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REVIEW

Classi cation and naming of dyes, stains and uorochromes

JA Kiernan
Department of Anatomy and Cell Biology, The University of Western Ontario, London, Canada
Accepted after review, September 19, 2001

Abstract
A classication of dyes and other colorants is proposed, based on the chemical features responsible for their visibility and generally consonant with the writings of modern color chemists. The scheme differs in several respects from that of the Colour Index (CI), but it retains some traditional small groups of dyes that include biological stains. Natural dyes, recognized as a group in the CI, are placed with or near synthetic dyes with identical or similar chromophores. The new scheme also provides categories for dyes and uorochromes that do not have places in the CI classication. Some CI categories, including lactones, aminoketones and hydroxyketones, are not recognized in this new scheme, which is adopted in the forthcoming 10th edition of Conns Biological Stains: a Handbook of Dyes and Fluorochromes for Use in Biology and Medicine. Some rules are also set out for the spelling of trivial names, which has long been inconsistent in scientic literature. The ending `-ine is used for compounds derived from organic bases (e.g., fuchsine and thionine, not fuchsin or thionin), and names ending in `-in are for compounds that are not bases or their derivatives (e.g., eosin and phloxin, not eosine or phloxine). Initial capital letters are used only for words that are names of people or places (e.g., Nile blue or Congo red) and for the `generic components of CI application names (as in Acid yellow 36). Other words, including trade names that have fallen into common usage are not capitalized (e.g., alcian blue, biebrich scarlet, coomassie blue). The recommended spellings of some dyes differ from those commonly seen in vendors catalogs and in biological publications, but they are generally consistent with English and American dictionaries, with recent writings in English by color chemists, and with the trivial names of other organic compounds. Key words: classication, colorants, Conns biological stains, dyes, uorochromes, nomenclature, probes, spelling, stains, taxonomy A new and completely rewritten edition of Conns Biological Stains, is soon to be published for the Biological Stain Commission by BIOS Scientic Publishers (Horobin & Kiernan, 2002). The many colorants used in biological work are placed in chapters and subordinate groupings principally on the basis of their chemistry. The arrangement of dyes and uorochromes conforms to writings in the eld of dye chemistry since about 1980 and differs in some respects from the scheme of classication in the Colour Index (CI), which was followed in the rst 7 editions of Conn (by H.J. Conn) and in the 8th and 9th editions (by R.D. Lillie). In the rst part of this article, I explain the categories of visible and uorescent stains. I then discuss the various names and numbers applied to dyes and similar substances, and present some rules for standardized spelling of the commonly used names. These are the rules adopted in the 10th
261

Address for correspondence: J.A. Kiernan, Department of Anatomy and Cell Biology, The University of Western Ontario, London N6K 1V5, Canada. Phone: 519 661 2111 (ext. 86822); FAX: 519 661 3936. E-mail: jkiernan@uwo.ca. Biological Stain Commission Biotechnic & Histochemistry 2001, 76(5&6): 261277.

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edition of Conn. Trivial names are widely used for dyes because true chemical names are practical only when short (as in picric acid). Furthermore, the CI numbers and application names are either not easily remembered or not available for many dyes and uorochromes used in laboratories.

do not occur. Resonance also exists across quinonoid rings and other chromophoric systems, often with delocalization of electrical charge among the conjugated atoms. Consequently there is always more than one way to draw the structure of a dye. For example, toluidine blue O, a stain commonly used for microscopy, is correctly represented by at least three formulae.

Some chemical structural considerations and terminology

Colored organic compounds contain chromophores. A chromophore is an arrangement of conjugated (alternating) double bonds providing a conguration of electrons that can absorb radiation in the visible part of the electromagnetic spectrum. Every chromophore includes a chain of one or more methine (2 2CH2 2) groups. Electron-accepting and electron-donating atoms (usually nitrogen or oxygen) are present at the ends of the chain in all colored compounds except some polyenes and large aromatic hydrocarbons. Aza- or indamine (2 2N2 2) groups may substitute for methines, and in some dyes the chromophore contains also oxygen (2 2O2 2) or sulfur (2 2S2 2) bridges. In almost every dye, the chromophoric chain of alternating double bonds extends through aromatic and quinonoid rings. An aromatic ring can be formulated with s classical alternating double bonds, as in Kekule formula for benzene, with single bonds to atoms attached to the rings. A quinonoid ring contains a pair of double bonds, conjugated with a pair of double bonds to atoms outside the ring.

Some of the electrons of an aromatic ring system cannot be explicitly shown in conventional Kekule formulae. The real situation is typically represented by organic chemists as two or more resonance forms representing extremes of the actual electron distribution. Electrons are delocalized across the structure, so that distinct single and double bonds
262 Biotechnic & Histochemistry 2001, 76(5&6): 261277

The alternating double bonds are all equivalent. The positive charge is shared by the nitrogen and sulfur atoms; in fact it is distributed across the whole cation and not localized at any particular atom. It is customary to draw, when possible, a structure with a para-quinonoid ring and the positive charge attached to the more strongly basic nitrogen atom, as in the rst of the three formulae above. This convention gives an incomplete picture of the chemistry, but it simplies the recognition of chromophoric systems in structural formulae. (Quaternary nitrogen atoms such as R4 N are the strongest nitrogenous bases because they exist only as cations. Tertiary amines such as R3 N are stronger bases than secondary, R2 NH, or primary, RNH2 , amines). Although it is partly aromatic and partly quinonoid, the skeleton of the toluidine blue molecule closely resembles the three-ring aromatic heterocyclic compound called thiazine (or phenothiazine). This dye is therefore included with similar ones in a group known as the thiazine dyes. Many other groups of dyes are similarly named from ring systems present within their molecules. A heterocyclic ring system like phenothiazine contains carbon atoms and heteroatoms (oxygen, nitrogen or sulfur). Carbocyclic rings like those of benzene or naphthoquinone contain only carbon atoms. The wavelength spectrum of absorbed light, which determines the color of the dye, is affected

