., Alzheimer's disease, Parkinson's disease, Type II diabetes) or from genetic disorder, although a few are actually functional. Some pathologic variants are transmissible (prions). All amyloids are detectable with high pH Congo red solutions containing salt and alcohol, using a combination of light, polarizing and fluorescence microscopy. Thioflavine T is another dye used to detect amyloid, but its mode of action, selectivity  and sensitivity are unknown.

Aniline blue. A mixture of triphenylmethane acid dyes, two of which are methyl blue and water blue, with sulfophenylamino side-chains. Commercial samples of the dye contain sirofluor, a by-product of manufacture that is a useful fluorochrome. The blue components of aniline blue are not fluorescent. This large acid dye is used in mixtures with other acid dyes that have smaller colored anions. A solution in saturated aqueous picric acid is similar to Van Gieson’s stain. In the trichrome family of staining methods, aniline blue is often the component that colors collagen fibers. It is soluble in water and much less soluble in ethanol.  Synonyms: CI 42780, Acid blue 93 (methyl blue), CI 42755, Acid blue 22 (water blue), aniline blue WS, cotton blue, ink blue, soluble blue. Commercial lots are available certified by the Biological Stain Commission.

Aniline dye. Old fashioned, but still strangely popular, term for a synthetic dye. This derives from the mid-19th century when aniline was perhaps the most frequent precursor of synthetic dyes.

Anion
, Anionic. A negatively charged atom or molecule, for instance an ionic species such as an acid dye or a chloride ion. Anions are attracted to the positive electrode (anode) in electrophoresis.

Antibodies
(singular: antibody). Immunoglobulin proteins produced by an animal in response to administration of an antigen. Antibody molecules bind specifically to parts of antigen molecules known as epitopes. Cf. monoclonal antibody, polyclonal.

Antigen. A substance administered to an animal that evokes the production of antibodies. See also epitope, immunoglobulin and marker.

Antigen retrieval (AR). The restoration of the native conformation of epitopes that have been directly altered by a fixative, or that were masked (hidden) by crosslinks or hydrophobic inversions. Procedures to achieve AR include citraconic acid treatment, enzyme retrievalglyoxal-specific antigen retrieval, and heat-induced epitope retrieval (HIER).

Antiserum (plural: antisera). Serum containing antibodies to an antigen. Commonly only the globulin fraction is used. An antiserum is polyclonal and also contains antibodies to antigens other than the one with which the animal was immunized. Diluted solutions containing affinity-purified antibodies are nevertheless often called antisera.

Aprotic solvent
. A liquid polar organic compound that lacks a hydrogen atom that can be released as a proton. Aprotic solvent molecules cluster around (solvate) cations, but leave anions relatively unimpeded, so that the latter will be more reactive than when dissolved in an ordinary (protic) polar solvent. Examples of aprotic solvents are dimethylsulfoxide and N,N-dimethylformamide.

Argentaffin
. A substance (e.g. melanin) able to directly reduce Ag+ ions to produce a deposit of metallic silver (Ag0) without the need for an added reducing agent. Argentaffin also describes cells containing such substances, e.g. the enteroendocrine cells. The term argentaffin reaction thus describes a type of silver staining.

Argentaffin reaction
. A silver stain wherein the tissue component reduces ionic silver to elemental metallic silver without the need of an external reducing agent like formaldehyde or hydroquinone. See Fontana-Masson argentaffin reaction.

Argyrophil
. A tissue element giving rise to deposits of metallic silver (Ag0) following uptake of Ag+ into the specimen, with the process driven by an added reducing agent. The term argyrophil reaction thus describes a type of silver staining.

Argyrophil reaction
. A silver stain wherein silver ions are first adsorbed onto tissue elements and subsequently reduced by an external reagent like formaldehyde or hydroquinone. See Bielschowskys silver, Bodian protargol stain, Gomori's method for reticular fibers, Grimelius argyrophil stain.

Aromatic hydrocarbon
. In a histotechnical context: any of several organic compounds,  containing only carbon and hydrogen atoms, with ring structures composed of alternating single- and double-bonded carbon atoms, e.g. benzene, toluene, xylene. Aromatic hydrocarbons are associated with high levels of toxicity. Contrast with aliphatic hydrocarbons.

Artifact. (1) Entity generated by a human being, in which sense all stained specimens are artifacts. (2) In histotechnical usage, something not naturally present, resulting from the preparative procedure; e.g. technical errors or lack of understanding of specimen, specimen preparation, or the staining procedure. Synonym: artefact.

Auramine O.  A diphenylmethane basic dye with small cations, used chiefly as a fluorochrome in sensitive staining methods for detecting leprosy and tubercle bacilli and other microorganisms. A fluorescent Schiff-type reagent is formed by reaction with sulfur dioxide. Auramine O is moderately soluble in water, more soluble in ethanol, and slightly soluble in xylene. Synonyms: CI 41000, Basic yellow 2. Commercial lots are available certified by the Biological Stain Commission.  

Azo dye. Dye containing one (monoazo, e.g. orange G), two (bisazo or disazo, e.g. Congo red) or more (polyazo, e.g. sirius red F3B) azo group(s), namely −N=N−.

Azocarmine B.  An acid dye of moderate size in the aminoazine group, used chiefly in the AZAN staining procedure, a trichrome method. This dye differs from azocarmine G by having an additional sulfonate group, hence its greater water solubility. The dye is soluble in water, and slightly soluble in ethanol. Synonyms: CI 50090, Acid red 103. Commercial lots are available certified by the Biological Stain Commission.

Azocarmine G. An acid dye of moderate size in the aminoazine group, used chiefly in the AZAN staining procedure, a trichrome method. The dye is soluble in water (less so than azocarmine B), and slightly soluble in ethanol. Synonyms: CI 50085, Acid red 101. Commercial lots are available certified by the Biological Stain Commission.

Azure A. A small thiazine basic dye, one of the products of polychroming methylene blue. It is used in the compounding of some of the Romanowsky blood stains. Azure A is soluble in water and ethanol. Synonym: CI 52005. Commercial lots always contain other thiazine dyes derived from methylene blue. Lots suitable for inclusion in blood stains are available certified by the Biological Stain Commission.

Azure B. A small thiazine basic dye, one of the products of polychroming methylene blue. It is present in all the Romanowsky blood stains and is indeed necessary for their correct performance. Azure B chloride is soluble in water and ethanol. The perchlorate, tetrafluoroborate and thiocyanate salts, which have the highest purity, are less soluble and require addition of an aprotic solvent to achieve concentrations high enough for use in blood stains. Synonym: CI 52010. Commercial lots of azure B chloride usually also contain other thiazine dyes derived from methylene blue. Lots suitable for inclusion in blood stains are available certified by the Biological Stain Commission.

Azure C. A small thiazine basic dye, one of the products of polychroming methylene blue. It is used in the compounding of some of the Romanowsky blood stains. Azure C is soluble in water and ethanol. Synonyms: CI 52002. Commercial lots always contain other thiazine dyes derived from methylene blue. Lots suitable for inclusion in blood stains are available certified by the Biological Stain Commission. 

Baker. Dr John Randal Baker (1900–1984), was an English zoologist and anthropologist at the University of Oxford His book Principles of Biological Microtechnique (1958) is a classic in the field of mechanisms of fixation and staining.

Basic dye. A dye with a cationic colored component, e.g. alcian blue, hemalum, or neutral red. Such basic dyes are not bases; the term derives from textile dyeing usage, in which they were originally used to color silk and wool fibres from alkaline (“basic”) dyebaths.

Basic dyeing. The uptake of the cation of a basic dye into those cell and tissue structures where biopolymers which are anionic predominate. These biopolymers are most commonly DNA and RNA (with phosphate anions), or glycosaminoglycans and polysaccharides (with sulfate anions and, at higher pH, carboxylate anions). The affinity of a basic dye for such biopolymers is only partly due to coulombic forces between dye cations and tissue anions, and is substantially due the various short range dye–biopolymer van der Waals forces. However, the coulombic forces do control the selectivity of the staining.

Basic fuchsin. See basic fuchsine.

Basic fuchsine. A rather small basic dye, in the aminotriarylmethane class, which stains basophilic structures. Major uses of basic fuchsine are for detection of acid-fast bacteria (with the Ziehl-Neelsen stain), and for preparation of Schiff’s reagent. Traditionally, basic fuchsine was a mixture containing four similar dyes: pararosaniline, rosaniline, magenta II and new fuchsine. Most of the basic fuchsine currently manufactured is the acetate or chloride salt of pararosaniline, but at least one major American vendor still supplies basic fuchsine composed of mixed dyes. The dyes comprising basic fuchsine are moderately soluble in water (heating needed) and ethanol. Synonyms: basic fuchsin, magenta, CI 42500, Basic red 9 (pararosaniline), CI 42510, Basic violet 14 (rosaniline), CI 42520, Basic violet 2 (new fuchsine).  Note: there disagreement between dictionaries, handbooks, vendors and end-users on the spelling of this dye name, with Google Ngram indicating that books currently favor fuchsine over fuchsin. Commercial lots are available certified by the Biological Stain Commission.

Basic fuchsine, special for flagella. A name applied by the Biological Stain Commission to batches of the dye that work well in staining bacterial flagella. It may be either a 3:1 mixture of the acetate and chloride salts of pararosaniline or another mixture of basic fuchsine components that works equally well in Leifson’s flagella stain. Soluble, albeit slowly, in water and ethanol.  Commercial lots are available certified by the Biological Stain Commission.

Basophilia, basophilic. Literally, basic dye lover. Basophilic tissue components, with affinity for basic dyes, include cartilage matrix, nuclear chromatin, and mast cell granules.

Best’s carmine. Traditional selective glycogen stain, comprising a natural dye, namely carminic acid, dissolved in alkaline, salty aqueous-methanol. Intriguingly, all these components of Dr Best's stain are required for effective staining; and the technique provides an example of dye-tissue hydrogen bonding.

Biebrich scarlet. An acid dye in the disazo class, with colored anions of moderate size, used in some trichrome staining methods. It is soluble in water and slightly soluble in ethanol. Useful in some experimental work because the color does not change over a wide pH range. Synonyms: CI 26905, acid red 66, ponceau B.

Bielschowsky. Max Bielschowsky (1869–1940) was a German neuropathologist. He learnt histological staining techniques from Weigert at the Senckenberg Pathology Institute, Frankfurt-am-Main; but was later purged from German academia in the anti-Semitic pogrom following 1933, eventually emigrating, via Spain, to the UK. The eponymous silver stain bearing his name was an improvement of a procedure developed by Ramon y Cajal.

Bielschowsky's silver. Perhaps the original argyrophil reaction to demonstrate axons, dendrites, neurofibrils; as well as the neurofibrillary tangles and senile plaques of Alzheimer's disease. Sections are treated with silver nitrate, after which bound silver Ag+ is reduced with formaldehyde to precipitate metallic silver Ag0, and finally gold toned to heighten contrast. This silver staining procedure was highly capricious and so has been modified numerous times by many authors.

Biological Stain Commission (BSC). A not-for-profit corporation that ensures the quality of dyes through independent assays and other tests, promotes cooperation and dialogue among manufacturers, vendors and users of dyes in all fields of biological and biomedical sciences, educates users of biological stains about sources of reliable dyes and how they might best be used, and publishes information about innovations and improvements in biological staining and histochemistry. Dye batches that pass the BSC’s tests are certified stains. The BSC also publishes a journal, Biotechnic & Histochemistry, a book, Conn’s Biological Stains, and conducts correspondence and annual meetings, maintaining dialogue among scientists, manufacturers and vendors concerned with biological stains.  For more information about the BSC, go to http://biostain.com.

Biopolymer. High molecular weight compound character­istic of living organisms: nucleic acids, glycosaminoglycans, polysaccharides, proteins etc.

Biotin. An easily attached organic label that can be detected by virtue of its specific affinity for avidin or streptavidin, large proteins that can themselves be labeled with either fluorochromes or enzymes.

Bismarck brown Y.  basic dye of the disazo class, with cations of moderate size that stains basophilic structures.  It has been used to stain mucus, amyloid, plant cell walls and bacteria, and it is a component of some Papanicolaou stain variants that are used for cancer diagnosis. It is soluble in water and ethanol.  Synonyms: CI 21000, Basic brown 1, vesuvine. Commercial lots are available certified by the Biological Stain Commission.

Blockade, blocking reaction. Conversion of tissue sites able to bind or generate stain into non-binding, non-generating forms. Examples include: attachment of unlabeled antibodies to antigenic tissue sites to block subsequent binding of labeled immunoglobulins; enzyme inhibition following exposure to glutaraldehyde; and esterification of tissue acids to reduce basophilia.

Bluing agent or reagent. A mildly alkaline solution following a hemalum stain, used to shift the reddish violet color to blue-violet or blue. Hard tap water, a dilute lithium carbonate solution or Scott's tap water substitute are the agents most commonly used.

Bodian. David Bodian (1910-1992) was an American medical scientist. In 1936, while a student of comparative neuroanatomy in Chicago, he published a silver staining method for axons and nerve endings using protargol, a technique with which his name is associated. Bodian later became professor of anatomy at Johns Hopkins University in Baltimore, where he conducted important research into the pathogenesis of poliomyelitis. See also: Bodian's protargol stain and protargol.

Bodian's protargol stain. Demonstrates nerve fibers in paraffin sections. Certain silver proteinate solutions, called protargol-S, and certified by the Biological Stain Commission, are used to impregnate axons. Neurofilaments within the axons bind the silver Ag+, which is then reduced with hydroquinone in an argyrophil reaction. The section is then gold toned to enhance contrast.

Bound water. Water molecules hydrogen-bonded to specimen molecules, effectively insulating them spatially and electronically from one another. Contrast with free water.

Brilliant cresyl blue. A basic dye in the oxazine class with small cations, used for vital staining. Two major applications are the detection of reticulocytes in blood, and the assessment of the suitability of oocytes of various species for in vitro fertilization purposes. The dye is soluble in water and ethanol. Synonyms: CI 51010, brilliant cresyl blue ALD. Commercial lots are available certified by the Biological Stain Commission. Note: one major vendor currently sells brilliant cresyl blue lots which are stated to be a “mixture of toluidine blue and water blue”.

Brilliant green. A basic dye in the triphenylmethane class with cations of moderate size that stains basophilic structures, and has been used for coloration of bacteria and of fungal hyphae. Its principal use, however, is to inhibit growth of coliform bacteria in cultures for detection of Salmonella infection. Brilliant green is soluble in water, and more soluble in ethanol. Synonyms: CI 42040, Basic green 1, aniline green, diamond green G, emerald green, fast green J, malachite green G, solid green JJO. Commercial lots are available certified by the Biological Stain Commission. 

Brown-Brenn Gram stain. A Gram stain variant for smears, that uses basic fuchsine as the stain for Gram negative bacteria and picric acid as a background stain.

Brown-Hopps Gram stain. A Gram stain variant similar to the Brown-Brenn procedure that was modified for tissue sections instead of smears.

Buffer. A solution that maintains some physicochemical property constant, typically pH, resisting the addition of small amounts of acid or base.

Cajal. Santiago Felipe Ramon y Cajal (1852–1934) was a Spanish physician and anatomist, best known for providing microscopic evidence in support of the neuron theory but also for investigation of the retina and central visual pathways, and the degenerative and regenerative changes that follow injury to the central and peripheral nervous systems. For this he received the Nobel Prize in Physiology or Medicine, together with Golgi, in 1906.  The name Cajal is associated with a variety of cell-types in the nervous and digestive systems; with several silver staining methods, a method for astrocytes; and with a combination of carminepicric acid and indigocarmine (Cajal's trichrome) that provides red nuclei, yellow erythrocytes, green muscle and blue collagen fibers.

Cajal's gold sublimate. This demonstrates neuroglia, particularly astrocytes. Tissues are fixed in a mixture of ammonium bromide and formaldehyde, with release of hydrogen ions to lower the pH to 1.5.  Sections are stained in an aqueous solution of chloroauric acid ("gold chloride") and mercuric chloride ("sublimate") to impregnate the cells. Sodium thiosulfate halts the reaction. Astrocytes are dark purple or black; other cells acquire lighter shades of purple. See also Cajal.

Callose. A plant cell-wall polysaccharide composed of β‑1,3‑glucosyl units. It occurs normally in the sieve plates that separate cells of the phloem  and is added to cell walls at sites of injury. Aniline blue and sirofluor are useful fluorescent stains for callose.

Canonical forms. Different structures of an organic compound in which resonance occurs. In drawing canonical structures, only bonds and sites of electric charge may be varied; the positions of the atoms may not be changed.

Carmine. An aluminum complex of carminic acid, of variable composition, manufactured from cochineal. This dye-metal coordination complex is used in staining solutions devised to provide red coloration of cell nuclei, chromosomes, glycosaminoglycans or glycogen. Solutions containing carmine or carminic acid plus aluminum salts are termed carmalum.  Synonyms: CI 75470, Natural red 4. Commercial lots are available certified by the Biological Stain Commission.

Carminic acid. A red anthraquinone natural dye derived from cochineal, which can form metal coordination complexes. Some of these, especially with aluminum or iron, are used as biological stains, in particular for chromosomes.  Synonyms: CI 75470, Natural red 4.

Cation, Cationic. A positively charged atom or molecule, an ionic species such as a basic dye, or a sodium ion. Such species are attracted to the negative electrode (cathode) in electrolysis or electrophoresis.

