Safranin
Encyclopedia
Safranin is a biological stain
used in histology
and cytology
. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei
red. This is the classic counterstain in a Gram stain. It can also be used for the detection of cartilage
, mucin
and mast cell
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 the ortho- position of the lower ring. Both compounds behave essentially identically in biological staining applications, and most manufacturers of safranin don't distinguish between the two. Commercial safranin preparations often contain a blend of both types.
Safranin is also used as redox indicator
in analytical chemistry
.
s of symmetrical 2,8-dimethyl-3,7-diamino-phenazine. They are obtained by the joint oxidation of one molecule
of a para-diamine with two molecules of a primary amine; 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 solids showing a characteristic green metallic luster
; they are readily soluble in water and dye
blue or violet. They are strong bases and form stable monacid salt
s. Their alcohol
ic solution shows a yellow-red fluorescence
. Phenosafranine is not very stable in the free state
; its chloride
forms green plates. It can be readily diazotized, and the diazonium salt when boiled with alcohol yields aposafranine or benzene induline, C18H12N3. F. Kehrmann showed that aposafranine could be diazotized in the presence of cold concentrated sulfuric acid
, and the diazonium salt on boiling with alcohol yielded phenylphenazonium salts. Aposafranone, C18H12N2O, is formed by heating aposafranine with concentrated hydrochloric acid
. These three compounds are perhaps to be represented as ortho- or as para-quinone
s. The "safranine" of commerce is a ortho-tolusafranine. The first aniline
dye-stuff to be prepared on a manufacturing scale was mauveine
, which was obtained by Sir William Henry Perkin by heating crude aniline with potassium
bichromate
and sulfuric acid. Mauveine was converted to parasafranine (1,8-dimethyl Safranine) by Perkin in 1878 by oxidative/reductive
loss of the 7-N-para-tolyl group.
Staining (biology)
Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes...
used in histology
Histology
Histology is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining cells and tissues commonly by sectioning and staining; followed by examination under a light microscope or electron microscope...
and cytology
Cell biology
Cell biology is a scientific discipline that studies cells – their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division and death. This is done both on a microscopic and molecular level...
. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
red. This is the classic counterstain in a Gram stain. It can also be used for the detection of cartilage
Cartilage
Cartilage is a flexible connective tissue found in many areas in the bodies of humans and other animals, including the joints between bones, the rib cage, the ear, the nose, the elbow, the knee, the ankle, the bronchial tubes and the intervertebral discs...
, mucin
Mucin
Mucins are a family of high molecular weight, heavily glycosylated proteins produced by epithelial tissues in most metazoans. Mucins' key characteristic is their ability to form gels; therefore they are a key component in most gel-like secretions, serving functions from lubrication to cell...
and mast cell
Mast cell
A mast cell is a resident cell of several types of tissues and contains many granules rich in histamine and heparin...
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
Methyl group
Methyl group is a functional group derived from methane, containing one carbon atom bonded to three hydrogen atoms —CH3. The group is often abbreviated Me. Such hydrocarbon groups occur in many organic compounds. The methyl group can be found in three forms: anion, cation and radical. The anion...
in the ortho- position of the lower ring. Both compounds behave essentially identically in biological staining applications, and most manufacturers of safranin don't distinguish between the two. Commercial safranin preparations often contain a blend of both types.
Safranin is also used as redox indicator
Redox indicator
A redox indicator is an indicator that undergoes a definite color change at a specific electrode potential....
in analytical chemistry
Analytical chemistry
Analytical chemistry is the study of the separation, identification, and quantification of the chemical components of natural and artificial materials. Qualitative analysis gives an indication of the identity of the chemical species in the sample and quantitative analysis determines the amount of...
.
Safranines
Safranines are the azonium compoundChemical compound
A chemical compound is a pure chemical substance consisting of two or more different chemical elements that can be separated into simpler substances by chemical reactions. Chemical compounds have a unique and defined chemical structure; they consist of a fixed ratio of atoms that are held together...
s of symmetrical 2,8-dimethyl-3,7-diamino-phenazine. They are obtained by the joint oxidation of one molecule
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...
of a para-diamine with two molecules of a primary amine; by the condensation of para-aminoazo compounds with primary amines, and by the action of para-nitrosodialkylanilines with secondary bases
Base (chemistry)
For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...
such as diphenylmetaphenylenediamine. They are crystalline solids showing a characteristic green metallic luster
Lustre (mineralogy)
Lustre is a description of the way light interacts with the surface of a crystal, rock, or mineral. The word lustre traces its origins back to the Latin word lux, meaning "light", and generally implies radiance, gloss, or brilliance....
