Safranin (Safranin O or basic red 2) is a biological stain used in

histology Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...

and

cytology Cell biology (also cellular biology or cytology) is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living and ...

. Safranin is used as a counterstain in some staining protocols, colouring

cell nuclei The cell nucleus (pl. nuclei; from Latin or , meaning ''kernel'' or ''seed'') is a membrane-bound organelle found in eukaryotic cells. Eukaryotic cells usually have a single nucleus, but a few cell types, such as mammalian red blood cells, h ...

red. This is the classic

counterstain A counterstain is a stain with colour contrasting to the principal stain, making the stained structure easily visible using a microscope. Examples include the malachite green counterstain to the fuchsine stain in the Gimenez staining technique a ...

in both

Gram stain In microbiology and bacteriology, Gram stain (Gram staining or Gram's method), is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. The name comes from the Danish ...

s and

endospore staining Endospore staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample. Within bacteria, endospores are protective structures used to survive extreme conditions, including high temperatures making them h ...

. It can also be used for the detection of

cartilage Cartilage is a resilient and smooth type of connective tissue. In tetrapods, it covers and protects the ends of long bones at the joints as articular cartilage, and is a structural component of many body parts including the rib cage, the neck an ...

mucin Mucins () are a family of high molecular weight, heavily glycosylated proteins (glycoconjugates) produced by epithelial tissues in most animals. Mucins' key characteristic is their ability to form gels; therefore they are a key component in most ...

and

mast cell A mast cell (also known as a mastocyte or a labrocyte) is a resident cell of connective tissue that contains many granules rich in histamine and heparin. Specifically, it is a type of granulocyte derived from the myeloid stem cell that is a par ...

granules. Safranin typically has the chemical structure shown at right (sometimes described as dimethyl safranin). There is also trimethyl safranin, which has an added

methyl group In organic chemistry, a methyl group is an alkyl derived from methane, containing one carbon atom bonded to three hydrogen atoms, having chemical formula . In formulas, the group is often abbreviated as Me. This hydrocarbon group occurs in many ...

in the ''ortho-'' position (see

Arene substitution pattern Arene substitution patterns are part of organic chemistry IUPAC nomenclature and pinpoint the position of substituents other than hydrogen in relation to each other on an aromatic hydrocarbon. ''Ortho'', ''meta'', and ''para'' substitution * I ...

) of the lower ring. Both compounds behave essentially identically in biological staining applications, and most manufacturers of safranin do not distinguish between the two. Commercial safranin preparations often contain a blend of both types. Safranin is also used as

redox indicator A redox indicator (also called an oxidation-reduction indicator) is an indicator which undergoes a definite color change at a specific electrode potential. The requirement for fast and reversible color change means that the oxidation-reduction e ...

analytical chemistry Analytical chemistry studies and uses instruments and methods to separate, identify, and quantify matter. In practice, separation, identification or quantification may constitute the entire analysis or be combined with another method. Separati ...

Safranines

Safranines are the azonium

compound Compound may refer to: Architecture and built environments * Compound (enclosure), a cluster of buildings having a shared purpose, usually inside a fence or wall ** Compound (fortification), a version of the above fortified with defensive struct ...

s of

symmetrical Symmetry (from grc, συμμετρία "agreement in dimensions, due proportion, arrangement") in everyday language refers to a sense of harmonious and beautiful proportion and balance. In mathematics, "symmetry" has a more precise definiti ...

2,8-dimethyl-3,7-diaminophenazine. They are obtained by the joint

oxidation Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a d ...

of one

molecule A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioch ...

of a ''para''-diamine with two molecules of a

primary amine In chemistry, amines (, ) are compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are formally derivatives of ammonia (), wherein one or more hydrogen atoms have been replaced by a substituent such a ...

; by the condensation of ''para''-aminoazo compounds with primary amines, and by the action of ''para''-nitrosodialkylanilines with secondary bases such as diphenylmetaphenylenediamine. They are

crystalline A crystal or crystalline solid is a solid material whose constituents (such as atoms, molecules, or ions) are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. In addition, macrosc ...

solids showing a characteristic green metallic

lustre Lustre or Luster may refer to: Places * Luster, Norway, a municipality in Vestlandet, Norway ** Luster (village), a village in the municipality of Luster * Lustre, Montana, an unincorporated community in the United States Entertainment * '' ...