by variations in the chromophoric system and by other components of the molecule, traditionally called auxochromes. The latter include charged and uncharged groups of atoms that substitute for hydrogens in the chromophoric skeleton of the dye molecule. These substituents may also inuence the way the dye attaches to a substrate. In toluidine blue, the primary and tertiary amine groups are the principal auxochromes. Note that the system of conjugated (alternating) double bonds includes the parts of the molecule traditionally designated as the chromophore and auxochromes. Whole colored structures of this kind are sometimes called chromogens. The methyl group attached by a single bond to the phenothiazine system is not part of the chromogen because it has little or no effect on the color of the dye. Azure A, which lacks this methyl group, has an absorption spectrum almost identical to that of toluidine blue (Green 1990). Chromogens commonly include charged atoms that are neutralized either by oppositely charged groups within the molecule or by a separate ion, most frequently chloride or sodium. Charged groups are the substituents most obviously involved in attracting a colored molecule to a substrate that is being dyed. Toluidine blue is a typical example of a cationic or basic dye; its positively charged colored ions are attracted to anionic groups in the material being dyed. There are other dyes that have sulfonate (2 2SO3 ) groups, conferring an overall negative charge to the chromogen. Applied from an acidic solution, such dyes can be expected to target oppositely charged protonated amino groups (2 2NH 3 ) of proteins. Dyes with negatively charged substituents are called anionic or acid dyes. Such dyes are used on protein bers such as wool, and many are used as stains for microscopy preparations. Traditional accounts of staining (Mann 1902, Baker 1958) emphasized electrostatic attraction between charged auxochrome groups and the substrate. In reality, the attraction of oppositely charged ions is only one of several mechanisms of dye binding both in industrial applications (Bird and Boston 1975) and in the biological laboratory (Horobin 1982, 1988, Prento 2001). Some dyes contain arrangements of atoms that can form bonds to metal atoms; aluminum, chromium and iron are most often associated with biological stains. The presence of the metal enhances both the color and the strength of binding to some textiles. The traditional belief was that metal ions served as bridges or teeth that held the dye to the substrate. This mechanism is not currently favored by dye chemists, but the name mordant

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(from the French mordre, to bite) is still applied to metals used in conjunction with dyes. A uorescent compound or uorochrome emits light of longer wavelength than the radiation it absorbs. For example, UV absorption may be associated with blue emission, or green absorption with red emission. Many uorescent compounds contain chromophores that also occur in ordinary dyes; however, there are other uorescent compounds with arrays of conjugated double bonds (called uorophores) that do not absorb visible light. The uorescence of a compound may be enhanced or suppressed by atoms or side chains attached to the hydrocarbon or heterocyclic skeleton of the chromophore or uorophore (Bridges 1968). Some complexed metal ions, notably europium and terbium, can enhance the emission of visible light by otherwise weakly or nonuorescent UV-absorbing molecules. There is no easy way to predict uorescent properties by looking at a structural formula. Some important uorochromes do not t into any of the recognized groups of dyes and are allocated to a ``miscellaneous category.

Classi cation of dyes

Chemical structures determine the colors, properties and uses of dyes, and provide the only rational basis of a classication of these compounds. The categories of colorants recognized in the CI and in the rst nine editions of Conn are set out in Table 1.
Table 1. Constitution (chemical) classes of dyes recognized in the Colour Index . Nitroso dyes Nitro dyes Azo dyes Azoic dyes Stilbene dyes Carotenoid dyes Diphenylmethane dyes Triarylmethane dyes Xanthene dyes Acridine dyes Quinoline dyes Methine dyes Thiazole dyes Indamine dyes Indophenol dyes Azine dyes Oxazine dyes Thiazine dyes Sulfur dyes Lactone dyes Aminoketone dyes Hydroxyketone dyes Anthraquinone dyes Indigoid dyes Phthalocyanine dyes Natural organic coloring matters Oxidation bases Inorganic coloring matters

Some of these groups contain similar dyes with distinctive structural features, but others, e.g., hydroxyketones or natural coloring matters, are chemically heterogeneous. A more rational classication of dyes and related compounds requires
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Table 2. Synopsis of the classication of dyes and other colored and uorescent substances adopted in the 10th edition of Conns Biological stains (Horobin and Kiernan 2002) compared with the 9th edition (Lillie 1977). Group in 10th edition Inorganic coloring agents Nitroso and nitro dyes Chapter in 10th edition 29 11 Chapter(s) in 9th edition 17 5 Group in 9th edition Natural dyes etc. Nitroso and nitro dyes, some indicators, and miscellaneous chromogenic reagents Monoazo dyes and uorochromes Dis- tris- and polyazo dyes Azoic diazo compounds, phenolic and other azo coupling reagents and some related chromogenic reagents Tetrazolium salts and related chromogenic reagents Ketoneimines, diarylmethanes and hydroxyketones Aminoarylmethanes Hydroxytriaryl-methanes etc. Hydroxytriaryl-methanes etc. Hydroxytriaryl-methanes etc. Hydroxytriaryl-methanes etc. Xanthenes and acridines Xanthenes and acridines Anthraquinone and other polycyclic dyes: phenanthrolines Quinone imine dyes etc. Quinone imine dyes etc. Quinone imine dyes etc. Natural dyes Ketoneimines, diarylmethanes and hydroxyketones (None in 9th ed.)

Azo dyes: Monoazo dyes Dis- tris- and polyazo dyes Diazonium salts and their reaction products Tetrazolium salts and formazans Arylmethane dyes: Diarylmethanes Aminotriarylmethanes Hydroxytriarylmethanes Simple hydroxytriaryl methanes (rosolic acids) Phthaleins Sulfonphthaleins Xanthene dyes Acridine dyes Phenanthridines Azine dyes Oxazine dyes Thiazine dyes Polyene dyes Carotenoids Stilbene dyes and uorescent brighteners Other polyenes (Diphenylhexatrienes and benzofuranyl uorochromes) Polymethine dyes and uorochromes Cyanines and azamethines

12 13 14

6 7 8

15

16 16 17 12

10 11 12

18 19

12 12 13 13 16 15 15 15 17 10

20 21 22 24 24 24 24

Quinoline, polymethine, and thiazole dyes; Tetrazolium salts and related chromogenic reagents (Subgroups include cyanines, hemicyanines, streptocyanines, neutrocyanines, merocyanines, azacyanines and oxonols. An individual dye can belong to more than one of these subgroups.) Cyanine-like uorochromes 25 16 Anthraquinone and other polycyclic (Benzimidazoles and indoles) dyes Styryl dyes 26 0 (None in 9th ed.) Indamine and indophenol dyes 26 15 Quinone imine dyes Quinophthalone dyes 26 14 Quinoline, polymethine and thiazole dyes Thiazole dyes 26 14 Quinoline, polymethine and thiazole dyes Anthocyanines 26 17 Natural dyes etc. Coumarin dyes and uorescent 26 0 (None in 9th ed.) brighteners

25

14 & 9

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Group in 10th edition Flavonoid and similar neutrocyanine dyes Carbonyl dyes: Indigoid and thioindigoid dyes and pigments Naphthoquinone dyes Anthraquinone dyes

Chapter in 10th edition 26

Chapter(s) in 9th edition 10 & 17

Group in 9th edition Hydroxyketones; natural dyes

27 27 27

16 10 & 17 16 17 10 16

Naphthalimide dyes and uorochromes Aza[18]annulene colorants (includes porphyrins and phthalocyanines) Other organic colorants

27 28

Anthraquinone and other polycyclic dyes Hydroxyketones; natural dyes Anthraquinone and other polycyclic dyes; Natural dyes (carmine, tetracyclines) Aminoketones etc. Anthraquinone and other polycyclic dyes (Natural ones not in 9th ed.) In various chapters in 9th ed.