; they are readily soluble in water and dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....
blue or violet. They are strong bases and form stable monacid salt
Salt
In chemistry, salts are ionic compounds that result from the neutralization reaction of an acid and a base. They are composed of cations and anions so that the product is electrically neutral...
s. Their alcohol
Alcohol
In chemistry, an alcohol is an organic compound in which the hydroxy functional group is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms....
ic solution shows a yellow-red fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
. Phenosafranine is not very stable in the free state
Free state
Free state may refer to:* Free state , a loosely defined term used by various states at different times and places to describe themselves...
; its chloride
Chloride
The chloride ion is formed when the element chlorine, a halogen, picks up one electron to form an anion Cl−. The salts of hydrochloric acid HCl contain chloride ions and can also be called chlorides. The chloride ion, and its salts such as sodium chloride, are very soluble in water...
forms green plates. It can be readily diazotized, and the diazonium salt when boiled with alcohol yields aposafranine or benzene induline, C18H12N3. F. Kehrmann showed that aposafranine could be diazotized in the presence of cold concentrated sulfuric acid
Sulfuric acid
Sulfuric acid is a strong mineral acid with the molecular formula . Its historical name is oil of vitriol. Pure sulfuric acid is a highly corrosive, colorless, viscous liquid. The salts of sulfuric acid are called sulfates...
, and the diazonium salt on boiling with alcohol yielded phenylphenazonium salts. Aposafranone, C18H12N2O, is formed by heating aposafranine with concentrated hydrochloric acid
Hydrochloric acid
Hydrochloric acid is a solution of hydrogen chloride in water, that is a highly corrosive, strong mineral acid with many industrial uses. It is found naturally in gastric acid....
. These three compounds are perhaps to be represented as ortho- or as para-quinone
Quinone
A quinone is a class of organic compounds that are formally "derived from aromatic compounds [such as benzene or naphthalene] by conversion of an even number of –CH= groups into –C– groups with any necessary rearrangement of double bonds," resulting in "a fully conjugated cyclic dione structure."...
s. The "safranine" of commerce is a ortho-tolusafranine. The first aniline
Aniline
Aniline, phenylamine or aminobenzene is an organic compound with the formula C6H5NH2. Consisting of a phenyl group attached to an amino group, aniline is the prototypical aromatic amine. Being a precursor to many industrial chemicals, its main use is in the manufacture of precursors to polyurethane...
dye-stuff to be prepared on a manufacturing scale was mauveine
Mauveine
Mauveine, also known as aniline purple and Perkin's mauve, was the first synthetic organic chemical dye.Its chemical name is3-amino-2,±9-dimethyl-5-phenyl-7-phenazinium acetate...
, which was obtained by Sir William Henry Perkin by heating crude aniline with potassium
Potassium
Potassium is the chemical element with the symbol K and atomic number 19. Elemental potassium is a soft silvery-white alkali metal that oxidizes rapidly in air and is very reactive with water, generating sufficient heat to ignite the hydrogen emitted in the reaction.Potassium and sodium are...
bichromate
Chromate
Chromate salts contain the chromate anion, CrO42−. Dichromate salts contain the dichromate anion, Cr2O72−. They are oxyanions of chromium in the oxidation state +6. They are moderately strong oxidizing agents.- Chemical properties :...
and sulfuric acid. Mauveine was converted to parasafranine (1,8-dimethyl Safranine) by Perkin in 1878 by oxidative/reductive
Redox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....
loss of the 7-N-para-tolyl group.
External links
- Safranin O Staining Protocol
- Safranine has recently been employed as the cationic component of a tecton used to build a porous anionic Hydrogen BondHydrogen bondA hydrogen bond is the attractive interaction of a hydrogen atom with an electronegative atom, such as nitrogen, oxygen or fluorine, that comes from another molecule or chemical group. The hydrogen must be covalently bonded to another electronegative atom to create the bond...
ed Network.