Celestine blue.  An oxazine basic dye with small colored cations that are also able to form larger cationic dye-metal complexes. The complex formed with ferric salts has staining properties similar to those of hemalum. Celestine blue is soluble in water and ethanol. Synonyms: CI 51050, Mordant blue 14, celestin blue, celestine blue B, coelestin blue, coreine 2R, gallo sky blue B.

Certified stain. Currently the Biological Stain Commission (BSC) offers testing and certification for over 60 stain powders. The tests and required standards are published, and are revised from time to time as quality improves and applications change. For a batch of dye that passes the tests, the BSC issues a certificate to the vendor. The certificate identifies the vendor’s batch number, the BSC’s identification code for the batch, and the uses for which the dye is certified. Small labels, which are difficult to fake, also carry the BSC’s identification code; they are made available for affixing to bottles of BSC-certified stains. The BSC laboratory keeps samples of all certified (and rejected) dye batches, and records of sales of certified dyes from submitting vendors to other vendors.  Parts of the same batch often have different vendors’ numbers, but the BSC’s identification code never changes.  BSC certificates are valid for 10 years for all stains except alcian blue, for which the time is 5 years. There are vendors claiming to sell “certified stains” who have not had their products independently tested by the BSC.  The web page  http://biologicalstaincommission.org/vendors-list/  shows manufacturers and vendors who have recently submitted samples that received BSC testing and certification.

Chelating agent. Some are mordant dyes which react with metal ions giving rise to stains. Examples include carminic acid, celestine blue, gallocyanine, hematein and nuclear fast red. Some are used to generate colored products from tissue constituents, e.g.: demonstration of calcium ions by chelation with alizarin red S; or of nickel ions by chelation with dimethylglyoxime. Another chelating agent, EDTA, is used for decalcification of tissues. 

Chelation. Formation of a metal coordination complex by the reaction of a metal ion with a chelating agent. This involves a compound carrying two or more metal binding groups (such as CO2-, OH or C=O) able to form a complete ring which includes the metal ion. Such chelate rings often are chemically stable structures.

Chlorazol black E. An acid dye of the polyazo class, with large anions, which can stain all components of tissues in black and shades of gray. Colored impurities impart other colors to some materials including chitin (green) and glycogen (pink). It is used with animal (especially insect) and plant tissues, and to detect parasitic protozoa in fecal smears. Synonyms: CI 30235, Direct black 38, Erie black GXOO, pontamine black E. Commercial lots are available certified by the Biological Stain Commission.  

Chromaffin reaction. A fixation method that also demonstrates adrenaline and noradrenaline in the adrenal medulla. Potassium dichromate in the fixative oxidizes these monoamines to brown quinones.

Chromatin. Material in the nucleus of a cell that is stained by cationic dyes and by some dye–metal complexes such as carmine and hemalum (aluminum–hematein). Chromatin comprises DNA and the nucleoprotein (histone) of the chromosomes.

Chromic acid. In a biological staining context, this term describes acidified dichromate (Cr2O72¯) solutions. Chromic acid been used inaccurately as a synonym for the anhydrous form (chromium trioxide, CrO3), which is used to make such solutions.

Chromogen. A substance that reacts to give a coloured product, such as DAB and other compounds used in histochemical methods for localization of peroxidase activity.

Chromotrope 2R.  A hydrophilic red acid dye of moderate size that can also form metal coordination complexes. Very soluble in water; slightly soluble in ethanol. Used as a cytoplasmic stain, especially in trichrome methods, including Gomori's one-step trichrome technique. Synonyms: CI 16570, Acid red 29, Mordant blue 80. Commercial batches have dye contents up to 75%.

Churukian. Charles J. Churukian (1929–2011) was a member of the Biological Stain Commission and served as a histological technician in the Pathology Laboratory of the University of Rochester Medical Center, where he wrote numerous articles and a manual on improved techniques for special stains.

Churukian-Schenk method for argyrophil granules. See Grimelius argyrophil stain.

Citraconic acid or citraconic anhydride. The acid HOOC–CH2–CH(CH3)–COOH predominates in dilute solutions. An antigen retrieval agent used at elevated temperature, that removes the formaldehyde adduct from crosslinks. Said by some to be a universal antigen retrieval method, but may simply be widely effective because of its temperature and mild alkalinity.

Clearing agent. A solvent used to remove the dehydrant and unbound lipids from a tissue specimen during tissue processing, and to prepare the specimen for immersion into paraffin wax; typically an aromatic hydrocarbon (xylene or toluene), an aliphatic hydrocarbon (various proprietary mixtures), or d-limonene.

Cochineal. The dried, sometimes also powdered, bodies of gravid female insects (Dactylopius coccus, also called Coccus cacti), which contain carminic acid. Synonyms: confusingly, carmine and carminic acid are used as synonyms, especially in food and cosmetics.

Colloid. A substance composed of either macromolecules or aggregates of smaller molecules, dispersed in a liquid medium. Sizes of colloidal particles range from 1 to 500 nm. Individual suspended particles as small as 1 nm can be detected with visible light, but the distinction between one or two particles (resolution) cannot be made with a conventional light microscope if the size and separation are less than 200 nm.

Colloidal gold. A dispersion of tiny gold particles stabilized by their affinity for macromolecules. Particle size is determined by conditions for chemical reduction of the AuCl4 ion. In aggregate, the smallest gold particles provide a blue label and the largest particles show as red. Individual particles are clearly shown by electron microscopy.  Antibodies are macromolecules and gold can serve as a label useful in immunostaining. Colloidal gold particles can also serve as catalytic sites for amplification by physical development.

Colloidal iron. A method for demonstrating acid mucopolysaccharides. Ferric chloride is converted to a colloidal suspension of ferric oxide, whose large cationic particles bind to carboxylate and sulfonate anions in a manner analogous to basic dyeing. Potassium ferrocyanide is then applied to form Prussian blue pigment via the ferric-ferrocyanide reaction.

Colloid stabilizer. (1) Synonym for the protective colloids found in physical developers. (2) Water soluble, high molecular weight compound (for exam­ple, agar or polyvinyl alcohol) added to incubation solutions in enzyme histo­chemistry to prevent losses of biopolymers from the cells and tissues.

Color (US), Colour (elsewhere).  A quality of  sensation of light induced in the eye by electromagnetic radiation with wavelength in the range 350-800 nm, the   color being determined by the wavelength. Objects or materials that absorb in that complete range are seen as black. Absorption only of light with lower wavelengths (blue-violet) shows reflected or transmitted light with longer wavelengths (yellow, orange or red) and vice versa. Green materials absorb light in the blue and red  parts of the spectrum. See also: dye and  fluorescence.

Colour Index (CI). A database jointly maintained by the Society of Dyers and Colourists (SDC, in the UK) and the American Society of Textile Chemists and Colorists (ASTCC, in the USA). Information about some 13,000 different dyes and pigments is available online as the Colour Index International. Each compound has its unique CI number, which places it in a chemical category, and a unique generic name that includes indications of the usual industrial method of application and the color. The mode of synthesis is also given. Common names and trade names are also listed. For example, CI 26125 or Solvent red 27 is the dye commonly known as oil red O. The current version of the CI is available only to subscribers who can pay more than $700 per year. The last printed edition (3rd, 1973, in several volumes) is in libraries. The Colour Index Heritage Edition is a DVD publication that brings together almost 17,500 pages of information published in the three editions of the Colour Index between 1924 and 1999 prior to the publication of the 4th Edition online.  Again, this is expensive ($800), but may be available via an academic library. The CI database does not contain much information about fluorescent compounds that are used only as biological stains.

Common ion effect. A salt becomes less soluble when the concentration of one of its ions in a solution greatly exceeds that of the other ion.

Condensation reaction. In organic chemistry, combination of two molecules with elimination of a small molecule such as water. Contrast with adduct.

Conformation. For a macromolecule, this is its overall shape. For a protein this stems from its primary structure (i.e., the amino acid sequence) and the various intra- and inter-molecular interactions that bend or fold the molecules into higher order 3-dimensional shapes. See primary, secondary, tertiary and quaternary structure.

Congo red
. An acid dye of the disazo class, with large anions. It has been used in many histological staining methods, as a counterstain for blue stained nuclei, and in solutions devised to impart more selective coloration to cellulose, collagen fibers, elastic fibers and neurosecretion products. In recent decades the principal application has been in specific staining methods for amyloid.  Congo red is soluble in water, less soluble in ethanol. Synonyms: CI 22120, Direct red 28, Congo, Cotton red B, Kongoröt. Commercial lots are available certified by the Biological Stain Commission.

Congo red for amyloid. Various formulations of the solution exist, but the most reliable and selective is Puchtler's alkaline Congo red.

Conjugated bond number. Numerical structure parameter describing the extent of conjugation within a stain or staining reagent by a count of the number of conjugated bonds in a molecule. Usual abbreviation: CBN. Dyes vary widely regarding CBN values. Small molecules such as methylene blue and picric acid have values of 18 and 16 respectively; whereas large molecules such as alcian blue and sirius red F3B have values of 48 and 64, respectively.

Conjugated bonds, conjugated system. A chain of atoms linked by alternating single and double covalent bonds in which spatial localization of all the bonding electrons is not possible. For instance, the benzene carbon skeleton is conventionally drawn as comprising three carbon–carbon single bonds plus three carbon–carbon double bonds. Because the double bonds are conju­gated (alternating), all six bonds are identical, due to the delocalized π‑electrons. Such delocalized electrons are mobile, and electrical influences are readily propa­gated from one part a conjugated molecule to another, enhancing dipoles and polarizability, and favoring van der Waals forces.

Conn. Harold J Conn (1886–1975). A senior figure in the Society of American Microbiologists, based at the New York State Agricultural Laboratory in Geneva NY. He was part of the group whose efforts, in the early 1920s, established the Biological Stain Commission as a focus of efforts to standardize dyes used as biological stains.

Coulombic forces. Electrical attractions and repulsions due to positively and negatively charged species in tissues and stain molecules, such as –NH3+ and –SO3¯. Named from the coulomb, the SI unit for a quantity of electricity: 1.036×10−5 moles of 7018624200000000000♠protons or 7018624200000000000♠6.242×1018   electrons. Also called electrostatic forces.

Counterstain. A staining step carried out to provide a contrasting background to the staining of some specific structure or component. Use of such a process is termed counterstaining.

Covalent bond. Link between two atoms resulting from the sharing of, usually, an electron pair as in a C–C σ bond. Double bonds involve sharing two pairs of electrons, for instance C=O and C=C. Although drawn as equal, one electron pair form a σ bond while the second pair constitute a π (pi) bond in which the electrons are more loosely held between the two atoms than in the first pair.

Coverslip
. A thin piece of glass (rarely plastic) that fits over a tissue section on a microscope slide to prevent damage to the specimen. A coverslip is glued down with mounting mediumCoverslipping describes the act of applying a coverslip to a slide, either by hand or through the use of a coverslipping machine.

Cresyl violet. This name has been used for at least three basic dyes of the oxazine series with the same ring structure but different attached amino and methyl groups. These dyes bind to basophilic materials; applied from suitably acidified solutions, they strongly stain cell nuclei and Nissl substance (rRNA in the cell bodies of neurons).  Most dyes sold as cresyl violet are soluble in water and in ethanol.  Dyes sold as cresyl violet perchlorate are not soluble enough to be used as biological stains. Synonyms: cresyl echt violet, cresyl fast violet, cresyl violet acetate. Names of commercial products generally do not correspond to chemical entities. These dyes do not have Colour Index numbers and names.  Commercial lots designated as cresyl violet acetate are available certified by the Biological Stain Commission.

Cresyl violet for Nissl substance. An acidified cresyl violet solution which demonstrates Nissl substance in neurons by basic dyeing. This stain may be combined with Luxol fast blue when the latter dye is used to stain myelin. Synonyms: Cresyl echt violet, cresyl violet acetate.

Crocein scarlet. An acid dye in the disazo class, with colored anions of moderate size, used in the Movat pentachrome stain. It is soluble in water and ethanol. Synonyms: CI 27290, Acid red 73, woodstain scarlet.

Crosslink. A connection or bridge, commonly involving covalent bonds, between polymeric chains or lower molecular weight molecules. (1) Generated by fixative agents, e.g. crosslinking of proteins by formaldehyde, or of unsaturated lipids by osmium tetroxide. (2) Present in native proteins, most usually as disulfide bridges within or between polypeptide chains. (3) Occurring in some plastic embedding media after polymerization of a monomer.

Cryogenic spray. A gas (usually freon or carbon dioxide) under pressure in a can, that, when sprayed onto a specimen, causes freezing due to evaporative cooling. Used in cryotomy.

Cryosection. A frozen section produced on a cryostat. See also tissue section.

Cryostat. A microtome mounted in a freezing cabinet, used to produce frozen sections (see cryotomy).

Cryotomy. The production of tissue sections from frozen specimens using a cryostat. The advantage of frozen sections is that slides can be produced in minutes rather than hours, often while the patient is still in the surgical suite. Typically used for biopsies. See also Mohs surgery.

Crystal violet.  A moderately large basic dye, of the aminotriarylmethane class, used as an antiseptic and in a variety of staining methods for animal and plant tissues, notably the Gram stain for bacteria. Crystal violet is soluble in water and much more soluble in ethanol. Synonyms: CI 42555, Basic violet 3, gentian violet (in USA only; elsewhere this name refers to methyl violet), hexamethyl pararosaniline, methyl violet 10B. Commercial lots are available certified by the Biological Stain Commission.

Cyanoacrylate. An ester of cyanoacrylic acid, such as ethyl-2-cyanoacrylate, CH2=C(CN)COOC2H5, the principal ingredient of adhesives with names like crazy-glue and super-glue. Cyanoacrylates polymerize rapidly when exposed to traces of moisture. The polymerized glue is insoluble in water but (fortunately) soluble in many organic solvents.

Cytocentrifuge. Mechanical device used for preparing uniform layers of peripheral blood cells and other cell suspensions. Synonym: spinner.

DAB.  See Diaminobenzidine.

Darrow red.  A small basic dye of the oxazine series that binds to basophilic materials. Applied from a suitably acidified solution, it stains both DNA and Nissl substance (rRNA in the cell bodies of neurons). It was introduced in 1960 and is named for Mary A. Darrow, the technologist in charge of the Biological Stain Commission’s laboratory from 1926 to 1959. The dye dissolves slowly in water (heating needed) and is poorly soluble in ethanol. Commercial lots are available certified by the Biological Stain Commission.

Debye forces (dipole-induced dipole forces). A type of van der Waals attraction in which a polar group or molecule induces a dipole moment in the conjugated system of an adjacent non-polar entity.

Decalcification. Removal of calcium salt deposits from tissue specimens prior to microtomy.  Reagents used include formic or hydrochloric acids, and the chelating agent EDTA (ethylenediaminetetraacetic acid).

Dehydrant, dehydrating agent. A solvent, such as methanol, ethanol, isopropanol or glycol ether, that replaces water in a specimen by diffusion; or a ketal, that chemically reacts with water.

Dehydration. The removal of free (unbound) water from a specimen prior to exposure to a clearing agent. See also over-dehydration.

Delafield's hematoxylin. A regressive version of hemalum containing hematoxylin and aluminium ammonium sulfate, as well as water and ethanol as solvents and glycerol as a stabilizer to prevent over-oxidation. The solution is oxidized slowly by air and sunlight over a period of months.

Delocalized π-electrons. Electrons that are shared by more than two atoms and therefore cannot be said to form part of any individual covalent bond. π-electrons are associated with double or triple bonds, and with resonant structures such as aromatic rings.

Denaturant. Something which can cause denaturation of proteins. For instance heat, organic compounds such as ethanol and urea, and chemically reactive compounds such as dichromates or formaldehyde. Fixatives are typically denaturants.

Denaturation. Destruction of secondary (or higher level) organization of biopolymers, typically proteins. In this latter case hydrophobic amino acid residues become exposed on the molecular surface, leading to insolubility and aggregation. Denaturation can be achieved by heating or by using a wide variety of chemical denaturants. Denaturation usually reduces enzymic activity of proteins and may diminish antigenicity, although some denaturant fixatives actually protect antigenicity. Curiously, heat (boiling buffered water) can renature macromolecules to the point where lost antigenicity is restored (see heat-induced epitope retrieval).

Dialysis. Passage of small, but not large, molecules through a membrane. Also technique for concentrating solutions of proteins (or other macromolecular substances) using tubing that is permeable only to small molecules.

Diaminobenzidine (DAB). A polyamino primary aromatic amine. The free base is only slightly soluble in water; the tetrachloride dissolves easily. Oxidation of DAB, which can be catalysed by the enzyme peroxidase, gives rise to an insoluble brown polymeric pigment.

Differentiation
. Histochemical usage: controlled de-staining of a stained section.

Dimer
. A compound formed by the union of two identical molecules.

Dimethylglyoxime
. A chelating agent giving rise to insoluble, brightly colored metal coordination complexes with several metal ions, including nickel.

Dipole. The occurrence of a partial negative charge on the most electronegative atom of a covalently linked pair, with the other atom carrying a partial positive charge. For instance, in a nitro group (–NO2) the oxygen atoms are more electronegative than the nitrogen atom, and thus the former carry partial negative charges.

Dipole-dipole interactions. See Keesom forces.

Dipole-induced dipole forces. See Debye forces.