29

Various

stricter adherence to chemical structure, placing chemically similar compounds near to one another. For example, the ``natural dyes belong more ``naturally to groups that contain chemically similar synthetic dyes. Within each major category or family, dyes can be arranged according to variations in the chromophore and the attached substituents. This classication also accommodates many chromogenic reagents. These substances participate in chemical reactions that form colored compounds, and many are used in analytical biochemistry and histochemistry. The works of Allen (1971), Venkataraman (19521978), Gordon and Gregory (1983) and most especially Zollinger (1991) have guided the placement of compounds in and within the groups summarized in Table 2. No classication of dyes can keep everyone happy because many compounds meet chemical criteria for inclusion in more than one group. The following account summarizes the general chemical structural features of each family of dyes.

category because their visibility is due to emission of visible photons by the metal ions rather than their organic ligands.

Nitroso dyes
These result from the reaction of phenols with nitrous acid, which places a nitroso (2 2NO) group on a carbon atom ortho or para to the phenolic 2 2OH. The C-nitroso compound is in equilibrium with a quinone oxime:

Inorganic colorants
Iodine and silver nitrate are among the inorganic substances used to induce color in microscopy preparations. Colored products may also be formed by the reactions of colorless inorganic reagents, as when ferrocyanide ions react with ferric ions to form the insoluble pigment known as Prussian blue. Certain metal ions, notably those of the lanthanides europium and terbium, not only have intrinsic uorescence, but can also convert energy absorbed by complexed organic molecules into emitted visible light. Fluorescent and phosphorescent lanthanide chelates are placed in the ``inorganic

If the oxygen atom is ortho to the nitroso, the oxygens can form bonds to a metal atom. The resulting strongly colored compound may be a pigment or, if there is a solubilizing substituent (typically sulfonate 2 2SO3 ), a dye. An example is naphthol green B, CI 10020.

Nitro dyes
These yellow dyes contain one or more nitro (2 2NO2 ) groups. The N2 2O and NO bonds of the nitro group are equivalent because of resonance, and they are conjugated with the resonating C2 2C and CC bonds of the aromatic ring. If the compound is a phenol, the aromatic compound exists in equilibrium with a quinonoidone. Exam265

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ples include picric acid, CI 10305, and naphthol yellow S, CI 10316.

Azo dyes
2N2 The azo group, 2 2N2 2, is present in more commercially available dyes than any other chromophore. There are many applications in both industry and the laboratory for the dyes themselves and for the intermediate compounds that react with one another to form azo dyes or pigments.

bases, whereas phenols react as anions. Both of these reactants exist at highest concentration in alkaline media:

A strongly alkaline medium, however, also changes a diazonium cation to a diazotate anion:

Synthesis

An azo group is formed when a diazonium ion, known as the diazo component, reacts with either a phenol or an amine, known as the coupling component. Diazonium ions are generated by the action of nitrous acid (from NaNO2 and HCl) on aromatic primary amines at about 0o C.

The diazotate ion cannot couple. The synthesis of the dye must therefore be carried out at a pH that is a compromise between the alkalinity needed to generate the coupling component and the acidity that preserves the diazonium ion. The optimal pH is different for every pair of reactants; most phenols require more strongly alkaline conditions than most amines. Colored azo compounds can be synthesized by several methods other than coupling of diazonium salts (Zollinger 1961), but such reactions are not often exploited commercially.

Structure and properties

Most diazonium salts are unstable, so they are used as soon as they have been made. In the azo coupling reaction, the end nitrogen atom of a diazonium ion displaces a hydrogen from the aromatic ring of a phenol or amine. The coupling occurs para or ortho to the hydroxyl or amine group. The para position is favored if it is not already occupied or hindered by other substituents. For example, benzene diazonium chloride couples with dimethylaminobenzene to form p-dimethylaminoazobenzene: As a dye, the product is known as oil yellow II or butter yellow. Coupling components derived from benzene often couple in the para position, but naphthalene derivatives usually couple ortho to their hydroxyl or amine groups. In the azo coupling reaction, amines such as dimethylaminobenzene react as unprotonated
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The azo chromophore is often drawn as a linear structure, but in fact the bonds between the azo

nitrogen atoms and the aromatic rings are angulated, so that cis and trans isomers are theoretically

possible. Azo dyes exist as trans isomers because of hydrogen bonding between an azo nitrogen atom and a hydrogen atom of a nearby polar substituent such as an O- hydroxyl or O- amino group:

A phenolic oxygen and an azo nitrogen atom may be suitably placed to form bonds to a metal ion, the presence of which brings about a major change in the color and other properties of the dye. In most azo dyes, the aromatic rings are benzene or naphthalene. Some of the dyes include a heterocyclic ring, most commonly pyrazolone. The aromatic rings carry a wide range of substituent groups that determine both the color and dying properties. Many azo dyes have large molecules that contain two or more azo linkages. The number of azo groups forms the basis of segregation of this large group of dyes: monoazo dyes have one azo linkage, whereas dis, tris, tetrakis and polyazo dyes have two, three, four or more azo groups, respectively. From their modes of industrial application and their properties as biological stains, azo dyes fall into seven groups. Some other classes of dyes have members that are functionally similar to the rst six of these groups. Cationic or basic azo dyes. These may have amine side chains, which are protonated in solutions at low pH, or quaternary nitrogen atoms, which are positively charged even under neutral or alkaline conditions. The charged group may be part of the chromogen (delocalized cationic dyes) or attached by a nonconjugated chain of carbon atoms (pendant cationic dyes). Anionic or acid azo dyes. These have sulfonic or carboxylic acid groups attached to their aromatic rings. Sulfonic acids are strong (ionized at any pH), so the colored ions are always negatively charged. In acidic media, they are attracted to protonated amino groups of protein bers (wool, silk). A carboxylic acid may not be entirely ionized at the pH of the dye bath. Direct azo dyes. These anionic dyes have large molecules (two or more azo linkages) that can assume a coplanar conformation. Direct dyes bind to cellulose (cotton, linen) by nonionic, noncovalent forces. Reactive azo dyes. These have pendant side chains that can combine covalently with the substrate. Attachment to the hydroxyl groups of cellulose is a major commercial application; the color has greater resistance to washing than can be obtained with any other type of dyeing.

Mordant azo dyes. In these, a hydroxyl group is adjacent to a ring carbon that is joined to either an azo nitrogen atom or a carboxyl group. Either arrangement can combine with a metal atom, such as chromium(III), to form a stable ve- or sixmembered chelate ring. The dye-metal complex can be bound rmly by various types of substrates. Solvent azo dyes. These lack ionized amino or sulfonic acid groups that would confer solubility in water. They are used for mass coloring of hydrophobic materials including waxes, some plastics and inks for ballpoint pens. Azoic dyes. These are insoluble colored compounds formed in or on the substrate by reaction of separately applied diazonium salts and azoic coupling components. As stated earlier, most diazonium salts are chemically unstable, but some can be manufactured as dry powders. These stable compounds are called azoic diazo components. Many have two diazonium ions per molecule and are therefore informally known as tetrazonium salts, not to be confused with tetrazolium salts. Azo coupling reactions are exploited in many histochemical techniques. For example, a section of tissue may be immersed in a solution at pH 9 containing a stable diazonium salt and the phosphate ester of a naphthol. Hydrolysis of the ester is catalyzed by an enzyme (alkaline phosphatase) present in some cells. This reaction releases the naphthol, which immediately ionizes and couples with the diazonium salt to form colored deposits in the cells that contained the enzymatic activity.