; they are readily soluble in water and

dye A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution an ...

red or violet. They are strong bases and form stable monacid

salt Salt is a mineral composed primarily of sodium chloride (NaCl), a chemical compound belonging to the larger class of salts; salt in the form of a natural crystalline mineral is known as rock salt or halite. Salt is present in vast quantitie ...

s. Their

alcohol Alcohol most commonly refers to: * Alcohol (chemistry), an organic compound in which a hydroxyl group is bound to a carbon atom * Alcohol (drug), an intoxicant found in alcoholic drinks Alcohol may also refer to: Chemicals * Ethanol, one of sev ...

ic solution shows a yellow-red

fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...

. Phenosafranine is not very stable in the free state; its

chloride The chloride ion is the anion (negatively charged ion) Cl−. It is formed when the element chlorine (a halogen) gains an electron or when a compound such as hydrogen chloride is dissolved in water or other polar solvents. Chloride salts ...

forms green plates. It can be readily diazotized, and the diazonium salt when boiled with alcohol yields aposafranine or benzene induline, C₁₈H₁₂N₃. Friedrich Kehrmann showed that aposafranine could be diazotized in the presence of cold concentrated

sulfuric acid Sulfuric acid (American spelling and the preferred IUPAC name) or sulphuric acid ( Commonwealth spelling), known in antiquity as oil of vitriol, is a mineral acid composed of the elements sulfur, oxygen and hydrogen, with the molecular formu ...

, and the diazonium salt on boiling with alcohol yielded phenylphenazonium salts. Aposafranone, C₁₈H₁₂N₂O, is formed by heating aposafranine with concentrated

hydrochloric acid Hydrochloric acid, also known as muriatic acid, is an aqueous solution of hydrogen chloride. It is a colorless solution with a distinctive pungent smell. It is classified as a strong acid Acid strength is the tendency of an acid, symbol ...

. These three compounds are perhaps to be represented as ''ortho''- or as ''para''-

quinone The quinones are a class of organic compounds that are formally "derived from aromatic compounds

uch as benzene or naphthalene Uch ( pa, ; ur, ), frequently referred to as Uch Sharīf ( pa, ; ur, ; ''"Noble Uch"''), is a historic city in the southern part of Pakistan's Punjab province. Uch may have been founded as Alexandria on the Indus, a town founded by Alexand ...

by conversion of an even number of –CH= groups into –C(=O)– groups with any necessary rearrangement of double ...

s. The "safranine" of commerce is an ''ortho''-tolusafranine. The first

aniline Aniline is an organic compound with the formula C6 H5 NH2. Consisting of a phenyl group attached to an amino group, aniline is the simplest aromatic amine In organic chemistry, an aromatic amine is an organic compound consisting of an aroma ...

dye-stuff to be prepared on a manufacturing scale was

mauveine Mauveine, also known as aniline purple and Perkin's mauve, was one of the first synthetic dyes. It was discovered serendipitously by William Henry Perkin in 1856 while he was attempting to synthesise the phytochemical quinine for the treatment of m ...

, which was obtained by

Sir William Henry Perkin Sir William Henry Perkin (12 March 1838 – 14 July 1907) was a British chemist and entrepreneur best known for his serendipitous discovery of the first commercial synthetic organic dye, mauveine, made from aniline. Though he failed in trying ...

by heating crude aniline with

potassium dichromate Potassium dichromate, , is a common inorganic chemical reagent, most commonly used as an oxidizing agent in various laboratory and industrial applications. As with all hexavalent chromium compounds, it is acutely and chronically harmful to health ...

and sulfuric acid. Mauveine was converted to ''parasafranine'' (1,8-dimethylsafranine) by Perkin in 1878 by

oxidative Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a d ...

/ reductive loss of the 7''N''-''para''-tolyl group. Another well known safranin is phenosafranine (C.I. 50200, 3,7-diamino-5-phenylphenazinium chloride) widely used as a histological dye, photosensitizer and redox probe.

References

{{Stains Azin dyes Redox indicators Vital stains Phenazines Aromatic amines Quaternary ammonium compounds Chlorides