Dipole moment. A dipole property: the product of the magnitude of the charge on the electronegative atom, and the distance between the electronegative and electropositive atoms; the unit of measure is the Debye.

Direct dyeing. A textile dyeing term, describing the coloration of cotton textiles using large, planar, hydrophilic acid dyes, which “directly” bind to the fibers. In the Colour Index these dyes fall into the CI Direct dye application class. Historically such “direct” dyes were so named to distinguish them from colorants which only colored cotton following a pre-treatment with a metal salt or tannin.

Disazo (also bisazo). A word indicating that an azo dye  has two azo groups (−N=N−) in its structural formula.

Dispersion forces. See London forces.

Dye. An organic ion or molecule that can absorb visible light (and so is seen as colored), that can attach to and impart color to other materials. Absorption of light is due to the chemical bonds forming an extended conjugated system.

Dyeing. See staining.

EDTA (ethylaminediamine tetraacetic acid). A chelating agent that forms soluble complexes with many different metal ions; the disodium salt, Na2EDTA, is the form usually used in histotechnology. Other names include edetic acid, (ethylenedinitrilo)tetraacetic acid, edathamil and versene.

Ehrlich. Paul Ehrlich (1854–1915) trained in four different medical schools, where he was considered a mediocre student. Ehrlich’s major achievements in stain technology were made in his early years, when he was the first investigator to appreciate the differences between acid dyes and basic dyes for staining biological material. He also prepared the first neutral dye, albeit it not a Romanowsky stain. 

Electronegative, electronegativity. The tendency of an atom to attract an electron. For instance, a chlorine atom is very electronegative, so Cl ions are stable. However, if the attractive tendency is very low the atom is termed electropositive, and such atoms can lose electrons to form stable cations, e.g. the Na+ ion.

Electropositive, electropositivity. See electronegative.

Electrostatic forces.  These attract electrons (negative) towards nuclei of atoms (positive). Thus, cations are attracted to anions. Also called coulombic forces.

Embedding. Cells and tissues are immersed in a liquid, such as molten paraffin wax or the monomer of a plastic embedding medium. After the liquid is solidified, by freezing or polymerization respectively, the embedded specimen is interpenetrated and supported by a solid matrix, the embedding medium.

Emission. The wavelength of light emitted by a fluorescent substance.
 

Enantiomers. Isomers with three-dimensional structures that are mirror images.

Enzyme histochemistry. The demonstration of enzymes in cells and tissues by utilizing the catalytic activities of these biopolymers. Specimens are immersed in enzyme substrates which are enzymatically converted – directly or indirectly – into colored final reaction products, marking the enzyme’s site. In the case of indirect conversion, intermediate reaction products are transformed into final reaction products by reaction with visualization agents.

Enzyme label. These may be visualized using the simple, reliable methods of enzyme histochemistry Horseradish peroxidase (HRP) and alkaline phosphatase are the most popular labels of this type. Usually the final reaction product is black, brown or blue, and is viewed by ordinary bright-field microscopy.
 
Enzyme retrieval
. A type of antigen retrieval utilizing enzymes (and sometimes adjuncts like calcium to improve enzyme activity) that opens access to masked epitopes; it is not effective against epitopes directly changed by fixation.

Enzyme substrate. The compound acted upon by a particular enzyme. See enzyme histochemistry.

Eosin B. A moderately large, hydrophilic dibromodinitrofluorescein (i.e. xanthene) acid dye. Sometimes used as the counterstain in Romanowsky stains, and originally specified as such for the Leishman and Wright variants. The dye is soluble in water and ethanol. Synonyms: 45400, Acid red 91. Commercial lots are available certified by the Biological Stain Commission.

Eosin Y. A moderately large tetrabromofluorescein (i.e. xanthene) acid dye. This dye is widely used in histology, notably in the hematoxylin and eosin, Papanicolaou and Romanowsky stains. Also used to stain various acidophilic structures such as eosinophil granules and Negri bodies; and as a cytoplasmic counterstain in various procedures, such as silver stains. Eosin Y is soluble in water, but poorly soluble in alcohol except under acidic conditions. Synonyms: CI 45380, Acid Red 87, eosin. Commercial lots are available in a fairly pure form certified by the Biological Stain Commission.

Epitope. The part of an antigen molecule to which an antibody molecule attaches. Typically an epitope consists of 5 or 6 amino acids, either in sequence (a linear epitope), or brought into proximity by the folding of a polypeptide chain into the conformation of a protein molecule (a discontinuous epitope).

Epitope retrieval. See antigen retrieval.

Erythrosin B. A large tetraiodofluorescein (i.e. xanthene) acid dye, whose major species at neutral and alkaline pH is a lipophilic anion. Despite an extensive range of reported applications as a cytoplasmic counterstain to various violet and blue nuclear stains, no single histological staining method is widely used. The dye is used to stain sperm in cytological preparations, and widely applied as a vital stain to assess cell viability. Highly soluble in water, soluble in ethanol. Synonyms: CI 45430, Acid red 51; see below for complications of nomenclature. Available commercially both as the free acid and the disodium salt, with typical samples containing the tetraiodinated compound plus smaller amounts of the lower and non-iodinated species. Commercial lots are available certified by the Biological Stain Commission. Related dyes which have been confused with this dye, either by vendors or by lab workers, are erythrosin Y (diiodofluorescein), erythrosine yellowish (a 1:9 eosin-erythrosin B mixture) and rose Bengal.

Ethyl eosin. A lipophilic acid dye, of the xanthene class, being the ethyl ester of eosin Y. Used with ethanolic solutions and differentiators; e.g. as a counterstain for hemalum, and for demonstration of Negri bodies. Hardly soluble in cold water, slightly soluble in ethanol. Synonyms: CI 45385, Solvent red 92, acid soluble eosin. Commercial lots of the potassium salt are available certified by the Biological Stain Commission.

Ethyl green. See methyl green.

Eukaryotic cell. A cell that has its DNA associated with histone in a membrane-bound nucleus, as in animals, plants, fungi and protozoans. Contrast with prokaryotic cell.

Evans blue. A large, strongly hydrophilic acid dye of the disazo class. Widely used as a vital stain, usually to assess cell viability, but also as a marker and tracer within intact living organisms. The dye is soluble in water, and slightly soluble in alcohol. Synonyms: CI 23860, Direct blue 53. Commercial lots of high dye content are available, usually containing a red monoazo dye contaminant.

Fab segment. A simple immunoglobulin (IgG) molecule is branched in the form of a letter Y. Each of two branches, named Fab segments, carries, at its end, amino acid sequences and conformations that will bind to a single epitope. Papain, a proteolytic enzyme, cleaves the peptide linkages that join the Fab segments to the common stem of the Y, liberating two Fab fragments and one Fc fragment derived from the Fc segment.  Fab fragments are used as reagents in some immunostaining methods; they are smaller than IgG molecules and diffuse more quickly into cells and tissues.  

Fading. Unwanted conversion of colored dyes, fluorochromes or other final reaction products in a biological specimen into colorless derivatives. This may be due to photobleaching from sunlight or illumination in a microscope, or to the chemical environment, such as the mounting medium.

Fast green FCF. Large, very hydrophilic, triphenylmethane acid dye. Widely used in histology, e.g. as a cytoplasmic counterstain, and to stain nuclear histones. Also as a fade-resistant alternative to light green, in methods such as Masson’s trichrome and the Papanicolaou procedure. Used in plant histology, and to stain proteins on gel electropherograms. Very soluble in water, soluble in ethanol. Synonyms: CI 42053, Food green 3. Commercial lots of high dye content, usually containing several minor colored contaminants, are available certified by the Biological Stain Commission.

Fc segment. A simple immunoglobulin (IgG) molecule is branched in the form of a letter Y. The stem of the Y is common to all immunoglobulins of the same species of animal, and can serve as an antigen for production of secondary antibodies or antisera such as rabbit anti(mouse IgG). See also Fab segment.

Ferric-ferrocyanide reaction
. See Prussian blue for ferric iron.

Ferro-ferricyanide reaction. See Turnbull's blue for ferrous iron.

Feulgen. Robert Feulgen (1884–1955), was the son of a cloth factory worker. The Feulgen reaction was first reported in 1923 at the Annual Meeting of the German Physiological Society, when Feulgen was Professor of Physiological Chemistry at Giessen.

Feulgen reaction. Demonstrates DNA. An initial mild hydrolysis with hydrochloric acid removes the purines adenine and guanine, creating aldehydes that react with Schiff's reagent. See Feulgen for biographical information on the originator of this procedure.

Final reaction product
(FRP). A colored terminal reaction product marking the enzymic sites following enzyme histochemical staining. The FRP is immobile, either because of adsorption onto tissue proteins, or because of pigment formation, or both. Currently, the more common FRPs include sulfides of some metals (Co, Pb), azo dyes, indigo derivatives,  formazans and the polymeric product resulting from oxidation of DAB.

FITC. An acid dye, a derivative of fluorescein, carrying a reactive isothiocyanate substituent, and thus able to form stable covalent bonds with primary amino groups. FITC is widely used to attach fluorescent labels to biological molecules, e.g. with immunoglobulins to generate labeled antibodies. FITC is soluble in dimethylformamide and ethanol, but almost insoluble in water. Synonyms: fluorescein isothiocyanate. Commercial lots of high purity, containing predominantly the 5-isomer, are available; the dye has been certified by the Biological Stain Commission. FITC is typically used by mixing a dye solution in DMF, or other solvent, with water; such largely aqueous solutions are fairly stable but do slowly degrade.

Fite's acid fast stain for Mycobacterium leprae and Nocardia. All acid-fast organisms are stained, including the aforementioned bacteria which are likely to be missed with some other proceduress of the acid-fast stain type.

Fixation. This involves transformation of constituents of cells and tissues into insoluble forms no longer subject to dissolution, destruction by endogenous enzymes (autolysis) or destruction by exogenous enzymes (microbial decomposition). This preserves the native morphology and chemical reactivity of the biological specimen during processing, staining, and microsco­pic observation. Mechanistically, fixation is complex, and may involve crosslinking of biopolymers and unsaturated lipids, denatu­ration of proteins resulting in hydrophobic inversion, and trapping of nucleic acids, polysaccharides and some small molecules within a mesh of fixed proteins.

Fixatives. Reagents used to achieve cell and tissue fixation. Common fixatives are reactive aldehydes or other small molecule organic compounds, e.g. formaldehyde, glyoxalglutaraldehyde or picric acid; reactive metal ions or derivatives, e.g. Cr2O72¯, Hg2+, OsO4, Zn2+; or organic solvents such as acetic acid, acetone, ethanol or methanol. Most fixatives are protein denaturants and some also form crosslinks. Solvents less polar than water cause hydrophobic inversions. Many useful fixatives are mixtures of compounds with different actions, such as combinations of formaldehyde (a crosslinker) with protein precipitants such as ethanol, picric acid or a zinc salt.

Flow cytometry. A method of determining the number of particles, usually cells, within a population. It is mostly used in hematology to count different cell types in peripheral blood. Cells are suspended, and flow in a narrow jet of fluid past optical detectors of, e.g. absorption, fluorescence, and light scattering.

Fluorescein. A small fluorescent, weakly acidic xanthene acid dye. It is hydrophilic as the dianion and lipophilic as the non-ionic species. Main use in biological staining is to make FITC for immunohistochemistry, with infrequent use in microscopy as a tracer. Synonyms: CI 45350, Solvent yellow 94 (for the free acid), Acid yellow 73; the disodium salt is also known as uranin). Commercial lots of high purity are available.

Fluorescence. A process whereby light (or other electromagnetic radiation) previously absorbed by the fluorescent molecule is rapidly (in less than 25 nanoseconds) re-emitted. This fluorescent light is of longer wavelength than the radiation previously absorbed by the fluorescent molecule. The Stokes shift is the magnitude (usually given in nanometers) of the wavelength difference between absorption and emission. Cf. phosphorescence.
 
Fluorescent label
. More than one molecule of a fluorochrome can be conjugated with an antibody molecule. The resulting emitted light stands out against a dark unstained background. Absorption and emission spectra of different fluorochromes are exploited when two or more labeled antibodies are applied to the same preparation.

Fluorescent probe. See probe.

Fluorochrome. A dye or other stain exhibiting fluorescence when present in solution, cells or tissues, e.g., acridine orange. Note that fluorescence is often enhanced in the latter locations. Synonym: fluorophore.

Fontana-Masson argentaffin reaction. Demonstrates melanin & other argentaffin materials such as neurosecretory granules. The staining solution is an alkaline aqueous solution of silver ammine. The quinone groups of melanin reduce silver cations salts in the dark, without an added reducing agent, to form deposits of metallic silver. Gold toning is sometimes used to give amplification of staining. Unlike the Grimelius argyrophil reaction, an external reducing agent is not needed.

Formaldehyde. A gas (CH2=O), typically sold as formalin, a 40% w/v (37% w/w) solution in water, with methanol added to inhibit formation of paraformaldehyde. Formaldehyde is the active ingredient in many fixatives.

Formaldehyde fixation. Given sufficient time, formaldehyde fixes tissue first by addition to form hydroxymethyl adducts, and subsequently by crosslinking via methylene bridges. The latter process may continue for many months. Tissue groups attacked by formaldehyde include amides, amines (primary and secondary), hydroxyls, reactive hydrogen atoms on aromatic amino acids, and sulfhydryls.

Formalin.
A concentrated solution (37% w/w or 40% w/v) of formaldehyde in water, usually with 10-15% methanol to inhibit polymerization. Synonym: formol.  So-called “10% formalin” is a 10% v/v dilution containing 4% w/v formaldehyde. This is the most common fixative for specimens taken from animals and people. The dilution is usually into either saline or a sodium phosphate buffer at neutral pH that is approximately isotonic with extracellular fluids. 

Formalin pigment. See hemosiderin.

Formazan. A compound produced by oxidation – chemically or enzymically – of a water-soluble, colorless tetrazolium salt. Thus the MTT formazan is produced by reduction of the MTT tetrazolium salt. Histochemically relevant formazans, like other final reaction products, are immobile, either because of adsorption onto tissue proteins, or due to pigment formation, or both.

Formic acid. An organic acid, HCOOH. This is frequently used in decalcification; it also occurs as an unwanted oxidation by-product of formaldehyde solutions that are not buffered.

Free water. Water that in vivo and ex vivo diffuses through tissue spaces; it must be removed completely during tissue processing for successful subsequent infiltration of clearing agents and of embedding media such as paraffin wax and some plastic monomers. Contrast with bound water.

Frozen section. A thin slice cut from a frozen block of tissue, usually with a cryostat but sometimes with another type of microtome devised for the purpose. See also tissue section.

Furanose. A sugar with a ring structure comprising four carbons and one oxygen atom; named for furan, C4H4O, a five-membered aromatic compound.

Gallocyanine. A small oxazine mordant dye whose colored species can be a cation, or a zwitterion or an anion, depending upon pH. Used to make gallocyanine chrome alum. Synonyms: CI 51030, Mordant blue 10. Commercial lots of gallocyanine, of high dye content and containing a single major component, are available.

Gallocyanine chrome alum. A large, hydrophilic, cationic metal coordination complex consisting of two molecules of the mordant dye gallocyanine chelating a chromium ion. Gallocyanine chrome alum has been used to stain DNA and RNA is a variety of cell types. Soluble in acidic aqueous solution, insoluble in ethanol. Synonyms: Gallocyanine is CI 51030, Mordant blue 10. Commercial lots of gallocyanine, of high dye content and containing a single major component, are available. However the metal complex has to be prepared in the laboratory.

Gel. A colloidal solution with a semi-solid consistency due to extensive hydrogen bonding between the suspended macromolecules and the “solvent”, which is usually water.

Gel filtration. A technique for separating molecules of different sizes by virtue of their diffusion into and out of pores in beads of a suitably designed polymer. Typically, the polymer is packed into a chromatography column and a solution containing molecules of varied size is applied at the top. When the column is eluted with a suitable solvent, the larger molecules move down the column most rapidly while the smaller molecules are retarded because they spend more time within the pores in the beads.

Gentian violet. See crystal violet and methyl violet.

Giemsa. Berthold Gustav Giemsa (1867–1948) was a German chemist at the Institute for Maritime and Tropical Diseases in Hamburg. He studied the products formed by polychroming the dye methylene blue, and published a series of papers on the Romanowsky stain from 1902 to 1934. Giemsa’s stain is made from “azure II” (impure azure B), methylene blue and eosin Y.

Gill. Gary Wesley Gill, CT, an American cytotechnologist who developed the half-oxidized hemalum  known as Gill hematoxylin and wrote books and numerous papers on cytological technique.

Gill hematoxylin. A hemalum solution containing aluminium sulfate and hematoxylin, which is half oxidized with sodium iodate, and acidified with acetic acid. The original version, published by Gary Gill in 1973, became commercialized as Gill II Hematoxylin and is widely used in histology and cytology. Other commercial variants are Gill I, a weaker solution mostly used in cytology; and Gill III, an extra-strength solution for rapid staining.

Glare. Out of focus light within the microscope, originating from the biological specimen or elements of the optical system, which becomes diffusely spread across the image owing to multiple reflections within the microscope. Glare results in visual degradation of the image.

Globulin. A protein that is insoluble in pure water but soluble at neutral pH in dilute aqueous solutions of simple salts (such as sodium, potassium or ammonium chloride or sulphate). Globulins are precipitated by half-saturation with (NH4)2SO4. Examples include the immunoglobulins and many other proteins of animals and plants.