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Tetrazolium salts and formazans

Many colored compounds can be generated by oxidation of colorless substances, and most colored compounds can be bleached by reducing agents. The production of color by reduction is an unusual and useful reaction exhibited by tetrazolium salts. A tetrazolium cation contains a ve-membered ring with four nitrogen atoms and one carbon. Reduction of this ion yields an insoluble colored formazan with an open chain in which two pairs of nitrogen atoms are attached to the same carbon. Conjugated double bonds extend through the formazan chromophore into the aromatic substituents (R, R0 , R00 in the formulae below) derived from the original tetrazolium salt.

Classication and naming of dyes, stains and uorochromes

267

In formazans the paired nitrogen atoms, conjugated with aromatic rings, are reminiscent of the chromophoric system of the azo dyes. Some formazans are used as metal complex dyes for cotton. One metal-complexing formazan, zincon, is used histochemically to detect zinc. The chromogenic reduction of tetrazolium salts to formazans is exploited in the study of enzymes that catalyze biological oxidations. A tetrazolium salt can replace one of the links in a cells chain of cytosolic and mitochondrial electron acceptors. Insoluble formazans are the end products of most of the histochemical methods that localize dehydrogenase activities. A monotetrazolium salt has a single tetrazolium ring and is oxidized to a formazan that typically is insoluble in water, but soluble in alcohol and nonpolar solvents. A ditetrazolium salt has two tetrazolium rings joined by a bridging group, commonly biphenyl, that greatly extends the chain of alternating double bonds. Reduction of a ditetrazolium ion proceeds in two stages. The monoformazan typically is red and has solubility properties similar to those of a formazan derived from a monotetrazolium salt. Further reduction yields the blue or brown diformazan, which typically is insoluble. My comments on solubility apply to formazans derived from tetrazolium salts used for the histochemical detection of enzymes that catalyze biological oxidations. Some tetrazolium salts have hydrophilic side chains and yield soluble formazans suitable for use in spectrophotometric assays of enzymes in solution.

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also a continuous range of intermediate conditions in which the bonds could not be represented by either single or double bonds. The three bonds to the central carbon are all the same, and are quite unlike the C2 2H bonds of methane. For this reason, Zollinger (1991) prefers the name arylmethine dyes. Other modern authors retain the traditional -methane sufx despite its chemical inaccuracy. The planes of the rings in a triarylmethane dye are tilted, giving a somewhat propeller-like shape to whole molecule. The analogy to a propeller is imperfect because one ring is more twisted than the other two, even in triphenylmethanes with identical substituents on the three rings. The most common substituents on the rings, which determine the properties of the dyes, are amine and hydroxyl groups. Thus, four families of arylmethane dyes are recognized: diarylmethanes, aminotriarylmethanes, hydroxytriarylmethanes and hydroxyaminotriarylmethanes.
Diarylmethanes

This is a small group of cationic dyes with the general structure:

Auramine O, CI 41000, is the only diarylmethane commonly used as a biological stain.

Aminotriarylmethanes

Arylmethane dyes
The general formula for this large group of dyes is:

The simplest member of this series is pararosaniline, a dye whose colored cation can be drawn correctly in various ways:

where R and R0 are benzene or naphthalene rings. R00 is an amino group in the diarylmethanes or another ring in the much more numerous triarylmethanes. The system of conjugated bonds includes all three bonds to the central carbon atom and all the rings, one of which is drawn as part of a quinoid conguration. In the general formula, R0 is shown in quinoid form, but this is only one condition of the molecule. Delocalized electrons are present throughout the conjugated system, so that no particular conguration of alternating single and double bonds is more valid than another. There is
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In other dyes of this group, the rings may be naphthalene rather than benzene, the basic groups may be secondary or tertiary amines, or the basic

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groups may be outnumbered by sulfonate ions to give anionic dyes. Bulky side chains on one or more of the rings can greatly increase the size of the dye molecule. These dyes are notable for their strong, bright colors.
Hydroxytriarylmethanes

In these anionic dyes, at least two of the carbocyclic rings attached to the central carbon bear phenolic or quinonoid oxygen atoms. Other substituents on the aryl rings (carboxyl, sulfonic acid, alkyl groups, halogen atoms) determine the effect of pH on the chromophore and may allow the formation of metal complexes. The hydroxytriarylmethane series includes many important pH indicators, and also dyes that can bind metal ions (mordant dyes). Three subgroups of hydroxytriarylmethane dyes are recognized (CI, Lillie 1977).
Simple hydroxyarylmethanes

Sulfonphthaleins

Sulfonphthaleins have a sulfonic acid group ortho to the central carbon of the triarylmethane structure, and it also can form a lactone-like linkage with the central carbon. An example is phenol red:

Simple hydroxyarylmethanes or rosolic acids are analogs of rosaniline and related aminotriarylmethanes in which oxygen replaces nitrogen. Thus, pararosolic acid is a triphenylmethane that has 2O substituents para to the central carbon 2 2OH or 2 atom. A quinonoid structure can be attributed to two or three oxygen-bearing rings.

The lactone forms of sulfonphthaleins are white and exist only in the solid state.

Xanthene dyes
The chromophore in these dyes contains the planar skeleton of the oxygen-containing heterocyclic compound xanthene.

Phthaleins

Phthaleins are triarylmethanes with para oxygen atoms in two rings; one oxygen is phenolic and one is quinoid. The third ring lacks a para oxygen atom, but it has a carboxyl group ortho to the central carbon. In a basic environment, this exists as a colored carboxylate anion. Acidic conditions suppress ionization, and the carboxyl group forms a lactone (an internal cyclic ester) that incorporates the central carbon atom. The lactone, which does not contain the triarylmethane chromophore, is colorless.

In the general formula, R may be a hydrogen atom or an aliphatic or aromatic group, and X is nitrogen in the aminoxanthenes or oxygen in the hydroxyxanthenes. The formula of an aminoxanthene dye is usually shown with a positively charged nitrogen attached to the p-quinoid ring, though resonance allows an alternative structure with a positive charge associated with the xanthene oxygen. In many of these dyes, R is a phenyl group with one or more carboxyl or sulfonic acid side-chains. The structure is that of a triphenylmethane with an oxygen bridge joining two of its phenyl groups. Halogens commonly replace hydrogens in the xanthene and other parts of the molecule.
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Xanthene dyes used for biological staining are yellow or red, and many are also uorescent. There are blue and violet xanthenes that are used as textile dyes. The pyronines and rhodamines are examples of aminoxanthene dyes; uorescein and the eosins are well known hydroxyxanthenes.

