Dyes and Dyeing Flashcards
Why does tissue need to be dyed?
Tissue all has the same refractive index so tissue sections appear dull and it is difficult to define structures within the tissue.
How are dyes classified?
Dyes can be classified as natural or synthetic. Natural dyes are derived from natural sources and synthetic dyes are derived from hydrocarbons, most often from benzene.
Describe what natural dyes are and give examples.
Natural dyes are derived from natural sources. Haematoxylin is derived from the heart-wood of Haematoxylon campechianum, a South American tree. Carmine is derived from the fat-body of the female Cochineal beetle. Orcein is derived from lichen (an organism comprised of fungi and algae), such as those containing the fungus Lecanora tinctora.
Using an example, describe how a dye is formed from benzene.
To make a dye from benzene, both a chromophoric (colour-bearing) group and auxochromic (binding) group need to be substituted onto the benzene ring. The dye, Picric Acid, is formed when nitrogen dioxide (NO2) undergoes a substitution reaction with benzene to form trinitrobenzene. This gives a chromogen - an aromatic with a chromophoric group attached. NO2 is the chromophoric group in this case. A hydroxyl (OH) group undergoes a substitution reaction with trinitrobenzene to form the dye, Picric Acid. The auxochrome is the hydroxyl group.
What is an acid dye? Give an example.
An acid dye is coloured in the anion i.e. the chromophoric group is negatively-charged. The auxochrome can be sodium or potassium. They are negatively-charged (anionic) and bind electrostatically to positively-charged (cationic) groups in the tissue, which is often NH3. Eosin is an acid dye; it binds to basic proteins in the cytoplasm.
What is a basic dye? Give an example.
A basic dye is coloured in the cation i.e. the chromophoric group is positively-charged. The auxochrome is often NH2. They are positively-charged (cationic) and bind electrostatically to negatively-charged (anionic) groups in the tissue. These tissue groups can be chloride, sulphate or acetate. Methyl Green is a basic dye and has an affinity for phosphate groups in DNA.
What sort of bonding do acid and basic dyes form with groups present in the tissue? Is this an example of direct or indirect binding?
Acid and basic dyes bind to tissue using electrostatic binding, which refers to the attraction between negatively- (anionic) and positively-charged (cationic) groups in the dye and tissue. This is direct binding because the dye can directly attach to the tissue.
What is an auxochrome? Give examples.
An auxochrome is a combining group which can bind to tissue end-groups through electrostatic or covalent bonding. An auxochrome, as well as a chromophore, is necessary to form a dye from benzene. An auxochrome can be positively-charged (cationic) e.g. amino group (NH2) or negatively-charged (anionic) e.g. hydroxyl (OH), carboxyl (COOH) or sulphonic (SO3). The charge of an auxochrome gives the chromogen the characteristics of a salt.
Define chromophore.
A chromophore is a colour-bearing group, which is substituted onto a benzene ring to produce a chromogen (an aromatic compound with a chromophore attached).
What are the classifications of chromophoric groups?
Chromophores can be classified as azo dyes, nitro dyes and quinonoid dyes. Azo dyes contain a N=N group, nitro dyes contain a NO2 group and quinonoid dyes contain either a para- or ortho-quinoid ring.
What is an azo dye? Give an example.
An azo dye has a N=N chromphoric group. They can be categorised based on the number of chromophoric groups present in their chemical structure i.e. monoazo, diazo, triazo and tetrakisazo. Congo Red is a diazo dye, which stains amyloid red. Sirius Red is a tetrakisazo dye and it stains collagen red.
What is a nitro dye? Give an example.
Nitro dyes contain a NO2 chromophore. Picric Acid is a component in the Van Gieson stain and it stains collagen yellow. Martius Yellow stains red blood cells yellow.
What is a quinonoid dye? Give an example.
A quinonoid dye contains either a para- or ortho-quinoid ring as a chromophore. The quinoid ring strongly absorbs visible light, making it the most important chromophoric group. Quinoid dyes can be cationic or anionic. Crystal Violet is a cationic quinonoid dye, which is used in the Gram stain to differentiate Gram-negative and Gram-positive bacteria. With the use of a trapping agent (iodine), Crystal Violet is trapped within Gram-positive bacteria but washed out of Gram-negative bacteria in subsequent steps. Gram-positive bacteria stain purple and Gram-positive stain pink. An anionic quinonoid dye is Eosin, which stains cytoplasm shades of pink.
What is an auxochrome?
An auxochrome is a combining group, which can bind to tissue end-groups by electrostatic or covalent bonding. An auxochrome can be cationic or anionic, thereby giving the chromogen the characteristics of a salt. N.B. In order to make a dye, benzene needs both a chromophoric group and an auxochrome substituted onto its structure.
What is a chromogen?
A chromogen is an aromatic compound, such as benzene, which has a chromphoric group attached.