Glutaraldehyde. O=CH(CH2)3CH=O. A fixative for electron microscopy that crosslinks proteins and polyhydroxy (carbohydrate) materials.
 

Glycocalyx. Carbohydrate-containing material (usually glycoproteins or glycolipids) present on the outside surfaces of all cells.

Glyoxal. The third smallest aldehyde (after formaldehyde and acetaldehyde) and the smallest dialdehyde: O=CH–CH=O. This is used as a safer alternative to formaldehyde. Fixes by addition reactions, and under certain conditions of pH, may crosslink. Glyoxal may be intentionally inhibited from crosslinking to improve preservation of immunoreactivity, again by adjusting pH. Glyoxal reacts with arginine to form cyclic imidazoles (the imidazole reaction), creating a histochemical blockade that also suppresses immunoreactivity on arginine-rich epitopes (but the latter can be reversed by glyoxal-specific antigen retrieval.

Glyoxal-specific antigen retrieval. A method to reverse the imidazole reaction, involving high temperature and pressure at pH 8.6. See also antigen retrieval and glyoxal.

Gold toning.  Treatment of a silver stained preparation with a solution containing a salt of gold, usually HAuCl4 or NaAuCl4 (“gold chloride”). The reaction 3Ag0(s) + [AuCl4]    Au0(s) + 3AgCl(s) + Cl  makes a yellow or light brown background paler, increasing contrast for the more strongly stained objects. In some methods, the gold chloride is followed by aqueous oxalic acid; this greatly intensifies the color of specifically silver-stained material, probably by reducing tissue-bound [AuCl4]  to colloidal Au0, which is dark red or black. The final step in the procedure is immersion in a sodium thiosulfate solution, which forms soluble complexes with any remaining gold or silver ions in the preparation. The word toning comes from methods used to change the colors of black-and-white photographs.

Golgi. Camillo Golgi (1843–1926) was an Italian pathologist and histologist. He experimented with silver staining and, in 1873, noticed blackening of occasional whole neurons in pieces of nervous tissue that had been stored in potassium dichromate solution (a commonly used fixative at the time) and then immersed in a solution of silver nitrate. He used this method to describe sensory organs in tendons and various types of neurons in the brain, which are associated with his name. In 1898, using a similar method, he described the “internal reticular apparatus”, now known as the Golgi complex and recognized as an organelle present in all eukaryotic cells. Golgi shared the Nobel Prize in Physiology or Medicine with Ramon yCajal, in 1906.

Gomori. György Gömöri (1904–1957) was a Hungarian physician who moved to the USA in 1938. He developed several new histochemical staining methods and wrote Microscopic Histochemistry (1952), one of the earliest books in the field.

Gomori's aldehyde fuchsine. This procedure is most often used for demonstrating elastin, but other elements also stain. The solution is made by first by depolymerizing paraldehyde to acetaldehyde, which is then combined with pararosaniline to form a variety of reactive products that may covalently bond to suitable substrates and are also cationic and contain large conjugated systems. The stain bonds to elastin by van der Waals forces and possibly also through covalent bonds with aldehydes. Sulfated mucopolysaccharides in mast cell granules and cartilage matrix stain due to basic dyeing while carboxylic acids are inhibited from staining by the low pH. With prior oxidation by potassium permanganate, lipofuscin and other sulfur-rich proteins, including those of pancreatic beta cell granules, also stain by basic dyeing. However, these granules also stain without prior oxidation through an as yet unknown mechanism.

Gomori's method for reticular fibers. This silver staining method involves potassium permanganate oxidation of the section, followed by bleaching; treatment with iron alum; then treatment with silver ammine, followed by aqueous formaldehyde as a reducing agent. These steps ensure that silver cations, which are taken up non-specifically into the tissues, are only reduced to metallic silver in the reticular fibres. Stain amplification by use of gold toning is often used to enhance contrast.

Gomori's one-step trichrome. This histological oversight stain colors cytoplasms and collagen fibers in contrasting colors. The acidic, aqueous staining solution contains fast green FCF, chromotrope 2R and phosphotungstic acid. The possible mechanism is rate control, with the larger green dye dominating in the more rapidly stained collagen fibres. Hemalum is a suitable nuclear counterstain. See also trichrome stain.

Gram. Hans Christian Joachim Gram, MD (1853–1938), a Danish bacteriologist and Professor of Medicine. He invented the Gram stain while in Berlin in 1884. A modest man, concerning this stain he said “I have therefore published the method, although I am aware that as yet it is very defective and imperfect; but it is hoped that also in the hands of other investigators it will turn out to be useful.”

Gram stain. Typically this stains Gram-positive bacteria blue with gentian violet (termed crystal violet outside the USA) and Gram-negative species red with basic fuchsine, neutral red or safranine O. Basic dyeing with gentian violet colors all bacteria, after which treatment with Lugol’s iodine generates a poorly soluble triodide salt. This is selectively removed from Gram-negative organisms, and background, by differentiation with an organic solvent such as acetone or alcohol. The thick cell walls of Gram-positive bacteria, however, retard the extraction. Gram-negative organisms are then counterstained with the red basic dye. Many variants exist using different counterstains. Others are designed for specific types of specimens (sections versus smears): Brown-Hopps, Brown-BrennTwort's stain is another variant for bacteria in tissue sections.  See Hans CJ Gram for biographical information.

Gridley's stain
. A method to demonstrate fungi in tissues. An initial oxidation with chromic acid generates aldehydes from the chitin and other polysaccharides of fungal cell walls. Treatment with metabisulfite results in partial sulfonation of the aldehydes, and residual aldehydes are then stained by treating with Schiff's reagent. Subsequent basic dyeing with aldehyde fuchsine amplifies staining of fungal cell walls, and counterstaining by 
with metanil yellow visualizes the tissue background. 

Grimelius argyrophil stain. This procedure demonstrates cells of the dispersed neuroendocrine system (DNS). Monoamines in these argyrophil cells bind silver ions from silver nitrate solution, but cannot reduce them (unlike argentaffin cells; see Fontana- Masson argentaffin reaction). Reduction to black metallic silver is achieved using an external reducing agent, hydroquinone. Improvements in this technique were made by Charles Churukian and Eric Schenk.

Grocott's methenamine silver.  A procedure for demonstrating fungi. Methenamine (synonyms: hexamethylenetetramine and hexamine) is created from formaldehyde and ammonia; this in turn is complexed with silver nitrate. Chromic acid oxidizes carbohydrates in fungal cell walls to aldehydes, which reduce the Ag+ in the methenamine complex to black metallic silver (Ag0).

Harris hematoxylin. A nuclear stain based on hemalum, often used for regressive staining in routine histopathology, and progressive staining of diagnostic exfoliative cytology specimens. Hemalum comprises an acidified solution containing Al3+ (from aluminum ammonium sulfate) and hematein (from hematoxylin), which deposits a blue metal coordination complex on the chromatin of cell nuclei. Upon standing, the solution forms a precipitate from the ammonium alum, which must be filtered before staining. Modern Harris hematoxylin, unlike the original version, has iodate as the oxidant and acetic acid-alcohol for differentiation.

Häutchen preparation. A thin layer of cells peeled or otherwise removed from a surface to make a whole-mount for microscopy. From German for film or membrane.

Heat-induced epitope retrieval
(HIER). The freeing of altered or masked epitopes by heating in buffered solutions, either passively, under microwave irradiation or under elevated pressure. The heat and consequent molecular vibration breaks bonds, while the buffer guides the now-disordered molecule back to its native (or nearly native) conformation. See also antigen retrieval.

Heidenhain. Two Heidenhains devised and applied staining reagents, namely Rudolf (1834–1897) and his son Martin (1864–1949, see Heidenhain's iron hematoxylin). On at least one occasion they both contributed to the development of the same stain, namely the Erhlich-Biondi-Heidenhain procedure, a modification the triacid stain for blood cells, developed by Ehrlich, for tissue sections.

Heidenhain's iron hematoxylin. Tissues are pre-mordanted with iron alum (ferric ammonium sulfate), which attaches to carboxyl groups of proteins. A solution of hematoxylin, naturally ripened by atmospheric oxygen, is applied, forming a metal coordination complex with the iron. The section becomes almost uniformly black. A second iron alum solution is then used to differentiate the tissue under the microscope until only denser objects like mitochondria and muscle striations retain color. See Michael Heidenhain.

Hemalum (US) or Haemalum (elsewhere). A staining solution made by dissolving hematoxylin, an oxidizing agent (usually NaIO3, but sometimes O2 from the air), an aluminum salt and usually also a weak acid (acetic or citric), in water. A hydrophilic organic solvent (ethylene glycol or glycerol) is often included in the solution.  The staining component is a complex of aluminum with hematein. There are many hemalum formulations; most are intended for staining cell nuclei. Synonym: hematoxylin (in the second sense).

Hematein (US) or Haematein (elsewhere). A yellow-brown compound formed by oxidation of hematoxylin, which is white when pure. Hematein forms intensely coloured dye-metal coordination complexes with aluminum, iron and other metal ions; these complexes are the colorants in staining solutions with names such as hemalum, hematoxylin, and iron hematoxylin. 

Hematin (US) or Haematin (elsewhere). A pigment formed when hemoglobin is degraded in acid conditions. Also known as acid hematin or formalin pigment when the degradation is caused by acidic formaldehyde. It is seen as granular deposits around blood vessels in sections of tissue stored in formaldehyde solutions that have not been buffered to neutrality.

Hematoxylin (US) or Haematoxylin (elsewhere). A phenolic compound extracted from logwood (Haematoxylum campechianum L.), a tree originally from Central America, now growing in many tropical and subtropical regions. Hematoxylin is the starting material for making many solutions that are used as biological stains. Preparation of most “hematoxylin” staining solutions involves oxidation to hematein, and subsequent complexation of the hematein with a metal. The hematein-metal coordination complex is the compound with specific staining properties. The name “hematoxylin” is commonly applied to solutions for which hemalum would be more appropriate; examples are Delafield’s hematoxylin, Gill hematoxylin, Harris hematoxylin.

Hematoxylin and eosin (H&E).  This sequence stain is the most widely used oversight stain for animal and human tissue sections, in which cell cytoplasms and nuclei are contrasted red and blue respectively. In this context hematoxylin implies hemalum, which acts as a basic dye staining nuclei and cytoplasmic ribosomes. Eosin implies eosin Y, a red acid dye which stains intra- and extracellular proteins.

Heme (US) or Haem (elsewhere). The non-protein portion of the hemoglobin molecule. Often used more generally to describe iron–porphyrin prosthetic groups of proteins, present in many enzymes and cytochromes.
 
Hemiacetal. A compound formed by condensation of one molecule of an aldehyde with one molecule of an alcohol to give the structure: R–O–CH(Rʹ)–OH in which R, Rʹ are alkyl or aryl groups. The hemiacetal configuration occurs in the ring structures of sugars.

Hemosiderin. A brown intracellular pigment containing Fe3+, derived from the heme of red blood cells that have been phagocytosed.

Hydration. Water molecules can bind strongly to various groupings on biopolymers. For instance to –OH in glycogen and –NH3+ and –CO2- in proteins. Such water (bound by dipoles and hydrogen bonding) can form a substantial hydration shell around a biopolymer. Indeed water can amount to a significant proportion of the weight of a sample of apparently dry protein. 

Hydrogen bond. A directed, weak bond between a hydrogen atom and an adjacent electronegative atom (usually nitrogen or oxygen), there being no covalent bond between the atoms. Partial charges range from +0.15 to +0.30 on the hydrogen atoms and -0.15 to -0.50 on the electronegative atoms.

Hydrophilic. Literally, water loving. (1) Of compounds with an affinity for water, e.g. alcian blue, glycogen, NaCl. (2) Of molecular fragments such as –SO3¯ and –CONH– whose presence results in such affinity. Organic compounds are often hydrophilic because they contain groups such as –OH and –NH2 which can form hydrogen bonds with water. Hydrophilic compounds are also polar. The hydrophilic nature of a reagent may be assessed using a structure parameter such as log P or the hydrophilic/lipophilic index.  See also hydration; contrast with hydrophobic.

Hydrophilic/Lipophilic Index (HLI). An alternative to log P as a structure parameter, measuring the tendency of a molecule to favor a hydrophilic or lipophilic (hydrophobic) environment. HLI = Σ (0.155¯Abs C), where Abs C is the absolute partial charge on each atom. Negative values are hydrophilic, positive values are lipophilic.

Hydrophobic. Literally, water hating. (1) Of compounds with little or no affinity for water, e.g. oil red O, lipids. (2) Of molecular fragments whose presence results in such lack of affinity, e.g. methyl and phenyl groups. Hydrophobic compounds have few or no atoms that can form hydrogen bonds, and are typically non-polar. Such compounds are often lipophilic, though some, such as perfluorocarbons, are both hydro- and lipophobic. The hydrophobic nature of a reagent may be assessed using a structure parameter such as log P or the hydrophilic/lipophilic index.

Hydrophobic bonding. This describes the tendency of hydrophobic molecules or groupings originally dispersed within an aqueous environment to come together. Pheno­mena driven by hydrophobic bonding include folding of polypeptide chains, where hydrophobic amino acid residues come together in the core of the protein; and the use of a hydrophobic dye to stain a hydrophobic tissue substrate such as suberin or lipid. Although described as hydrophobic bonding, the process is driven by changes in the hydrogen bonding pattern of the aqueous solvent, and not by attractive bonds between hydrophobic groups. Once in position, groups or molecules may actually bond by dispersion forces.

Hydrophobic inversion. When insufficiently fixed (stabilized) tissue specimens are exposed to the higher concentrations of alcohol during tissue processing, hydrophilic areas formerly on the surfaces of macromolecules shift inward while hydrophobic regions, once hidden from the aqueous environment, are twisted outward. Commonly manifested as a nuclear bubbling artifact.

Hydroxyalkylation
. Addition of an aldehyde or ketone to an aromatic ring. This reaction is exploited in a histochemical method for proteins rich in tryptophan that makes use of the reagent p‑dimethylaminobenzaldehyde  (PDAB).

Hypertonic. Having a higher osmotic pressure than blood or extracellular fluid.
 
Hypotonic. Having a lower osmotic pressure than blood or extracellular fluid.

Imidazole reaction. Addition reaction between glyoxal and arginine that results in loss of charge, formation of cyclic imidazoles and significant change in molecular shape; readily reversed by glyoxal-specific antigen retrieval; also used in histochemistry to blockade arginine.

Imide. A compound formed by condensation of two carboxyl groups with the nitrogen of ammonia or a primary amine.

Imine. A compound containing a double bond between carbon and nitrogen atoms: R–CRʹ=N–Rʹʹ. They are formed by condensation of aldehydes with primary amines. Synonyms: anil, azomethine, Schiff’s base. The term “imino” is sometimes applied to the –NH– group of secondary amines.

Immunization. Administration of antigen to an animal to induce production of antibodies. The word ordinarily means stimulating immunity to infectious diseases. Among scientists it is more liberally used.
 
Immunocytochemistry. Widely used as synonym for immunohistochemistry but correctly applicable when the objects of interest are cells (rather than tissues), as with immunostaining of smears or monolayer cultures.
 

Immunofluorescence. Secondary fluorescence introduced into cells or tissues by the application of immunocytochemical or immunohistochemical methods in which fluorochromes are used as labels.
 
Immunoglobulin. An antibody protein in the gamma-globulin class. Five types of immunoglobulin are IgA, IgD, IgE, IgG and IgM. Only IgG (which has 4 sub-types) and IgM are used as immunostaining reagents. An IgM antibody molecule comprises 5 IgG units linked to form a cyclic pentamer. Some monoclonal antibodies are IgMs. As immunostaining reagents, IgMs diffuse into tissues and cells more slowly than IgGs. See also Fab fragment, Fc segment. Immunoglobulins in plasma are produced by cells in lymph nodes that respond to contact with protein molecules not previously encountered.

Immunohistochemistry. Histochemical staining of sections of tissue resulting from the use of labeled anti­bodies. Cf: immunocytochemistry, immunostain.

Immunorecognition. The ability of an antibody to identify and bind to its antigen.

Immunostain. A useful verb, meaning to carry out an immunocytochemical or immunohistochemical procedure to generate a visible product at the site of an antigen.

Immunostaining. Histochemical staining resulting from use of labeled anti­bodies.

Indigo carmine. See indigocarmine.

Indigocarmine. An acid dye of the indigo class, which is small and hydrophilic. This has been used in histology to stain collagen, and in botany to track stages in the cell cycle. The dye has various applications as a clinical vital stain, e.g., in chromoendoscopy and for identifying sentinel lymph nodes. Soluble in water, very slightly soluble in ethanol. Synonyms: CI 73015, Acid blue 74, indigo carmine.  Commercial lots of high dye content, usually containing a minor colored contaminant, are available certified by the Biological Stain Commission.

Intermediate reaction product. See enzyme histochemistry.

Ionic crystal. A regular, three-dimensional array of anions and cations whose overall charge is zero. An example is lead sulfide, the final reaction product in Gomori-style enzyme histochemistry, where the constituent ions are Pb2+ and S2.

Ionic weight. The nominal molecular weight of an ion, ignoring its counterion. For example, the dianionic form of the acid dye eosin Y is available both as the potassium and sodium salts, with the molecular weights of the two salts being 724 and 692 respectively. The ionic weight of the colored eosin Y anion however is the same in both salts, namely 646.