Acridine dyes
The structure of acridine resembles that of xanthene except that the heteroatom is nitrogen instead of oxygen.

A wide variety of substituents (R) may be attached to one of the phenazine nitrogens, and in many azine dyes one or both of the six-carbon rings in the general formula may be part of a larger system of fused rings. Although the chromophore is intrinsically cationic, some azine dyes are anionic because sulfonate substituents outnumber the cationic nitrogen atoms. Neutral red, CI 50040, (R H), and safranine O, CI 50240, (R phenyl or otolyl), are azine dyes used as biological stains.

Oxazine dyes
In these dyes, a nitrogen and an oxygen atom make bridges between two six-carbon rings. R may be hydrogen or an alkyl or aryl group. R0 is usually hydrogen, but may be an alkyl group. A pquinonoid structure is shown above. The acridines are strongly uorescent yellow cationic dyes. Acridine orange, CI 46005, and acriavine, CI 46000, are examples of dyes in this class.
Phenanthridines

Phenanthridines are related compounds, with a chromophore that is an isomer of acridine. The phenanthridines include ethidium bromide, propidium iodide and related uorochromes, which bind to nucleic acids and are excited optimally by green light with red emission.

The chromophore is shown here in p-quinonoid form; other structures include o-quinoid forms with the positive charge on either of the bridging heteroatoms. Most oxazine dyes are either colored cations or metal-binding phenolic compounds. They include important pH indicators and microscopy stains. The oxazine group includes compounds present in litmus and orcein, which are substances obtained by applying simple chemical treatments to compounds that occur in some lichens. The triphenodioxazine dyes and pigments have two oxazine groups in each molecule, making a ve-ring chromophore.

Thiazine dyes
In the name of a heterocyclic compound, the prex ``thia indicates a sulfur atom, just as ``aza indicates a nitrogen and ``oxa an oxygen atom. In the three-ring chromophore of the thiazine dyes, nitrogen and sulfur make bridges between two sixcarbon rings that may be drawn as benzenoid or quinonoid structures. These are all cationic dyes, and electron delocalization results in partial positive charges located on both nitrogen atoms and on the sulfur. This is indicated visually in the resonance formulation below. In these general formulae, R represents hydrogen or an alkyl group. In the simplest thiazine dye, thionine, all four R groups are hydrogen atoms. In methylene blue, CI 52015, all four are methyl groups, and other dyes of the series contain

Azine dyes
The azine chromophore contains the skeleton of phenazine, a planar heterocyclic aromatic compound consisting of two benzene rings linked by two nitrogen atoms. A delocalized positive charge and an alternation of aromatic and quinonoid structures constitute the chromophoric system.

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Carotenoids
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These colorants are notable for having no aromatic rings. More than 300 carotenoids occur in plants, and a few are synthesized industrially for coloring foodstuffs. The simplest member of the series is lycopene, named for its presence in Lycopersicon, the tomato.

combinations of hydrogen, methyl and ethyl groups. The chromophore readily accepts an electron (or loses a proton), and is reduced to a colorless compound.

This formula shows the carotenoid structure, comprising eight isoprene units arranged so that there is a long conjugated chain in the middle part of the molecule (Kienzle and Isler 1978). In other carotenoids, the ends of the chains are folded into rings, which may be alicyclic or quinonoid, and may bear such substituents as 2 2OH and 2O, 2 2 2OCOCH3.
Stilbene dyes and uorescent brighteners

The reaction is readily reversed by atmospheric oxygen or other oxidizing agents. Commerce in thiazine dyes owes its existence almost entirely to their applications in the laboratory. These dyes are used everywhere for staining bacteria and blood cells, and for biochemical studies of oxidation-reduction reactions.

These are manufactured compounds in which the chromophoric system includes stilbene with aromatic substituents (Ar and Ar0) at positions 4 and 40 .

Polyene dyes and

uorochromes

The polyene dyes have the simplest of all chromophores, a chain of alternating double and single bonds. If the compound is a hydrocarbon, the conjugated chain must contain at least 11 double bonds to bring its absorption spectrum into the visible range. This number is reduced to ve if the chain has hydrophilic oxygen-containing groups (free or esteried carboxyl) at its ends. Aromatic and quinonoid rings also enhance visibility. The polyene group could include all dyes and uorochromes, but its denition excludes compounds in which a positive or negative charge might exist at one end of the conjugated chain. This constraint consigns to other categories all but a few dyes and uorochromes, the carotenoids and stilbenes.

The group includes some direct cotton dyes of uncertain composition and many compounds used as uorescent brighteners. Some of the latter have been used as uorochromes, especially in botanical microscopy. An example is calcouor white M2R, CI 40622.
Other polyenes

Certain uorescent compounds used to trace transport within living cells are placed in this category for convenience. They include certain diphenylhexatrienes and the cationic benzofuranyl uorochromes known as fast blue and true blue.

Polymethine dyes and

uorochromes

In these compounds, the chromophore consists of 2 (methine) groups with an one or more 2 2CH2
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electron donor at one end of the chain and an electron acceptor at the other end. In most of these dyes, the donors and acceptors are nitrogen atoms, at least one of which is within a heterocyclic ring. The hydrogen atom of a methine group may be replaced by another atom, such as carbon in a methyl group or as part of a ring. Related dyes included in the polymethine group have one or 2 2 (azamethine or indamine) rather than more 2 2N2 2 methine groups within the conjugated chain. Dye chemists differ in their use of terms and placement of these dyes into groups (Brooker 1966, Gregory 1990). Here, nine major groups are recognized within the diverse family of polymethine dyes based on conspicuous chemical features such as particular ring systems. There are, however, many dyes that qualify for inclusion in more than one of these groups.
Cyanines and azamethines

The general formula for this group of dyes is shown below.

ring neutrocyanines, are here treated as a separate group of polymethine dyes; they include some important biological stains. The merocyanines are synthetic neutrocyanine dyes. In the azamethine dyes, either X or Y of the general cyanine formula is a nitrogen atom. Indamines and indophenols (see below) are azamethine dyes in which a single 2 2N forms the bridge between rings A and B of the general cyanine formula. An oxonol dye has methine (or azamethine) bridges between ring systems that contain oxygen atoms, such as ketone or hydroxyl groups, as the electron donor and acceptor. Oxonols are notable anionic polymethine dyes. Some are used as sensitizers in photographic emulsions; others serve as voltage-sensitive uorescent indicators in neurophysiological experiments. If in the general cyanine formula n 0, there is no methine bridge connecting ring A with ring B. Such a dye is sometimes called an apocyanine, because of its resemblance to a cyanine. The thiazole and quinophthalone dyes (see below) are examples of apocyanines. Cyanines and azamethines have many industrial uses, especially in the manufacture of photographic materials, and some are used as biological stains and uorochromes.
Cyanine-like uorochromes

A and B are aromatic rings, at least one of which is heterocyclic with a positively charged (usually quaternary) nitrogen. Several subgroups are recognized, but they are not mutually exclusive. For example, a particular hemicyanine might also be an azamethine. The wavelength of maximum absorption increases with the number of bridging methine groups (n in the general formula). Cyanine dyes proper have n > 0, and X and Y are carbon atoms in the general formula. Rings A and B, which contain the donor and acceptor nitrogen atoms, may be the same (symmetrical cyanines) or different (asymmetrical cyanines). In a hemicyanine dye there is only one charged nitrogen-containing ring; the nitrogen at the other end of the polymethine bridge is not a part of ring B. In a streptocyanine dye, the chain of methine groups is terminated at both ends by nitrogen atoms that are not in rings; A and B may be noncyclic groups such as 2 2N(CH3)2. The word carbocyanine is sometimes applied to dyes in which n 1, but it is also used for cyanines that are not azamethine dyes (see below). Neutrocyanines have a potentially anionic acceptor atom, such as oxygen in a carboxyl or phenolic hydroxyl group, and the molecule has no net charge. The avonoids, which are naturally occur272 Biotechnic & Histochemistry 2001, 76(5&6): 261277

This arbitrary group contains two groups of cationic uorochromes that are used in biology for their property of binding to nucleic acids, typically with a resultant increase in the intensity of uorescence. Benzimidazoles and indolenines include, respectively, the `Hoechst dyes and some other uorochromes that bind to DNA and are excited by near ultraviolet or blue, with blue, green or yellow emissions.
Styryl dyes

These have molecules containing a styryl group, usually conjugated with a tertiary amine group. The properties of the dyes are determined by the substituents (R, R0, X and Y):

The structure resembles that of hemicyanines and other polymethines, but the chromophoric system does not end in a heterocyclic ring. For this reason,

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Gregory (1990) placed styryl dyes in a major class of their own. Some have small molecules that readily penetrate the hydrophobic domains of polyester textiles. Fluorescent styryl dyes with larger molecules are used as probes that enter and are retained in the membranes of cell surfaces or organelles.
Indamine and indophenol dyes

nines. The methine that joins the two rings is partly included in benzothiazole.

An indamine (2 2N2 2) group forms a bridge between two rings, one aromatic and the other quinonoid. In the indamine dyes, nitrogen atoms terminate the conjugated chain in both ring systems, whereas in an indophenol dye the chain is terminated by phenolic hydroxyl or a quinonoid carbonyl group at one end. These dyes are unimportant as biological stains, but some are used as analytical reagents. Colored compounds with indamine and indophenol structures are the end products of some histochemical reactions.
Quinophthalone dyes

Ar is an aromatic ring, which may be benzene or another benzothiazole. The chromophoric system can be positively charged from protonation or methylation of the thiazole nitrogen. Some dyes in this group are made anionic by sulfonation of carbon atoms in either benzothiazole or the Ar component. A few thiazole dyes, including primuline, CI 49000, and the thioavines, are used as uorescent tracers and as stains.
Anthocyanines

These dyes, also known as quinoline dyes, have quinoline joined to phthalic anhydride. A methine and an azamethine group are present in the center of the molecule. An earlier formulation of this type of dye (CI,

Many compounds in plants are glycosides in which sugars such as glucose are joined, with elimination of water, to phenolic hydroxyl groups. The linkages can be hydrolyzed in acid conditions to yield the sugar and the nonsugar or aglycone components. The aglycones of many plant pigments are anthocyanines. These compounds have phenolic 2 2OH groups attached to a three-ring cationic structure that can be formulated with the positive charge on an oxygen or carbon atom.

Lillie 1977) showed the components more clearly, but it was incorrect because it did not include a methine group in the chromophore:

The oxonium ion is usually depicted in structural formulae. The ring system of the anthocyanines has features in common with those of the avones and coumarins.
Coumarin dyes and uorescent brighteners

Quinophthalone dyes are moderately hydrophobic, so they are used industrially as colorants for plastics and polyester textiles. A few of these dyes are used in biological research because they have greater afnity for cell membranes than for extra- or intracellular uids.
Thiazole dyes

These uorescent compounds contain coumarin with a wide variety of substituents.

These dyes, like the quinophthalones, are apocya-

Depending on the nature of the substituents, these compounds can also be classied as neutrocyanines or hemicyanines. They also are structurally similar to the avonoids.
273

Classication and naming of dyes, stains and uorochromes

Flavonoid dyes
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synthetic indigoid dye used in some staining procedures.

Naphthoquinone dyes

Flavone is a ketone that forms part of many colored compounds that occur in plants.

Just as simple phenols can be oxidized to quinones, naphthols can be oxidized to naphthoquinones.

The arrangements of aromatic and quinoid rings vary greatly among these dyes, which also meet the denition of the neutrocyanines. Brazilein and hematein, formed by oxidation of the naturally occurring substances brazilin and hematoxylin, are two examples of avone dyes. They are sometimes included, together with naphthoquinones and some anthraquinones, in a group known as ``hydroxyketone dyes (Allen 1971, Lillie 1977). Hematein, the oxidation product of hematoxylin, CI 75290, is a avone dye widely used as a biological stain.

This is not a large group, but several naphthoquinones have been used as textile dyes (Tilak 1971), and a few serve as biological stains or histochemical reagents.
Anthraquinone dyes

Carbonyl dyes
The carbonyl group occurs in aldehydes (R2 2CHO) and ketones (R2 2CO2 2R0 ). Quinones are cyclic diketones in which the C2 2O double bond is conjugated with carbon-carbon bonds within a ring. The family of carbonyl dyes recognized by Zollinger (1991) includes compounds in which two carbonyl groups form parts of a chain of conjugated bonds interposed between an electron donor and an electron acceptor.
Indigoid and thioindigoid dyes and pigments

Anthraquinone dyes are built around the anthraquinone structure.

The chromophore is

in which X is 2 2NH in the indigo series (blue) and 2 2S2 2 in the thioindigos (red). Indigo and thioindigo are vat dyes, i.e., insoluble colored compounds formed by oxidation of soluble, colorless precursors. There are also dyes that are made soluble by addition of sulfonate groups to the indigoid structure. In addition to traditional uses as biological stains, indigoid compounds are the end products of some reactions exploited for enzyme histochemistry. Indigocarmine, CI 73015, is a
274 Biotechnic & Histochemistry 2001, 76(5&6): 261277

There can be many substitutions, including junctions with other fused ring systems. This is by far the largest group of carbonyl dyes including hundreds of compounds that are applied to textiles in many ways. The most notable from an industrial standpoint are the anthraquinone vat dyes for cotton, disperse dyes (Table 3) and pigments (Greenhalgh 1976). Mordant dyes in this group include the natural colorants carmine, CI 75470, and alizarin, CI 58000. The former has many uses as a histological stain. Synthetic anthraquinones include anionic, solvent, mordant and reactive dyes. Nuclear fast red, CI 60760, is an example of a synthetic anthraquinone mordant dye.
Naphthalimide dyes and uorochromes

These were classied as ``aminoketones by Lillie (1977). Here (following Zollinger 1991) they cons-

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titute a small group within the carbonyl dyes. The central structure of the molecule is shown below;

4-bora-3a,4a-diaza-s-indacene (BODIPY) uorophore, which, with a variety of reactive substituents at positions 1, 2, 3, 5, 6 or 7, can be incorporated into a wide range of uorescent probes and other reagents.

various side-chains may be attached. Naphthalimides are used as disperse dyes and as uorescent brighteners. Some uorescent compounds of this type, such as the lucifer yellow dyes, can be injected into living cells to study diffusion within the cytoplasm and through gap junctions into adjacent cells.