Iron hematoxylin. A group of stains, all of which contain or produce a metal coordination complex or complexes involving Fe3+ and hematein, whose precise chemical structures are uncertain. Such stains can demonstrate a variety of tissue components. Examples are Heidenhain’s iron hematoxylin, which uses a ferric iron salt is followed by naturally oxidized hematoxylin, Verhoeff’s hematoxylin and Weigert’s hematoxylin in which the iron oxidizes hematoxylin and bonds to the tissue elements.

Isomers. Compounds that have the same elemental composition and the same molecular weight but different structures.

Isotonic. Having osmotic pressure the same as that of blood or extracellular fluid.

Janus green B. A basic dye of lipophilic character and moderate size in the monoazo class. A traditional vital stain of mitochondria in living cells. Soluble in water, and somewhat less so in ethanol. Synonym: CI 11050. Commercial lots, typically containing multiple components, are available certified by the Biological Stain Commission.

Keesom forces. A type of van der Waals attraction in which two dipolar groups or molecules align with the partial positive charge of one bonding to the partial negative charge of the other. Also known as dipole-dipole interactions.

Ketal. A compound formed by condensation of a ketone with an alcohol. DMP (2,2-dimethoxypropane) is a ketal that can be used to dehydrate specimens by virtue of its chemical reaction with water.

Kinyoun's acid fast stain. Used for selective staining of bacteria in the genera Mycobacterium and Nocardia. Specimens are initially stained with carbol-fuchsine, an aqueous-alcoholic solution of basic fuchsine or pararosaniline and phenol. This imparts a red coloration to all bacteria, due to basic dyeing of their nucleic acids. Subsequent differentiation in acid alcohol removes dye from all but the acid-fast bacteria, whose thick lipid cell walls render them less permeable. A contrasting counterstain, namely methylene blue, is then applied, which colors bacteria of other types, also by basic dyeing. The preparations are not heated, as in the related Ziehl-Neelsen stain. Kinyoun’s stain can also be used to demonstrate protozoa of the genus Cryptosporidium.

Kluver and Barrera luxol fast blue, see Luxol fast blue for myelin.

Label. A molecule artificially attached to a macromolecule (such as an antibody or a nucleic acid probe), typically by covalent bonding. The type of labeling used in immunostaining determines the amount of amplification, and therefore the sensitivity of the technique. Frequently used labels are biotin, colloidal gold, enzyme labels, fluorescent labels, and quantum dots.

Labeled antibodies. Reagents used as stains in immunohistochemical (IHC) staining. The affinity and selectivity of the binding these reagents with tissue antigens is provided by the antibody. The label provides visualization. Some labels (e.g. the fluorochrome FITC and colloidal gold) provide microscopic visualization directly. Other labels, which are themselves invisible, can give rise to a visible final reaction product. For instance, when the enzyme peroxi­dase is used as a label, this can be visualized using enzyme histochemistry. Synonym: labeled antibody. Cf. immunoglobulin.

Le Chatelier’s principle. When a constraint is applied to any system in equilibrium, the system will always react in a direction to oppose the constraint. For chemical equilibria, the constraint may be a change in concentration of a reactant, or a change of temperature, etc. The law of mass action and the common ion effect are examples of this principle.


Leifson’s flagella stain. Smears containing bacteria are stained with an aqueous solution containing  basic fuchsine, tannic acid and sodium chloride at pH 5.0. Deposition of a dye-tannic acid complex enlarges the flagella sufficiently to make them visible with ordinary light microscopy. Without such staining, an electron microscope is needed to resolve individual bacterial flagella.
 
Leishman. Sir William Boog Leishman (1865–1926) was a Scottish pathologist and a Lieutenant General in the Royal Army Medical Corps. In 1900 he described a simple and reproducible Romanowsky stain for blood cells, which is still widely used. He discovered the parasitic protozoan that causes kala azar, a common tropical disease that now is more often called leishmaniasis. The causative organism in East Africa and India was later named Leishmania donovani. Another species, L. infantum occurs elsewhere in Africa and in tropical regions of the Americas.

Light green SF. Large, very hydrophilic triphenylmethane acid dye. A collagen fiber stain in methods such as Masson’s trichrome; a component of the Papanicolaou procedure, and the Twort stain for microorganisms in tissue sections. Light green is also used as a cytoplasmic counterstain, in animal and plant histology. In botanical work the dye also stains cellulose cell walls. Highly soluble in water, soluble in ethanol. Synonyms: CI 42095, Acid green 5, light green SF yellowish. Commercial lots of high dye content are available certified by the Biological Stain Commission. Typical lots contain a single major component plus minor colored contaminants, probably of higher and lower degrees of sulfonation.

Lillie. Ralph D. Lillie (1896–1979). Born in Cucumonga, CA, graduated in medicine at Stamford in 1920. Although attracting the epithet “a master magician of histochemistry” it was not until that he became a Trustee of the Biological Stain Commission that his major contributions were made, including the 4 editions of his encyclopedic text Histopathologic Technic & Practical Histochemistry (1947, 1954, 1965, 1976).

Lipofuscin. A yellow or light brown autofluorescent pigment containing lipids and proteins, seen in the cytoplasm of cells, especially in old animals. It may be the indigestible remains of phagocytosed material.

Lipophilic. Literally, lipid loving. (1) Of a molecular species with an affinity for lipids, e.g. a Sudan dye. (2) Of molecular fragments favoring such behavior, e.g. a methyl or naphthyl group. (3) Of a solvent of lipids, such as chloroform or xylene.

Log P. The logarithm of the water-octanol partition coefficient of a molecule. Used as a structure parameter to model lipophilicity. See also hydrophilic/lipophilic index.

London forces (dispersion forces). A type of van der Waals attraction in which the oscillations of π electrons in one hydrophobic group or molecule entrain similar electrons in a neighboring entity. Halogen atoms and large conjugated systems can in this way provide significant bonding power.

Lugol’s iodine
. Formulations vary, but originally 6% w/v I2 in 4% w/v aqueous KI. This solution contains elemental I2 in equilibrium with triiodide (I3¯) and other polyiodide anions. Lugol’s iodine has been used as a stain for a wide variety of protozoa, animal and plant materials; and to form a poorly soluble salt of crystal violet in the Gram stain. Gram’s and Weigert’s iodine are formulations containing differing amounts and proportions of I2 and KI.

Luxol fast blue. The dye currently sold under this name is a large lipophilic acid dye, being the diarylguanidine salt of a sulfonated copper phthalocyanine. Routinely used to stain myelin. Soluble in ethanol, very slightly soluble in water. Synonyms: CI 74180, Solvent blue 38. Commercial lots are available, as this is an industrial colorant. Note that dyes of a different chemical nature have been sold under this name, e.g., luxol fast blue G, which is the diarylguanidine salt of a trisazo acid dye.

Luxol fast blue for myelin. The staining solution comprises an acid dye with unusual hydrophobic counter ions, see luxol fast blue, in warm, acidic ethanolic or methanolic solution. Initially, myelin is stained by partitioning of hydrophobic ion-pairs, with electrostatic attraction of the colored anions to the cations of basic proteinsof myelin. Non-specific background, also due to acid dyeing, is selectively removed by differentiation under microscopic control, using alternating aqueous alkali and alcohol.

Macromolecule. A large molecule, typically assembled from one or several repeating units. Biological macromolecules (e.g., peptides, proteins, large carbohydrates, DNA, RNA) typically have secondary or higher molecular conformations.  Such macromolecules are also termed biopolymers. Synthetic macromolecules include plastic embedding media.

Malachite green. A small lipophilic triarylmethane basic dye. Has been used routinely as a stain for bacterial spores, in the Gimenez stain for Rickettsia and Helicobacter pylori, and in staining methods for fungal and plant tissues. Soluble in water and ethanol. Synonyms: CI 42000, Basic green 4. Commercial lots of high dye content, usually containing a single colored component, are available certified by the Biological Stain Commission.

Mallory. Frank Burr Mallory (1862–1941) was a pathologist in Boston, MA. He devised several staining techniques, notably acid fuchsine followed by a mixture of orange G, aniline blue and phosphomolybdic or phosphotungstic acid, for differential coloration of cytoplasm and collagen; and a phosphotungstic acid-hematoxylin combination for, neuroglial scar tissue. Mallory bodies are acidophilic cytoplasmic inclusions in degenerating liver cells. He played a major role in the standardization of biological stains in the USA, and was one of the founders, and a long time Trustee, of the Biological Stain Commission (BSC). He was also author, with J. H. Wright, of the classic text Pathological Technique (8 editions from 1897 to 1938), and collaborator with H.J. Conn in the first 4 editions (1925-1940) of the BSC's reference book Biological Stains.

Mallory's trichrome. Utilizes moderate size acid fuchsine to stain nuclei and some cytoplasmic elements, large aniline blue to stain collagen, cartilage matrix and mucus, and small orange G to stain cytoplasm, erythrocytes and myelin. The latter two dyes are in the same solution with phosphotungstic acid. See Trichrome stain for mechanism.

Marker. This word is used for an antigen characteristic of a particular cell-type. Immunostaining for markers provides specific staining of different cell-types. Markers are usually glycoproteins of cells’ surfaces, with abbreviated names that do not relate to their locations or functions. For example, CD3 is a marker for T-lymphoctes, and Nogo-A is used to identify oligodendrocytes.
 
Masking
. Inhibition of staining of one cell or tissue component by a second component. For example, DNA inhibits the staining of nuclear histones by acid dyes.

Masson. Pierre Masson (1880–1959) was a French (and later Canadian) pathologist now best known for trichrome methods in which a black nuclear stain with iron‑hematoxylin is followed by counterstaining with a red acid dye, followed by differentiation in phosphomolybdic or phosphotungstic acid and then staining of collagen with a blue or green dye.

Masson’s trichrome. Utilizes Biebrich scarlet as a small acid dye that stains nuclei and cytoplasm, followed by a solution of phosphotungstic acid  (sometimes with phosphomolybdic acid), and fast green FCF as the larger collagen dye. See Trichrome stain for mechanism.

Mayer. Paul Mayer (1848–1923) was a German zoologist, whose most significant work was carried out in the Zoological Station in Naples. Whereas Mayer's mucicarmine stain and Mayer's hemalum indicate his ability to devise new stains, his significant contributions were in systematizing histological staining techniques, including a complete reworking and extension of the staining compilation, Lee’s The Microtomist's Vade-mecum, to produce a staining handbook widely used for many years.

May-Grunwald stain. This is a Romanowsky stain variant, most commonly used to stain blood and marrow smears, or cytological specimens such as sputum or urine sediment. The procedure involves an initial staining with the May-Grunwald solution, a mixture of methylene blue and eosin, followed by staining in another Romanowsky stain, usually Giemsa’s. The addition of the pre-Romanowsky staining ensures that basophilic cytoplasms are clearly seen as azure blue.

Metabolite. Any substance participating in a chemical reaction in, and native to, a living organism.

Metachromasia. Sometimes a single, pure, dye gives rise to different colors when bound to different cell and tissue components. The precise mechanisms of the process are varied, but usually involve tissue-mediated aggregation of dye.

Metachromatic dye. Dye whose staining exhibits metachromasia, e.g. methylene blue or toluidine blue. Fluorochromes such as acridine orange can also show metachromasia.

Metal coordination complex. A molecular species containing a metal ion to which ligands are bound by polar covalent bonds. There are several examples of histochemical interest, both soluble and insoluble. One is an aluminum-hematein complex present in hemalum, which has been the routine nuclear stain in histopathology for more than 100 years. See also mordant dye and ammine.

Metanil yellow. An acid dye in the monoazo class, with slightly lipophilic anions of small size, used in the Movat pentachrome stain. Soluble in water and ethanol. Synonyms: CI 13065, Acid yellow 36.

Methyl blue. See Aniline blue.

Methyl green. A basic dye which is hydrophilic, of the triarylmethane class. This dye is used as a nuclear counterstain for immunostaining, in situ hybridization, and enzyme histochemistry. Also as a component of the methyl green-pyronin method for distinguishing structures rich in RNA from those rich in DNA. Methyl green is very soluble in water, and slightly soluble in ethanol.  Synonym: CI 45290. Note: this dye was once termed ethyl green, but many years ago was substituted the dye originallysold as methyl green (CI 42585), which had become commercially unavailable. Commercial lots of high dye content and purity are available certified by the Biological Stain Commission.

Methyl green-pyronine stain. This colors DNA-rich nuclear chromatin blue-green with methyl green, and RNA-rich cytoplasms and nucleoli red or pink with pyronine Y. Red staining of other strongly basophilic structures – such as cartilage matrix, mast cell granules and some mucins – also occurs. Although many variants have been devised, sections are typically stained in a slightly acidic mixture of the two dyes, washed, and then dehydrated carefully with some organic solvent. Both colorants act by basic dyeing, with the methyl green having a greater affinity for DNA, the pyronine acting as a non-specific basic dye counterstain. The reliability of this method depends on using a pure lot of pyronine. 

Methyl violet 2B. A mixture of N-methylated pararosanilines, predominantly the higher homologues, consequently most components are slightly lipophilic basic dyes of the triarylmethane class. The dye has had a variety of applications, such as staining vascular plant tissues and as an oversight stain for epoxy resin sections. Soluble in water and ethanol, with considerable lot variation which probably reflects varying mixtures of homologues in different lots. Synonyms: CI 42535, Basic violet 1, gentian violet (in Europe; in the USA this name usually implies crystal violet), methyl violet. Commercial lots of high dye content, always containing several homologues, are available certified by the Biological Stain Commission.

Methylene blue. A small basic dye of the thiazine class. As well as providing the starting material for preparing Romanowsky stains, methylene blue has been used for a variety of other staining applications. Examples include oversight staining of epoxy resin sections of animal and plant material; as a general bacterial stain; and as a counterstain for Ziehl-Neelsen and other stains for acid-fast bacteria. Methylene blue is also used as a vital stain, e.g. for nerves and nerve terminals in muscle, and to detect sentinel lymph nodes. Soluble in water and ethanol. Synonyms; CI 52015, Basic blue 9. Commercial lots of high dye content, usually with a single major component, are available certified by the Biological Stain Commission.

Methylene violet. A small thiazine non-ionic dye which is lipophilic. The dye has no significant current staining applications but is included in some Romanowsky stains. Slightly soluble in ethanol, only soluble in water if acidified. Synonyms: CI 52041, methylene violet Bernthsen. Commercial lots, typically contaminated with other methylene homologues such as thionol and the thionolines, are available; the dye has been certified by the Biological Stain Commission.

Microtome. A device that first cuts a section from a paraffin or plastic block then advances the block by some set amount before another section is cut; may be manual or automatic, rotary or sliding in operation. An ultramicrotome prepares much thinner sections, needed for electron microscopy.

Microtomy (sectioning). The use of a microtome to cut thin sections of paraffin- or plastic-embedded specimens prior to mounting on a glass slide.

Mohs surgery. Small scale surgery, generally on skin cancer, in which tiny pieces are successively removed, sectioned with a cryostat and examined microscopically before the next sample is taken. This continues until all margins are found free of disease.  Named for American surgeon Frederic Edward Mohs (1910-2002).

Molecular dehydration. Undesirable action of an aggressive dehydrant like ethanol on tissue that has been inadequately fixed by formaldehyde. Ethanol combines with the hydroxymethyl adducts on amines, removing a hydrogen atom from the amine and a hydroxyl group from the adduct. The result is a highly reactive imine.

Molecular weight (MW). The weight of a single molecule. This is the sum of the atomic weights of the constituent atoms. MW is used as a structure parameter to model molecular size. Synonym: molar mass. Contrast with ionic weight.

Monoamine. A biologically significant compound with a single primary (–NH2) or secondary (–NH–) amino group. Examples are dopamine, noradrenaline (synonym:  norepinephrine), adrenaline (synonym: epinephrine) and serotonin (synonym: 5‑hydroxytryptamine).

Monoamine oxidase. An enzyme that catalyses the oxidative degradation of physiologically significant monoamines such as noradrenaline and serotonin.

Monoazo dye.  A dye with one azo (−N=N−) group per molecule.

Monoclonal antibody (or MAB). A single species of antibody globulin, produced in a cell cuture by the descendants (clone) of a single antibody-producing cell. A MAB recognizes a specific epitope. Most MABs are mouse immunoglobulins.

Mordant dye. 1. A dye capable of forming a metal coordination complex; or such a complex, e.g. hematein or gallocyanine chrome alum respectively. 2. A dye supposed by some to be bound to the tissues via dye–metal ion and metal ion–tissue bonds.

Mounting medium (mountant). A transparent liquid that preserves the object on a microscope slide and hardens, holding the coverslip in place. The high refractive index of the final material provides clarity to optical images of the specimen. Mountants may be organic solvent-based (permanent) or aqueous (for temporary mounts).
 

Movat. Henry Zoltan Movat (1923–1995) was a Romanian-Canadian pathologist, who developed his eponymous "pentachrome" stain in Toronto in the early 1950s.

Movat pentachrome stain.  A histological oversight stain, sequentially coloring connective tissue matrix and mucins blue by basic dyeing with alcian blue; elastic fibers and nuclei black with iron hematoxylin;  fibrin and muscle red by acid dyeing with a mixture of crocein scarlet (synonym: woodstain scarlet) and acid fuchsine; and collagen and reticular fibers yellow by acid dyeing with saffron. Other “named” variants are those of Russell and Silverman.

MTT tetrazolium and formazan
. MTT tetrazolium is a weakly lipophilic cation, which can be reduced chemically or biochemically to the strongly lipophilic MTT formazan. This reduction can be catalysed by tissue enzymes, and MTT tetrazolium has been used in enzyme histochemistry to demonstrate dehydrogenases. The major current application of MTT is as a vital stain, used to assess viability in bacteria and eukaryotic cells. The tetrazolium salt is soluble in ethanol and water; the formazan is insoluble in water, but soluble in dimethylsulfoxide and many lipids. Synonyms: methylthiazolyldiphenyl tetrazolium/formazan, thiazolyl blue tetrazolium/formazan. Commercial lots of the tetrazolium of high purity are available.

Mucicarmine for mucins.  This stain is prepared by heating carmine with aluminum salts in an acidic alcoholic solvent. The colorant is probably a large, cationic metal coordination complex of carminic acid with Al3+. If so, staining is basic dyeing of anionic glycosaminoglycans, with selectivity being due to restriction of the complex to the permeable, fast staining, mucins. Variants include those of Baker, Southgate and Mayer.  

Natural dye. A dye, or dye precursor, synthesized by a plant or animal. Examples include hematoxylin from the logwood tree Haematoxylum campechianumcarminic acid from the cochineal insect Coccus cacti, and saffron from the stigmas and styles of Crocus sativus flowers. Contrast with synthetic dye.

Negative stain. A stain deposited within an opening or against the surface of a cell or tissue, to visualize the morphology or presence of the biological structure. The entity of interest itself is not stained. Examples include Schmorl’s picro-thionine mixture for demonstrating bone canaliculi, and nigrosin for visualizing bacteria in smears.

Neutral buffered formalin (NBF). Generally a 10% v/v aqueous formalin solution in an approximately isotonic buffer at pH 6.8-7.4, which retards acidification by spontaneously generated formic acid, and prevents the formation of hemosiderin (formalin pigment). 

Neutral dye. An ionic dye whose anion and cation are both themselves colored dyes. Romanowsky stains provide an example, in which one key component is an azure B cation and the other is an eosin Y anion. A neutral dye is thus not non-ionic but a salt. Contrast with non-ionic dye. Synonym: Neutral stain.

Neutral red. A small basic dye of the aminoazine class, which is hydrophilic in acidic solution, from which it is used as a red nuclear stain, and as a counterstain for Gram stains. It forms a neutral stain with fast green FCF for bacteria (Twort's stain). It has been used as a fluorescent stain for nucleic acids in nervous tissue. In alkaline solutions, neutral red loses its charge and the resulting lipophilic dye stains hydrophobic material in plant cell walls. Neutral red is a vital dye, accumulating in acidic organelles such as lysosomes in living cells; thus it is used in flow cytometry, for testing cell viability, and as a marker for root growth.  Synonyms: CI 50040, Basic red 5, toluylene red (as the non-protonated form); sometimes confused with nuclear fast red. Commercial lots are available certified by the Biological Stain Commission.

Nigrosin WS. A complex mixture of dark blue or violet, high molecular weight acid dyes with large conjugated systems, of the azine class. Water soluble (hence the designation WS). Often sold blended with a yellow or orange dye to create a black color. Mostly used as a negative stain for microorganisms and for assessing cell viability. Synonyms: CI 50420, Acid black 2. Commercial lots are available certified by the Biological Stain Commission.

Nile blue A. An oxazine basic dye which is lipophilic, used to stain fatty acids and phospholipids. When the non-ionic hydrolysis product Nile red is present, neutral lipids stain pink. Nile blue A is also used as a fluorescent probe. Synonyms: CI 51180, Basic blue 12, Nile blue, Nile blue sulfate. Commercial lots are available certified by the Biological Stain Commission.

Non-ionic dye. A dye whose colored entities are not ionic. Some non-ionic dyes are strongly lipophilic (e.g. Sudan black B and oil red O) whereas others (notably hematein) are water soluble. Contrast with neutral dye.

Non-polar solvent. A hydrophobic liquid, not miscible with water, such as benzene or carbon tetrachloride. A molecule is non-polar because it either has no atom carrying a significant partial charge (as in hydrocarbons) or has polar bonds symmetrically oriented so that partial charges cancel out (as in CCl4).

Nuclear bubbling artifact. Occurrence of nucleoplasm with a soap bubble appearance, obscuring diagnostically important patterns of nuclear chromatin. A consequence of hydrophobic inversions.

Nuclear fast red. A small acid dye dye in the anthraquinone class. An aluminum metal coordination complex of this dye is often applied as a red nuclear counterstain for histochemical procedures that yield insoluble blue products. Alone, the dye has been used as a histochemical stain for calcium. Synonyms: CI 60760, Kernechtrot, Kernechtröt, calcium red. Sometimes erroneously confused with neutral red, a basic dye with different properties and applications.

Nucleoside. A molecule of ribose or deoxyribose joined at position 1ʹ to a purine or pyrimidine base. 

Nucleotide. A molecule of ribose or deoxyribose joined at position 1ʹ to a purine or pyrimidine base and at position 3ʹ or 5ʹ to a phosphate group. A nucleotide is a single unit of a DNA or RNA sequence.


Oil red O
. A hydrophobic non-ionic dye of the disazo class, commonly used to stain neutral lipids. It is darker in color than either Sudan III or Sudan IV. Synonyms: CI 26125, Solvent red 27, Sudan red 5B. Commercial lots are available certified by the Biological Stain Commission.

Oil red O for neutral lipids. Traditionally oil red O was applied as a saturated solution in 70% ethanol. Stronger staining is obtained with the Lillie modification, using a super‑saturated solution in an isopropanol-water mixture containing dextrin as a stabilizer. The uncharged hydrophobic dye molecules move into fat (in adipose tissue and in some diseased cells), but not into polar lipids such as those of the mitochondria or myelin.

Oligonucleotide
. A short sequence of nucleotides, typically 10 to 50 base pairs in length.

Orange G. A small acid dye of the monoazo class, used as a cytoplasmic counterstain, alone or in combination with other anionic dyes as in the Papanicolaou stain in cytology and various histological polychrome stains. Synonyms: CI 16230, Acid orange 10. Commercial lots are available certified by the Biological Stain Commission.

Orange II. A small acid dye of the monoazo class, most often used as a more yellow substitute for orange G in polychrome stains. Synonyms: CI 15510, Acid orange 7, tropaeolin OOO. Commercial lots are available certified by the Biological Stain Commission.

Orcein. A complex mixture of related dyes, some large in size with extensive conjugated systems, the mixture having a deep purple color. Originally synthesized from orcinol found in lichens, but all commercial dye lots today are derived from synthetic orcinol. Currently most commonly used in histology for demonstrating elastin, but also used to stain hepatitis B antigen and plant chromosomes. Synonyms: CI 1242 (ed. 1), Natural red 28. Commercial lots are available certified by the Biological Stain Commission.

Over-dehydration. The removal of bound water molecules from a specimen, causing shrinkage and unwanted chemical interactions (molecular dehydration and hydrophobic inversions) among adjacent, previously insulated, molecules.

Papanicolaou. George N. Papanicolaou, MD, PhD (1883–1962). Born in Greece, and educated there (MD) and in Germany (PhD), he was, in turn, a physician, zoologist, marine biologist, rug salesman, researcher at Cornell Medical School (NY), and endocrinologist and Professor of Anatomy. However, he is best known for founding the field of vaginal exfoliative cytology and developing training programs for cytotechnologists. The Pap smear and Papanicolaou stain are his legacy.

Papanicolaou stain. A sequential stain: hemalum, followed by orange G and phosphotungstic acid (PTA), and then eosin Ylight green SF and PTA. The stain was developed empirically by Papanicolaou to distinguish the various cell types in normal and abnormal human vaginal smears. It is also used on other smear preparations made for diagnostic purposes. Light green SF tends to fade and may be replaced with the more stable fast green FCF.

Paraffin
(wax). A mixture of long-chain (C20–C40) aliphatic hydrocarbons whose melting point is above room temperature; histological paraffin wax also contains resins or other additives to facilitate sectioning and ribboning. See also: embedding.

Paraformaldehyde. An insoluble, polymerized form of formaldehyde that can be returned to monomeric form by treatment with mild alkali. An aqueous fixative solution made with paraformaldehyde does not contain methanol, which is present at low concentration in a solution made by diluting formalin

Pararosaniline. A basic dye component of basic fuchsine, in the aminophenylmethane class. Pararosaniline is also commercially available as a single dye, as either the chloride or the acetate salt. Most commonly used to prepare Schiff's reagent. An acidified ethanolic solution binds to aldehyde groups produced in tissues by oxidation of neutral mucosubstances or partial acid hydrolysis of DNA, providing an alternative to Schiff reagent in the PAS or the Feulgen reaction. Diazotization of the three amino groups of pararosaniline gives hexazonium pararosaniline, which is a useful trapping agent in enzyme histochemistry. Synonyms: CI 42500, Basic red 9, magenta O, parafuchsin. Commercial lots are available certified by the Biological Stain Commission.  See also:  Basic fuchsine, special for flagella.

PAS.  See periodic acid Schiff.

Penetration. Diffusion of a liquid (fixative or processing fluid) into a tissue specimen. The rate of penetration is dependent upon whether the tissue is fresh or fixed, on the molecular size of the penetrating fluid, and on environmental factors such as agitation of the fluid, temperature, and use of vacuum. All other factors being equal, the rate of diffusion decreases as the diffusion pathway increases. A large specimen may take days or weeks before a fixative penetrates to its center.

Periodic acid Schiff (PAS). A histochemical technique in which adjacent –OH groups of neutral sugars (especially fucose, galactose, glucose and mannose) are oxidized to aldehydes by periodic acid. The aldehyde groups combine covalently with subsequently applied Schiff’s reagent, giving a pink to bright red product.  Proteoglycans are PAS‑negative because their acid sugar units resist oxidation by periodic acid. PAS-positive materials include basement membranes, cellulose, collagen, glycogen, and several types of mucus.

Perls. Max Perls (1843–1881) was a German pathologist who, in 1867, published a histochemical test for detecting Fe3+ in tissues by treatment with acid and potassium ferrocyanide.  Perls’ method detects hemosiderin but not hemoglobin, because the iron in heme is not released by treatment with acid. The visible product of this reaction is Prussian blue.

pH. The logarithm (to base 10) of the reciprocal of the concentration (in molar terms) of hydrogen ions (protons) in water that contains dissolved substances:  pH = −log[H+]. Solutions with low pH are acidic; those with high pH are alkaline. The pH of perfectly pure water is 7.0. Ordinary distilled or otherwise purified water has pH 5-6, owing to acidification by carbon dioxide taken up from the atmosphere. Because of the logarithmic nature of the scale, a solution at pH 2 is ten times more acidic than one at pH 3, and a hundred times more acidic than one at pH 4.

Phloxin B. A red lipophilic acid dye, in the xanthene class, with a moderately large conjugated system. Sometimes used in combination with eosin Y following hemalum; also followed by tartrazine to demonstrate dense granular cytoplasmic inclusions. Synonyms: CI 45410, Acid red 92, phloxine. Commercial lots are available certified by the Biological Stain Commission.

Phosphomolybdic acid (PMA). The solid compound H3PO4.12MoO3.24H2O. It dissolves in water to give an acidic solution containing large [PMo12O40]3− anions. It is used in trichrome staining methods. PMA is very soluble in water and also in ethanol. Synonyms: dodecamolybdophosphoric acid, molybdophosphoric acid.  

Phosphorescence. This is similar to fluorescence, but there is a longer time interval between the absorption of the exciting light and the radiation of the emitted light. Even when the delay lasts only for microseconds, electronic instruments can separate nonspecific background fluorescence from the emission of a phosphorescent label.

Phosphotungstic acid (PTA). The solid compound H3PO4.12WO3.24H2O. It dissolves in water to give an acidic solution containing large [PW12O40]3− anions.  Above pH 2, the anions in an aqueous solution aggregate into larger species, which decompose to phosphate and tungstate as the pH increases from 7 to 8.  PTA is used in trichrome staining methods, in the Papanicolaou stain, and as a contrast stain for electron microscopy. PTA is very soluble in water; and also in ethanol, in which the [PW12O40]3− anion is stable. Synonyms: dodecatungstophosphoric acid, tungstophosphoric acid.

Phosphotungstic acid hematoxylin (PTAH). This staining solution, introduced by Mallory in 1897, contains red and blue metal-hematein complexes. After 12–24h in PTAH, nuclei and muscle striations are blue; collagen fibers and bone matrix are red or orange. Various pre-staining treatments (iodine, dichromate, ferric salts, permanganate oxidation, oxalic acid) are commonly used, to enhance blue staining of fibrin deposits and neuroglial scar tissue. 

Physical development. One of several methods used to precipitate colloidal silver on submicroscopic particles (a few atoms) of the metal deposited at specific sites in a specimen. The tiny “nuclei” of Ag0 catalyze reduction of Ag+ in a physical developer, which is a solution containing a silver salt, a reducing agent, and other compounds that prevent the reduction from occurring in the solution.  This type of amplification technique was invented for intensifying pale images in black-and-white photography. The word physical seems inappropriate.

Picric acid. A small acid dye of the nitro class. Used in polychrome stains as the cytoplasmic staining component in mixtures with acid fuchsine (Van Gieson’s stain) or with other dyes, including aniline blue and sirius red F3B. Has also been used as a coagulative fixative, notably in Bouin’s fluid. Picric acid is conveniently kept as a saturated aqueous solution (1.28% w/v); it is more soluble in ethanol (8.3%) and other organic solvents. Synonyms: CI 10305, trinitrophenol. Hazard warning: solid picric acid must be stored under water because it explodes if its temperature reaches 300°C, which could arise from friction of dry powder in the thread of a screw-cap jar.

Pigment. A colored, insoluble substance. It may be either organic (e.g. a formazan) or inorganic (e.g. Prussian blue). Confusingly, the word is also applied to colored substances in animals and plants (e.g. hemoglobin, chlorophyll, melanin), some of which are soluble.  

Plasma. Blood without cells, obtained by preventing coagulation and then removing the cells by centrifugation. Plasma contains fibrinogen, albumin and globulins.  Cf. Serum.

Plastic embedding medium. An embedding medium generated by the infiltration of tissues with a liquid monomer, which is polymerized in situ to form a solid matrix. These media are used to preserve tissue fine structure, permit cutting of thin sections, and to facilitate the microtomy of hard structures. Some plastic media (e.g. methyl methacrylate) are routinely removed from tissue sections prior to staining. Other media – e.g. glycol methacrylate/GMA, and epoxy resins – are crosslinked and cannot easily be dissolved away. When plastic media are present during staining, protocols devised for paraffin or cryostat sections often require adjustment. Synonym: resin.
 
Polar.
A molecule is polar because it forms a dipole: electrons constituting the bonds between its atoms are unevenly distributed; one end of the molecule is relatively electropositive and the other end relatively electronegative. In histotechnology the electronegative atom is most frequently oxygen or nitrogen.

Polar solvent. A liquid miscible with water, and capable of dissolving ionized substances.

Polarizability
. The ease with which a dipole moment can be induced in a molecule or group  in the presence of an electric field, such as that due to the dipole of another molecule or group.

Polyanions. Polymeric molecules with repeating units that carry negative charges. Examples include DNA (whose anionic groups are phosphates) and glycosaminoglycans (GAGs), (whose anionic groups are carboxylate and/or sulphate.

Polychrome. (1) Of staining: the result of staining various cell or tissue structures in different colors, achieved by one stain or several; Romanowsky stains thus give rise to polychrome staining of peripheral blood smears. Synonyms: polychrome or polychromatic staining. (2) Of methylene blue: To create, by oxidation of this dye, a series of closely related thiazine dyes (azure A, azure B and azure C, thionine, methylene violet Bernthsen) termed polychrome methylene blue, which mixture provides polychrome staining.

Polyclonal. Describes an antibody composed of numerous monoclonal antibodies (MABs) to different epitopes in an antigen. The MABs are derived from an indefinite number of non-identical antibody-producing cells. An antiserum is polyclonal.

Polynucleotide. A strand of DNA or RNA composed of more than 50 nucleotides.

Ponceau 2R. An acid dye of the monoazo class, with colored anions of moderate size, used in some trichrome staining methods. It is soluble in water, and slightly soluble in ethanol. Synonyms: CI 16150, acid red 26, probably also ponceau de xylidine, xylidine ponceau 3RS.

Preservation.
Protection of s from destruction by either making the environment hypertonic with sugar or salts, by changing their molecular structure (fixation), or by freezing.

Primary antibody or antiserum. In immunostaining, the antibody or antiserum that combines specifically with the antigen of interest. Except in the direct immunofluorescence technique, a primary antibody or antiserum is unlabeled.
 
Primary structure. The amino acid sequence of a protein or polypeptide. See conformation.

Probe
. A fluorescent compound used for vital staining. Synonym: fluorescent probe.

Processing fluid. Any of various solvents used to prepare fixed, aqueous tissue specimens for infiltration with non-aqueous embedding media. Most typically, processing fluids include a graded series of ethanol (70%, 80%, 95% and 100%) as dehydrants followed by a clearing agent.

Progressive staining. Selective coloration of a specimen resulting from the staining of an initially colorless tissue preparation until the required staining intensity and staining pattern is achieved. Romanowsky stains are usually used progressively, with staining continued until purple nuclei are obtained. Contrast with regressive staining.