Aza[18]annulene colorants
A ring of methine and azamethine linkages included in and connecting four ring systems is the chromophore in many colored compounds of animals and plants including the porphyrins, cobalamins and chlorophylls.

Naming of dyes
The formal chemical names of most dyes are so long that they are never used in ordinary conversation or writing. Instead, each dye has one or more informal names. In addition, many dyes have standardized CI names based on their original industrial uses and colors.
Informal names

Thickened lines in each structural formula above indicate one of the possible rings of 18 conjugated bonds that constitutes the aza[18]annulene chromophore in each of these two examples. Phthalocyanine and related compounds are synthetic pigments and dyes with large coplanar molecules in which M is a metal, most frequently copper. In dyes, various solubilizing side chains are attached to the four benzene rings at the corners of the molecule. Alcian blue, CI 74240, is a cationic phthalocyanine dye.

Other organic colorants

This miscellaneous category contains substances that differ conspicuously in chemical structure from those in any of the other 15 groups. Several such compounds are used as uorescent stains or probes. Examples are cascade blue acetylazide, a reactive uorochrome for labeling macromolecules, and the

Most informal names include the color, and some also indicate a major property or the general class of compounds to which the dye belongs. For example, acridine orange is one of several dyes that contain the heterocyclic ring structure known as acridine. Names may be related to industrial applications, as with wool fast scarlet R and woodstain scarlet; they may commemorate scientists (Bernthsens methylene violet, Bindschedlers green, Meldolas blue), or include a former or current manufacturers name or trade-names (Hoechst 33258, sirius red F3B, procion brilliant red M2B). Dyes that are natural products, such as alizarin, hematein and carmine, have traditional names that are sometimes carried over to chemically related synthetic dyes, as in the case of alizarin red S. There are also synthetic dyes with names that suggest natural origins, even though the compounds are not related to natural products. Amaranth and primuline are examples of synthetic dyes with names that wrongly suggest botanical origins. The informal name of a dye commonly includes a sufx of one or more capital letters and sometimes also one or more numbers. Frequently the sufx distinguishes the compound from simi275

Classication and naming of dyes, stains and uorochromes

Table 3. Application classes of dyes as used in CI names

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Acid dyes Azoic dyes Basic dyes Direct dyes Disperse dyes Fluorescent brighteners Food dyes Ingrain dyes Mordant dyes Natural dyes Oxidation bases Pigments Reactive dyes Solvent dyes Sulfur dyes

Vat dyes

Colored anions applied at low pH to wool, silk and nylon. Insoluble azo dyes formed within the substrate by reaction of a diazonium salt (azoic diazo component) with a naphthol (azoic coupling component). Colored cations, used on polyacrylonitrile (acrylic) textiles, which have acid side chains, formerly also on proteinaceous bers and tannic acid-treated cotton. Acid dyes with large molecules that will bind to cellulose bers directly. Insoluble colored compounds in ne suspension, which enter hydrophobic materials (cellulose acetate, polyester) when applied with a ``plasticizer. Absorb ultraviolet and emit blue; used to offset yellowing of white textiles. Substances used to color foodstuffs. Temporarily solubilized colorants that change chemically and become insoluble after application to the substrate being dyed. Dyes applied in conjunction with metals. The metal may be applied before, with, or after the dye. Made or extracted from plants or animals. Colorless amines and aminophenols that polymerize to brown or black insoluble substances when oxidized (usually by hydrogen peroxide); used on fur and hair. Finely divided white, black or colored materials insoluble in water and organic solvents; used in paints and for mass coloration of plastics. Colored compounds with side chains that react to form covalent bonds with the substrate; used to provide very fast (resistant) dyeing of cellulose etc. Colored compounds soluble only in hydrophobic solvents; used for mass coloration of liquids and plastics. Cheap polymeric dyes made by heating aromatic amines and phenols with sulfur or sodium polysulde. A soluble (reduced) form is applied to the textile and the insoluble polymer is then regenerated by oxidation. Applied to cloth as a soluble compound that is then oxidized to the insoluble, fully colored form of the dye; used mainly on cotton.

larly named products of the same manufacturer, or it may be part of a nonproprietary name. For example, eosin Y and eosin B are closely related red dyes with yellowish and bluish hues, respectively. Often the informal name gives little or no information about the chemical constitution or colorant properties of a dye. An expression like methyl blue or Congo red might apply to any blue or red compound, and names like vesuvin and coriphosphine do not reveal even the colors. Nevertheless, informal names are in widespread use and it is unlikely that users of dyes would want to change them. Moreover, there are many dyes with more than one widely used name: chromoxane cyanine R, Mordant blue 3 and solochrome cyanine R are all applied to a dye used to stain nuclei and myelin. Analytical chemists, who use the same dye as a chromogenic reagent for determination of iron, call it eriochrome cyanine R.
Colour Index names and numbers

on its industrial method of application and its color. Thus, methylene blue, a basic or cationic dye, is CI 52015 or Basic blue 9. Sirius red 4B (CI 28160) is named Direct red 81 because it is one of the group of direct cotton dyes. For some dyes, a name of this kind derived from the CI is the only one used in the biological literature. The groups of colorants that form the basis of this naming system are summarized in Table 3. The ve-digit numbers assigned by the CI are based on the classication by chemical constitution adopted in that work. Many numbers have been left unused for dyes not yet introduced. The chemical groups of dyes recognized in the CI are listed in Table 1.
Choices of names and spellings

The Colour Index and its supplements, published by the Society of Dyers and Colorists (19711996), is an encyclopedic reference for dyes and other coloring agents. In addition to traditional names, it provides each dye with a unique number and a name based
276 Biotechnic & Histochemistry 2001, 76(5&6): 261277

The preferred informal names of dyes used here are those believed to be most widely used by biologists. For uniformity, all words in the informal names of dyes begin with lower case letters unless they are obviously names of people or places. The application names from the CI are treated in the same way as the generic and specic names of animals or plants, with an initial capital letter for the word denoting mode of application class and lower case

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for the color. Thus, CI 51180 is Nile blue or Basic blue 12. Many words in the informal names of dyes end in `-ine or `-in, and these endings have often been used wrongly, especially for microscopy stains. The ending -ine is appropriate for an organic base or a compound derived from an organic base. The ending -in is used for compounds that are not bases or their derivatives. Thus, acid fuchsine receives a terminal -ine because it is basic fuchsine modied by addition of sulfonic acid groups. Eosin and erythrosin, on the other hand, are heterocyclic phenols, not bases, so their names end in -in. Correct spellings are found in British (Shorter Oxford) and American (Websters Encyclopedic) dictionaries and are invariably used in modern works concerned with dye chemistry such as those of Gordon and Gregory (1983), Waring and Hallas (1990) and Zollinger (1991). In a classical text on staining, Baker (1958) reminded his readers of the rules that governed spelling of chemical and pseudochemical names. Other modern writers in the eld including Lillie (1977), Horobin (1982, 1988), Chayen and Bitensky (1991), and Green (1990) have been less consistent.