Prokaryotic cell. A cell in which the DNA is not contained in a membrane-bound nucleus. Archaea and bacteria are prokaryotes. Contrast with eukaryotic cell.

Protargol. The trade-name of a silver proteinate formerly used as a topical antiseptic (strong silver protein of pharmacopeias). It is a pale brown powder containing ~8% silver. It is used in Bodian's protargol stain for nerve fibers and also in staining methods to show internal structures of ciliated protozoa. Preparations effective for staining are certified and designated protagol-S by the Biological Stain Commission. Dark brown silver proteinates containing ~20% silver (mild silver protein of pharmacopeias) are sometimes sold as protargol but are not suitable for use in staining methods for either nervous tissue or protozoa.  

Prussian blue. This inorganic pigment is produced as the reaction product in the Perls procedure for demonstration of tissue Fe3+. Prussian blue is the ferric salt of ferrocyanide, an anionic metal coordination complex, Fe(CN)64-.

Prussian blue method for iron in tissue. Exposure to acid releases the iron of hemosiderin and ferritin (but not that of hemoglobin) as Fe3+ ions, which react with ferrocyanide ions to form a blue pigment Prussian blue. In the commonly used technique of Perls, sections are immersed in 2% potassium ferrocyanide in 0.2M HCl for 30 min, usually followed by a red counterstain for cell nuclei. See also Turnbull's blue for ferrous iron.

Puchtler
. Holde Puchtler (1920–2006). Born in Germany, Dr. Puchtler was a professor at the Medical College of Georgia (USA). She wrote numerous articles on histochemistry, was one of the first to use molecular models to study staining mechanism, and developed the most selective stain for amyloid.

Puchtler's alkaline Congo red for amyloid. A progressive staining method using a freshly made solution of Congo red in 50% ethanol with 0.43M NaCl and 2.5mM NaOH. Puchtler and her colleagues devised this method in 1962, following principles of dyeing of cellulose textiles such as cotton. Selectivity can be attributed to the inhibition of acid dyeing of most proteins by the salt and alcohol, with the amyloid staining occurring due to hydrogen bond formation between the amino groups of the rod-shaped dye molecules and sites within the grooves of the amyloid β-sheets. 

Pyranose. A sugar with a ring structure of five carbons and one oxygen atom, so that it can be thought of as a derivative of pyran, C5H6O.

Pyronine B. See pyronine Y. Synonym: CI 45010, pyronin B. Commercial lots are available certified by the Biological Stain Commission.

Pyronine Y. This and pyronine B are closely related basic dyes in the xanthene class, primarily used with methyl green to differentiate DNA from RNA. Synonyms: CI 45005, pyronin G, pyronin Y. Commercial lots are available certified by the Biological Stain Commission. See also methyl green-pyronine stain.

Quantum dots. Tiny crystals of semiconductor compounds that absorb UV and efficiently fluoresce at visible wavelengths that increase with crystal size. A quantum dot for immunohistochemical labeling has a CdSe core (1.5–2.5 nm diameter) surrounded by a ZnS layer about 1 nm thick, which increases stability and fluorescence efficiency. Reactive organic compounds attached to the ZnS shell permit covalent bonding to proteins. Advantages over conventional fluorescent labels are greatly reduced fading and the availability of dots that emit different colors in response to a single exciting wavelength.

Quaternary structure. The assemblage of two or more molecules (usually proteins) possessing tertiary structure to form a highly complex topological shape. See also conformation.

Quenching. (1) Of fluorescence: the non-occurrence of expected fluorescence, because electronic excitation energy is not re-emitted but rather transformed (e.g. into heat) or transferred (e.g. to solvent, tissue, or other dyes). (2) Of tissues: fast cooling of a biological specimen in a suitable medium (e.g. propane slush or liquid nitrogen) prior to cryotomy.

Quinhydrone. The darkly coloured substance formed when hydroquinone is half-oxidized to quinone; formed by hydrogen bonding of hydroquinone to p-quinone.

Regressive staining. Selective coloration of a specimen resulting from differentiating an initially over-stained tissue preparation until the required staining intensity and staining pattern is achieved. Harris’s hematoxylin is used regressively in many histopathology laboratories. Contrast with progressive staining.

Renaturation, renature. Return a denatured molecule back to its native or near native conformation.

Reserpine. An alkaloid from Indian snakeroot (serpentine wood), Rauwolfia serpentina that depletes cells and nerve-endings of biogenic monoamines (dopamine, noradrenaline etc). Administration of reserpine is a negative control test for histochemical staining of monoamines in organs taken from laboratory rodents.
 
Resin. See plastic embedding medium.

Resolution. The shortest distance apart for two different points to be seen as visibly separate. With an ideal light microscope using an apochromatic oil immersion objective of high numerical aperture, diffraction limits this separation to about 200 nm (0.2 μm). In routine work there is blurring of detail in areas less than 1 μm across, but strongly stained smaller objects, such as bacteria, may nevertheless be seen. Ultraviolet microscopy (seldom used) and confocal microscopy provide resolution to 100 nm, and several types of super-resolution microscopy allow resolution to 20 nm. The electron microscope provides resolution to the level of macromolecules (1 nm) but is poorly suited to the collection of histochemical data. The atomic force microscope can image individual atoms (0.1–0.5 nm) on some surfaces.

Resonance. The conception of rapid alternation of different types of bond within a molecule. For example, the traditional representation of the benzene ring shows alternating double and single bonds, but in reality all 6 bonds are equivalent. See also delocalized π-electrons.

Resorcinol-fuchsine for elastin.  See Weigert’s resorcinol-fuchsine for elastin.

Rhodamine B. A xanthene fluorochrome, which in neutral aqueous solution is largely present as a zwitterion of lipophilic character, whilst being a cation of lipophilic character at acid pH. Despite having an extensive range of reported applications in histology (e.g. as a green fluorescent stain for bacteria and lipids), and as a vital stain for lysosomes and other acidic organelles, no single staining method is widely used. Soluble in water and ethanol. Synonyms: CI 45170, Basic violet 10. Commercial lots of high dye content are available, usually containing a significant dye contaminant. Note: several modern staining manuals have confused this dye with rhodanine, a completely different compound.

Ribboning. In microtomy, the adhesion of adjacent paraffin sections to one another to form a continuous strip, or ribbon.
 

Ribosome. A granule in the cytoplasm, containing much RNA, which is the site of translation from messenger RNAs to proteins. Ribosomes are attached to membranes of rough endoplasmic reticulum and occur also as circular aggregates called polyribosomes. The RNA of ribosomes (rRNA) accounts for the cytoplasmic basophilia of plasma cells, neurons, and other cell types that synthesize large amounts of protein.

Romanowsky. Dmitri Leonidovich Romanowsky (1861–1921) was a Russian physician.  In 1891, in parallel to similar work by Malachowski, he developed a staining method for blood cells and malaria parasites, using a combination of eosin Y and aged (polychromed) methylene blue solutions, and so initiated the development of the eponymous Romanowsky stain. Synonym: Romanowski.

Roma­nowsky-Giemsa effect. This refers to purple staining of nuclear chromatin and certain cytoplasmic granules by a Romanowsky stain. This contrasts with the azure (blue) staining of blast cell and lympho­cyte cytoplasms, and the red staining of eosinophil granules and erythrocytes.

Romanowsky stain. A family of techniques using unstable solutions of eosin Y and polychrome methylene blue, developed for staining the cell-types of blood and bone marrow, and for identifying protozoa, especially malaria parasites.  In fact, the only necessary dye components of Romanowsky stains are azure B and eosin Y. Development of Romanowsky stains was initiated by Malachowski and Romanowsky who independently published the first such methods in 1890–1891. Subsequent work was carried out by, amongst others, Giemsa, Leishman and Wright. The mechanism of action involves acid dyeing by eosin, basic dyeing by azure B, and the formation of a complex between eosin and azure B in certain sites, giving rise to the Romanowsky-Giemsa effect.

Rose Bengal B. A deep pink acid dye, in the xanthene class, dianionic above neutral pH. Used to stain microorganisms in soil and sediment, and in histology as a cytoplasmic counterstain and for demonstrating cell inclusions. Synonyms: CI 45440, Acid red 94, rose Bengale. Commercial lots are available certified by the Biological Stain Commission.

Saffron. Naturally occurring yellow substance obtained from plant materials such as saffron (Crocus sativus) flowers and wongshy (Gardenia jasminoides) fruit. Lots may contain the yellow compounds crocetin, a polyene carboxylic acid; together with its glycosyl esters, the crocins. Soluble in water, especially if alkaline, and in alcohol. Used in the Movat pentachrome stain. Synonyms: CI 75100, Natural yellow 6. See also Natural dyes.

Safranine O. A red basic dye in the azine class. Commercial lots contain a mixture of 3 or more related compounds, one of which is lipophilic and the others mildly hydrophilic. Used in microbiology to stain bacteria and bacterial spores; in histology for nuclei and glycosaminoglycans, and in botany as a stain for lignin, cuticle and nuclei, followed by light green SF or fast green FCF (for cellulose and cytoplasm). Synonyms: CI 50240, Basic red 2, safranin O or T. Commercial lots are available certified by the Biological Stain Commission.

Saline. Immunological reagents are usually dissolved in a solution of sodium chloride, 0.7–0.9% in a buffer (such as 0.03 M phosphate) at pH 7.2–7.4. Phosphate-buffered saline is commonly called PBS. As a solvent in immunostaining procedures, saline may optionally also contain a surfactant to enhance penetration of immunoglobulin reagents, and also an indifferent protein (such as albumin or casein) to compete with the antibody for nonspecific (weak) binding to substances other than the intended antigen. PBS is not always the ideal diluent, especially for monoclonal antibodies, which often give superior results when applied at a pH above and below the usual range, without added NaCl.

Salt. A compound in which a metal or other cation is neutralized by an anion.
 

Salting out. Precipitation of an ionic compound (such as a dye) by adding an excess of one of its ions, such as Na+ or Cl, to the solution. Also applied to precipitation of protein by addition of an inorganic salt to its solution (see albumin, globulin).
 
Schenk. Eric A. Schenk (1927–1993) was a pathologist at the University of Rochester Medical Center, and a Trustee and Secretary of the Biological Stain Commission. He co-authored (with Charles Churukian) several papers concerning standardized staining procedures.

Schiff. Hugo Schiff (1834–1915). German chemist who lived most of his life in Italy and wrote a series of papers in French and German describing his extensive experiments with reactions between amines and aldehydes. Several of his protocols became analytical methods in histochemistry. See Schiff's reagent.

Schiff’s reagent. A reagent containing a primary amine group that is used to selectively stain aldehydes. Schiff’s reagents are made from dyes which in some, but not all, cases have been decolorized to a “leuco" form by reaction with sulphur dioxide. Upon reaction with aldehydes, a colored final reaction product is generated. The most commonly used Schiff’s reagent is generated from the dye basic fuchsine. Synonym: Schiff reagent.

Schmorl. Georg Schmorl (1861–1932) was a German pathologist who made several innovations in microtechnique including fixative mixtures, decalcifying fluids and staining methods such as a picro-thionine procedure for demonstrating bone canaliculi. His book, Die pathologisch-histologischen Untersuchungs-methoden had 15 editions, the last in 1928.

Schmorl’s picro-thionine for bone canaliculi. Frozen or nitrocellulose sections of bone are strongly stained with aqueous thionine that has been made alkaline by addition of ammonium hydroxide, resulting in basic dyeing of the anionic proteins. Sections are then immersed in aqueous picric acid, which forms a water-insoluble salt with thionine. Differentiation in 70% ethanol removes most of both dyes, leaving the dark brown precipitate in the bone lacunae and canaliculi, residual yellow picric acid staining of bone matrix, and red-purple (thionine  metachromasia) cartilage and nuclei. 

Scott's tap water substitute. A mildly alkaline buffer made with magnesium sulphate and potassium bicarbonate. Used as a bluing agent with hemalum.

Secondary antiserum. A secondary antiserum is one that combines with the Fc segments of previously applied primary antibody molecules whose Fab segments are bound to antigenic sites in a specimen. The secondary antiserum is labeled in most, but not all, immunostaining techniques.

Secondary structure. The folding of a macromolecule to form structural motifs such as α‑helices, ß-sheets and hairpin loops. See conformation.

Sectioning
, see microtomy.

Selectivity of staining. The degree to which a target structure or substance is stained compared with non-target background material.

Sensitivity of staining. The degree to which a staining procedure can detect small amounts of a cell or tissue component. Fluorochromes are usually more sensitive than otherwise similar non-fluorescent dyes.

Serum. Blood plasma from which the fibrinogen has been removed. Whole blood is centrifuged, after clotting and shrinkage of the clot. Mammalian sera contain about 8% w/v protein, consisting of approximately equal proportions of albumin and globulin.

Shrinkage (of specimens). Occurs in insufficiently fixed specimens when aggressive dehydrants (e.g., ethanol), remove bound water from macromolecules, allowing intra- and inter-molecular interactions (ionic and hydrogen bonding, as well as van der Waals forces) between once-insulated chemical groups.

Silver staining. This entails conversion of Ag+ derived from the staining solution into metallic silver microcrystals in the tissues. Such procedures generate a range of colors from yellow to brown to black. Staining mechanisms are diverse, including direct reduc­tion of Ag+ by tissue constituents, and catalysis of the reaction between the silver ions and reducing agent present in the staining reagent by tissue constituents. For more commonly used silver stains, see Bielschowsky's silver, Bodian's protargol stain for nerve fibers, Churukian-Schenk method for argyrophil granules, Grimelius argyrophil stain, Gomori's method for reticular fibers, Grocott's methenamine silver for fungi, Steiner's argyrophil stain for spirochetes, Helicobacter and Legionella, Von Kossa silver for bone, and Warthin-Starry for spirochetes. See also argentaffin reaction, argyrophil reaction, and physical development.

Sirius red F3B. One of the largest acid dyes used in histology, this polyazo dye has an extensive conjugated system. It is extremely hydrophilic with six sulfonate groups. Most commonly used as a substitute for acid fuchsine in the Van Gieson stain and as a substitute for Congo red in amyloid stains. Synonyms: CI 35780, Direct red 80, chlorantine fast red with various letter designations, Sirius fast red; do not confuse with Sirius red 4B (CI 28160, Direct red 81).

Sirofluor. A compound present in commercial aniline blue that imparts yellow fluorescence to the β1,3‑glucan callose. It is also available as a pure product deliberately synthesized as a stain. Synonym: 4'4-[carbonyl bis(benzene 4,1-diyl) bis(imino)]bisbenzensulphonic acid disodium salt.   
 

Smear.
A specimen preparation in which a suspension of cells is spread evenly and thinly across a glass slide. Such preparations are usually cell monolayers. Typically used for blood, gynecological specimens, and aspirates (e.g., pleural fluid). Thicker blood films, used when searching for parasites, must, before fixation and staining, be treated with water (a hypotonic medium) to release hemoglobin from the erythrocytes.

Sol. A colloidal dispersion of an inorganic substance, such as gold, ferric hydroxide or sulfur, in a “solvent”, which is usually water. Unlike a gel, a sol is a mobile liquid. The particles suspended in a sol are charged; the balancing opposite charge is carried by the polar solvent molecules surrounding each particle.

Solvent dyeing. An industrial term, describing coloration of organic solvents and a variety of other non-polar materials (e.g., hydrocarbon fuels, lacquers, lubricants, hydrophobic polymers, and waxes) with a lipophilic non-ionic dye; or occasionally with an acid dye with a very lipophilic counter ion. In the Colour Index these dyes fall into the CI Solvent dye application class. 
 
Specificity of staining. The staining of a single cell or tissue component, and no other. Specific stains are much less common than many believe. Even a reagent such as a labeled antibody often gives positive artifacts, due to non-immunological staining mechanisms. Contrary to common parlance, there is no such thing as “quite specific”; that phrase and its equivalents actually refer to selective.

Spinner. See cytocentrifuge.

Staining. Visual labeling of some cell or tissue entity (anything from a molecular fragment to a physiological process) by attaching or depositing in its vicinity a visual marker of characteristic color or shape. The term most commonly (but not exclusively) refers to the use of a solution of a dye to color a biological specimen. The transfer of dye from solution to specimen does not usually require any change in covalent bonds or oxidation state, although there are exceptions (e.g., Schiff reagent). Other types of staining include localization of pigments and mineral deposits in tissues, enzyme histochemistry and immunohistochemistry

Steiner argyrophil stain for spirochetes. Spirochetes (such as Borrelia, Helicobacter, Leptospira, and Treponema) and some small bacilli (including Bartonella, Campylobacter, and Legionella) are not easily detected by staining with dyes, especially in sections of tissues. Instead, this argyrophil silver staining technique is used. In the method of Steiner and Steiner (1944) the sections are treated first with a solution of uranyl nitrate and then with silver nitrate at 56°C. Invisible catalytic sites (Ag0) are subsequently amplified by physical development. The initial treatment makes the organisms favourable for reduction of Ag+ to Ag0. More recent variants use other metal salts in the sensitizing step, and microwave heating to accelerate some or all of the three components of the procedure.  See also: Warthin-Starry argyrophil stain for spirochetes.

Stokes shift.  The wavelength difference (in nanometers) between maximum absorption and maximum emission of a fluorescent or phosphorescent substance.

Structure parameter. A number used to specify or model a structural/physico­chemical feature of a staining reagent. Thus the size of a non-ionic dye could be modeled by molecular weight, the extent of its conjugated ­system by conjugated bond number, and its hydrophilic or lipophilic nature by a log P value.

Substantivity. See Affinity.

Substrate. A chemical entity with which a reagent interacts. (1) Part of the specimen that a dye or other staining reagent reacts with. (2) In enzyme histochemistry the substrate is present in the incubating solution, and is catalytically transformed by an enzyme in the specimen; a product of this transformation is trapped by another chemical reaction, which yields a visible final reaction product.

Sudan black B. A non-ionic dye in the disazo class, used to stain both neutral and acidic lipids (e.g., phospholipids); cholesterol (not ordinarily colored by solvent dyes) can also be stained after bromination of the specimen. Synonyms: CI 26150, Solvent black 3. Commercial lots are available certified by the Biological Stain Commission which are of high dye content, consisting mostly of two isomers and a number of minor contaminants.

Sudan black B for neutral lipids and phospholipids. Phospholipids not extracted by tissue processing solvents are not stained by oil red O or other red solvent dyes such as Sudan IV. However Sudan black B dissolved in aqueous ethanol (1:7 v/v) or propylene glycol does stain them, indeed more strongly than neutral fats. In frozen sections, this method stains all lipids except cholesterol. The partitioning of this strongly lipophilic dye into the neutral lipids is expected, but the peculiar advantage of Sudan black B for staining phospholipids is not understood. 


Sudan dye
. A lipophilic non-ionic dye. Derivation of the term: from the commercial names of many effective lipophilic dyes, which take the form “Sudan X” (where X = III, black B, Red 5B, etc.). Oil red O also belongs in this group of solvent dyes.

Sudan III. A non-ionic dye in the disazo class, sometimes used as a stain for fats and other hydrophobic materials, e.g. suberin in plants, and fat droplets in cryosections, blood and sputum smears, and feces. Sudan III is soluble in ethylene glycol, slightly soluble in ethanol, and insoluble in water. Synonyms: CI 26100, Solvent red 23, fat ponceau G. Commercial lots are available certified by the Biological Stain Commission; they have high dye content, but contain a number of minor contaminants.

Sudan IV. A non-ionic dye in the disazo class, sometimes used to stain lipids, e.g. atherosclerotic lesions and fat droplets in cultured cells. It is slightly darker than Sudan III. Also used botanically to stain a variety of other hydrophobic materials such as suberin and oil droplets. Sudan IV is highly soluble in ethylene glycol, slightly soluble in ethanol, and insoluble in water. Synonyms: CI 26150, Solvent red 24. Commercial lots are available certified by the Biological Stain Commission; they have high dye content, but contain a number of minor contaminants.

Sulfoamino. The radical –NHSO3H

Synthetic dye. A colorant synthesized by humans, from organic precursor compounds derived originally from coal tar, but currently from crude petroleum. Most dyes in current use are synthetic. See also: natural dye.  

Tartrazine. An acid dye of the monoazo type, of moderate size, used in the phloxin-tartrazine stain to demonstrate fibrin, and intracellular objects such as viral inclusion bodies. Tartrazine is highly water soluble, and slightly soluble in ethanol. Synonyms: CI 19140, Acid yellow 23. Commercial lots can be of high dye-content and consist mainly of a single compound. 

Tertiary structure. Further folding of secondary structures of protein molecules into barrels, globular shapes, rods, etc. See conformation.

Tetrazolium salt. Colorless compound containing one (monotetrazolium, e.g. MTT tetrazolium) or two (bistetrazolium, e.g. nitro blue tetrazolium) cationic five-membered ring(s), each comprising four nitrogen atoms and one carbon atom. Tetrazolium salts are converted into colored formazans by oxidation. Tetrazolium salts are used as trapping agents (2) in enzyme histochemical methods to localize dehydrogenases.

Theory of staining
. A useful story for guiding and justifying practical actions. Scientific theories are preferably predictive, couched in suitably sober language (or, even better, in mathematical symbols), and are amenable to disproof.

Thioflavine T. A fluorochrome widely believed to be a small basic dye of the monobenzothiazole class. Dye lots are actually mixtures of mono-, di- and tribenzothiazole dyes. The latter two species are larger molecules, whose rod-shaped characters facilitate their selective binding to amyloid. Soluble in water and ethanol. Synonyms: CI 49005, Basic yellow 1.

Thioflavine T for amyloid. Paraffin sections are first stained with hemalum to prevent the nuclear fluorescence due to the subsequent application of thioflavine T, which is from an acidified aqueous solution, then differentiated in dilute aqueous acid. With excitation by UV or blue light, the fluorescent emission is white to yellow. The staining of amyloid is probably due to adherence of rod-shaped components of the dye to sites within the grooves of the amyloid β-sheets. A positive control section known to contain amyloid must also be stained, because some normal intracellular granules, such as those of mast cells, are also stained by basic dyeing. 

Thionine. A small basic dye, in the thiazine class, used to stain basophilic structures such as mast cell granules and Nissl substance. Used as a botanical stain with metachromatic properties. Can be used to prepare a blue variant of Schiff’s reagent. Thionine is soluble in water and ethanol. Synonyms: CI 52000, Lauth’s violet, thionin. Commercial lots are available certified by the Biological Stain Commission

Tissue processing. The sequence of steps starting from a fresh specimen and culminating in the formation of an embedded tissue block. Major stages usually include fixation, dehydration, clearing and infiltration with paraffin wax or other embedding media. Tissue processing may be accomplished by hand, but in larger laboratories it is achieved using a mechanical device (tissue processor).

Tissue processor. A mechanical device that sequentially exposes specimens to the various fluids involved with tissue processing. The device may allow application of pressure, vacuum, heat or agitation to the fluids to hasten processing time.

Tissue section. A thin slice cut off the face of a specimen, usually by use of a microtome. A paraffin section comes from a specimen embedded in paraffin wax, a plastic section comes from one embedded in a plastic embedding medium, and a frozen section has been cut from a frozen tissue block, usually with a cryostat. Before staining, paraffin sections are dewaxed and methylmethacrylate plastic sections have their resin removed. Glycol methacrylate and epoxy plastic sections are routinely stained with the resin remaining in situ. Contrast with smear.

Toluidine blue. A small basic dye, in the thiazine class, used to stain a wide variety of basophilic structures, such as mast cell granules and cell nuclei. Due to its metachromasia it has been exploited in staining sulphated mucins. The dye has been used as an oversight stain with frozen sections and with plastic sections. Toluidine blue is soluble in water and ethanol. Synonyms: CI 52040, Basic blue 17, tolonium chloride, toluidine blue O – not to be confused with toluylene blue, which is chemically quite different. Commercial lots are available certified by the Biological Stain Commission

Trapping. (1) A fixation mechanism, involving catching soluble compounds of unaltered nature (e.g. glycogen or insulin) within a mesh of fixed proteins. (2) In enzyme histochemistry, conversion of a freely diffusing intermediate reaction product by reaction with a trapping agent into an insoluble derivative, resulting in its retention in the tissue. For example, phosphate ions released from an organic substrate by a phosphatase can be precipitated as insoluble calcium or lead phosphate by Ca2+ or Pb2+ included in the incubation medium. See also visualizing agent.

Trichrome. This word refers to polychrome (1) staining procedures that give rise to three contrasting colors. It is used principally for methods in which anionic dyes are applied in conjunction with phosphomolybdic acid or phosphotungstic acid to enhance the uptake of different colors into cytoplasm and collagen, as in the trichrome stains of Mallory, Masson and Gomori.

Trichrome stain. A histological oversight stain, with numerous variants, giving contrasting colors to cytoplasms, collagen fibers and nuclei. Nuclei may be initially stained black or dark blue with a cationic metal coordination complex such as Weigert’s hematoxylin or a combination of celestine blue and hemalum. Cytoplasms are then colored red with a small acid dye that stains all proteins non-selectively by acid dyeing. Sections are next treated with phosphotungstic (PTA) or phosphomolybdic acid (PMA), which replaces the small acid dye only in collagen fibers. A second acid dye, of larger size and contrasting color (blue or green), is applied to stain collagen, either from the same solution as the PMA/PTA or afterwards. Finally the stained slides are washed in dilute aqueous acetic acid, which does not extract either the robust nuclear stain (if used) or any of the bound acid dyes. If the nuclear stain is omitted, as in most trichrome techniques other than that of Masson, nuclei are red but easily seen. See Gomori's one-step trichrome, Mallory's trichrome and Masson's trichrome.

Turnbull's blue for ferrous iron.  Stainable iron in animal tissues is present in the ferric, Fe(III), state and is complexed with proteins (ferritin, hemosiderin), from which Fe3+ ions are released by acid treatment. The ordinary staining method is the Prussian blue method for iron in tissue in which Fe3+ ions are precipitated as insoluble blue ferric ferrocyanide (Prussian blue). An alternative method, for which greater sensitivity has been claimed, involves immersing sections in a solution of ammonium sulphide (a reducing agent and precipitant) for up to 2 days; this changes the protein-bound Fe(III), and also any Fe(II) that might be in the tissue from  occupational exposure,  into insoluble  ferrous sulphide.  Subsequent treatment with an acidified potassium ferricyanide solution changes the brown FeS into ferrous ferricyanide (Turnbull’s blue).  The chemistry of the blue pigments is less simple; Prussian and Turnbull’s blues have long been known to be identical compounds! 

Twort. Frederick William Twort FRS (1877–1950), a British bacteriologist, the discoverer of bacteriophages. The eponymous stain demonstrates bacteria in tissue sections.

Twort’s stain. An alcoholic stock solution of neutral red and either light green SF or fast green FCF is diluted with water or acetate buffer (pH 4.9) to make the staining solution, which is stable for only a few hours. After staining for 5–10 minutes, the sections are rinsed in 2% acetic acid in ethanol, dehydrated, cleared and coverslipped. Nuclear chromatin and other basophilic materials are red; collagen and cytoplasm are green. Bone and cartilage matrix take up both dyes and are purple. Twort’s method originally was  a counterstain to follow crystal violet and iodine in the Gram stain for bacteria applied to tissue sections (Gram-positive bacteria are purple an Gram-negative bacteria are red).

Uranin. See fluorescein.

van der Waals forces. Various short-range, non-directed, attractive forces, including the polar Keesom and Debye forces as well as the non-polar hydrophobic London forces  that can hold molecules together. 

Van Gieson. Ira Thompson Van Gieson (1866–1913) was a USA neurologist, neuropathologist and psychiatrist. He introduced his eponymous stain for use in neurohistology in 1889, before the introduction of formaldehyde as a routine fixative.  

Van Gieson’s stain. After staining nuclei with Weigert’s iron-hematoxylin, sections are immersed in a solution of acid fuchsine in saturated aqueous picric acid. This is useful for giving contrasting colors to collagen (red) and muscle fibers (yellow), but the thinner collagenous structures fibers (reticular fibers, basement membranes) are unstained.  

Verhoeff. Frederick Herman Verhoeff (1874–1968) was a USA ophthalmic surgeon and pathologist, who developed the eponymous elastic fiber stain in 1908.

Verhoeff’s elastin stain.  After over-staining in a solution made up from ferric chloride, hematoxylin, iodine and potassium iodide (in 20% ethanol), sections are critically differentiated in aqueous ferric chloride.  Nuclei and elastic laminae and fibers are black. Following this iron hematoxylin stain, counterstaining sections with Van Gieson’s stain to show collagen and muscle in red and yellow respectively is often carried out. Musto’s staining method is a progressive variant of Verhoeff’s in which a more dilute staining solution is applied for a longer time. Synonym: Verhöff’s stain.

Visualizing agent. A reagent used in immunostaining or enzyme histochemistry to convert a colorless intermediate reaction product into a colored final reaction product. For instance, when demonstrating phosphatases, ammonium sulphide is often used to convert colorless lead phosphate into black lead sulphide. Compare with trapping (2).

Vital staining. The application of dyes to living cells, tissues or whole organisms. Uptake of the dye may be entirely due to “simple” physical chemistry, or may be achieved by a biological process such as active transport or some form of endocytosis. In any event, distribution of stain reflects the biochemical composition, or structure or physiological activities of the cell or creature. Although vital staining requires live cells, the resulting coloration may remain in place after death. Fluorescent compounds used as vital stains are often called probes for the organelles or other domains in which they lodge (e.g., neutral red in lysosomes), or for the property or process on which they report (e.g., Evans blue as a viability stain). Probably the most common current applications of vital staining involve the application of dyes to monolayers of cultured cells. In the past, administration of a dye to a whole animal or plant was termed intravital staining, whereas treating freshly excised tissues or cells with a dye was called supravital staining.

Von Kossa’s silver method for calcified material. Sections are immersed in an aqueous silver nitrate solution and left in sunlight or under a bright electric bulb for about 15 minutes. Deposits of calcium phosphate or carbonate are converted to the corresponding insoluble silver salts, which are photochemically reduced to black or brown metallic silver. The sections are then treated with a sodium thiosulfate solution, which removes protein-bound silver ions by complexation – a reaction similar to fixing a black and white photograph. In some variants of the technique, reduction is by a chemical mixture similar to a photographic developer rather than by light. The method with chemical reduction can be applied (over several days) to small (2 mm) pieces of bone. These are then decalcified in formic acid, and paraffin sections are obtained. Note that Von Kossa’s method is a histochemical test for phosphate or carbonate, not for the associated calcium.  See also silver staining.

Warthin-Starry argyrophil stain for spirochetes.  This probably is the most popular silver method for spirochetes (such as Borrelia, Helicobacter, Leptospira, and Treponema) in paraffin sections of tissues.  Immersion in a silver nitrate solution buffered to pH 3.6 at 55°C for 60 minutes is followed by 3–5 minutes of physical development, also at 55°C. The slides are then washed for 5 minutes in hot running tap water, dehydrated, cleared and coverslipped. Bacteria are dark brown to black; tissue components are nonspecifically colored yellow to light brown. See also Steiner argyrophil stain for spirochetes.

Water blue
. See Aniline blue.

Weigert. Carl Weigert (1845–1904) was born in Muensterberg [or Münsterberg], Silesia. Despite his contributions to histochemistry and pathology more generally, he never achieved a chair, perhaps because of his “almost punishable humbleness” or indeed his willingness to argue with the powerful — such as Virchow — and doubtless also because of antisemitism. He was an older cousin of Paul Ehrlich.

Weigert’s hematoxylin. An unstable iron hematoxylin generated immediately prior to use by mixing solutions of acidified ferric chloride with hematoxylin. Ferric ions oxidize hematoxylin to hematein. The major application of Weigert’s hematoxylin is as a nuclear stain when acidic staining solutions will be subsequently applied to the sections, as in van Gieson's stain. Weigert's name is associated also with a chromium-hematein stain for myelin in the central nervous system. See Weigert for biographical information on the originator of these procedures.

Weigert’s resorcinol-fuchsine for elastin. This cumbersome and unpredictable method uses the precipitate formed when basic fuchsine is mixed with water, resorcinol and ferric chloride (or ferric nitrate). The precipitate is dissolved in ethanol or 2-methoxyethanol, acidified with hydrochloric acid. Elastic fibers and laminae in skin and arteries stain brown to purple. A simpler, more reliable method, most likely involving the same staining mechanism, is Gomori's aldehyde fuchsine. See Weigert for biographical information on the originator of this procedure.

Woodstain scarlet. A former trade-name for crocein scarlet, a dye used in Movat's pentachrome stain.

Wright. James Homer Wright (1870–1928) was a USA pathologist. In 1902 he published a technically simple Romanowsky stain for blood cells: a solution of polychrome methylene blue and eosin B in 80% methanol.

Ziehl-Neelsen stain for acid-fast bacteria. This stain for the bacillus that causes tuberculosis was developed in the early 1880s. All bacteria contain nucleic acids, which can bind cationic dyes. Some bacteria, including Mycobacterium tuberculosis, have lipid-rich cell walls that admit cationic dyes only from solutions containing compounds such as phenol that can make holes that are permeable to dye molecules. An aqueous solution of basic fuchsine and phenol, usually heated, imparts a red color to all bacteria. Differentiation in acid-alcohol removes the dye from all but the acid-fast tubercle bacilli. A contrasting counterstain, namely methylene blue, is then applied to colour other bacteria in the preparation by basic dyeing. Variants of the Ziehl-Neelsen method, notably Fite's acid fast stain, are available for M. leprae (which causes leprosy), an organism less acid- and alcohol-fast than M. tuberculosis because its lipid envelope is less resistant to solvents.

Zinc formalin. Any of several formaldehyde solutions that contain a zinc salt (usually zinc sulfate or acetate); used to prevent hydrophobic inversions and to diminish loss of immunoreactivity caused by formaldehyde alone. The Zn2+ ions quickly make proteins and nucleic acids insoluble by forming coordination complexes with arginine, cysteine, cytosine, guanine, histidine, tryptophan, and with phosphate groups. Crosslinking of proteins by the formaldehyde in the mixture occurs more slowly. These fixatives may be buffered or not, and the solvent may be water or a water-ethanol mixture.

Zwitterionic. A zwitterion is an electrically neutral ion with equal numbers of positively and negatively charged groups. All amino acids are zwitterionic, as are some useful buffers such as HEPES. A solution of the dye rhodamine B is largely zwitterionic under physiological conditions, although that dye can be either anionic or cationic depending on the pH of the solution.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Revised 19th January 2019. 

.

.