Probes and labels

Dyes and uorochromes are used for both microscopy and analytical chemistry. Some are also used to impart color or uorescence to specic objects in living cells and tissues such as cell membranes, DNA molecules, mitochondria and other organelles. Substances used for this purpose are known as probes. If a probe is strongly colored or (more usually) uorescent, it may be visible directly. Examples include cationic phenanthridine and benzimidazole uorochromes that attach to nucleic acids, diphenylhexatrienes and lipophilic cyanines used to label membranes of living cells, and dyes such as janus green B (monoazo), neutral red (azine) and methylene blue (thiazine) that are used in traditional vital staining techniques. Green uorescent protein (GFP) provides an intrinsic uorescent label for the progeny of cells transfected with the gene that encodes the protein. Other probes are invisible compounds, such as proteins or drugs, and these must be made visible by labeling. A simple example of a labeled probe is the protein phalloidin, which attaches specically to actin laments in cells. If phalloidin molecules are made to form covalent bonds with a uorescent xanthene dye such as uorescein, the resultant labeled probe will impart uorescence to actin laments.

Not all labeling agents are colored or uorescent. Proteins, notably antibodies for use in immunohistochemistry and nucleic acid probes for use in situ hybridization, are frequently labeled by covalent conjugation to a histochemically demonstrable enzyme. Horseradish peroxidase is the enzyme most popular for this purpose, but alkaline phosphatase is also widely used. Glucose oxidase is somewhat less popular. The gene encoding b-galactosidase is often included in DNA sequences introduced experimentally into living cells. The transfected cells and their descendents can then be recognized by a staining method that localizes the activity of the enzyme. Two other invisible labeling agents are worth mentioning. It is easy to conjugate biotin to proteins, nucleic acids and other large molecules. This compound can be detected by virtue of its strong and specic afnity for avidin, a protein from egg white that can be labeled with any uorescent or enzymatic marker. Digoxigenin, a steroid of plant origin, can also be conjugated to large molecules. Commercially available antibodies are used for immunohistochemical detection of bound digoxigenin-labeled probes. Colloidal gold particles readily become coated with proteins and are used as a nonreactive label that can be detected by electron microscopy or by its red color.

Acknowledgments
I thank F.H. Kasten (East Tennessee University, Johnson City) for reading an early draft of this manuscript in 1996, and since then for verbal and email discussion of dye nomenclature and spelling of trivial names. We still do not agree on all aspects, but his helpful advice inuenced the eventual content of this review. The allocation of individual dyes and uorochromes to chemical groups was undertaken by R.W. Horobin (University of Glasgow, Scotland) in the planning of the 10th edition of Conns Handbook. Almost daily exchanges of emails for ve years have resulted in numerous modications to the denitions of groups and subgroups of dyes and uorochromes. After such a vigorous exchange of ideas, the number of compounds in the ``miscellaneous organic category is now quite small.

References
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Textiles. Dyers Company Publication Trust, Bradford, UK. Bridges JW (1968) Fluorescence of organic compounds. In: Luminescence in Chemistry. Bowen EJ, Ed., van Nostrand, London. pp. 77115. Brooker LGS (1966) Sensitizing and desensitizing dyes. In: The Theory of the Photographic Process. James TH, Ed., Macmillan, New York. pp. 198232. Chayen J, Bitensky L (1991) Practical Histochemistry, 2nd ed., Wiley, Chichester. Gordon PF, Gregory P (1983) Organic Chemistry in Colour, Springer-Verlag, Berlin. Green FJ (1990) The Sigma-Aldrich Handbook of Stains, Dyes and Indicators. Aldrich Chemical Co., Milwaukee WI. Greenhalgh CW (1976) Aspects of anthraquinone dyestuff chemistry. Endeavour 35: 134140. Gregory P (1990) Classication of dyes by chemical structure. In: The Chemistry and Application of Dyes. Waring DH, Hallas G, Eds., Plenum Press, New York. pp. 1747. Horobin RW (1982) Histochemistry: an Explanatory Outline of Histochemistry and Biophysical Staining. Gustav Fischer, Stuttgart. Horobin, RW 1988. Understanding Histochemistry: Selection, Evaluation and Design of Biological Stains. Ellis Horwood, Chichester. Horobin RW, Kiernan JA (2002) Conns Biological Stains: a Handbook of Dyes and Fluorochromes for Use in Biology and Medicine, 10th ed., Oxford: BIOS Scientic Publishers (in preparation).

Kienzle F, Isler O (1978) Synthetic carotenoids as colorants for food and feed. In: The Chemistry of Synthetic Dyes. Venkataraman, K., Ed., Academic Press, New York. Vol. 8, pp. 389414. Lillie RD (1977) H. J. Conns Biological Stains. 9th ed., Williams & Wilkins, Baltimore. Mann G (1902) Physiological Histology. Methods and Theory. Clarendon Press, Oxford. Prento P (2001) A contribution to the theory of biological staining based on the principles for structural organization of biological macromolecules. Biotech. & Histochem. 76: 137161. Society of Dyers and Colourists (19711996) Colour Index International. 4th revision of 3rd ed. Vols IIX (19711992); CD-ROM Version 2, with additions to 1996. Society of Dyers and Colourists, Bradford, England. Tilak BD (1971) Naphthoquinonoid dyes and pigments. In: The Chemistry of Synthetic Dyes. Venkataraman K., Ed., Academic Press, New York. Vol. 5, pp. 155. Venkataraman K (19521978) The Chemistry of Synthetic Dyes. Vols IVIII. Academic Press, New York. Waring DR, Hallas G, Eds. (1990) The Chemistry and Application of Dyes, Plenum Press, New York. Zollinger H (1961) Azo and Diazo Chemistry. Aliphatic and Aromatic Compounds (Transl. Nursten HE), Interscience Publishers, London. Zollinger H (1991) Color Chemistry. Synthesis, Properties and Applications of Organic Dyes and Pigments, 2nd ed., VCH Verlag, Weinheim.