Definition

 Stain and Dye

 * The term 'dye' is used to refer to a colouring agent that is used for general purposes, whereas the term 'stain' is used to refer to that dye which is used for biological purposes.

* A stain is any colouring organic compound that combined with another substance imparts a colour to that substance.

* Dye are used to colour non-biological materials whereas stains used for coloration of biological materials.

    Chemical makeup of stain

* Benzene = organic compound

* Chromophore (Gk. Chroma = colour; phoros = to bear) = colour 

* Auxochrome (Gk. auxein = to increase; chroma = colour) = ionization properties.

* Benzene + chromophore = chromogen 

- Chromogen is a coloured compound only.

* Auxochrome with chromogen allows the dye to form salt compounds that adhere to cells.

Stain - an organic compound composed of a benzene ring, a chromophore and auxochrome group.

              

              Benzene is a organic colourless solvent.

              Chromophore is the molecule that gives colour to benzene.

              (A CHROMOGEN - isn't a stain, just a colored molecule.

               It is made up of the benzene and the chromophore).

              Auxochrome ionizes the chromogen, gives it a charge.

              This helps the chromogen form salts and bind to substances like tissues or fibers.

* Every cell as well as stain exhibit some type of charge.

* Based on charge, stain can be ACIDIC and BASIC.

    Acidic Stain

* These are anionic.

* Their chromogen has negative charge on ionization.

* They have affinity for the positive components of a cell.

* Acidic stains do not stain bacterial cells. They stain background with contrasting color.

* Examples : Eosin, rose bengal and acid fuschsin, India ink, Nigrosin, Picric acid.

 Basic stain 

* These are cationic.

* Their chromogen has  a positive charge on ionization.

* They have affinity for the negatively charged molecules such as nucleic acids, many proteins, and the surfaces of bacterial and archaeal cells.

* Basic stains can stain bacterial cells as they possess negative charge.

* Examples : Methylene blue, basic fuchsin, crystal violet, safranin, malachite green.

 Mordant

 * Chemical which are required to bring about the staining reaction are called mordants.

* They helps to fix the color to the cell components. 

* Basic mordant reacts with acidic stains and acidic mordant reacts with basic stains.

* Examples : Phenol in carbol - fuchsin (Ziehl - Neelsen technique) and iodine in the Gram stainin.

 Leuco compound

* Chromophores are easily reduced by combining with hydrogen at the double bonds.

* Reduction of chromophore result is loss of color.

* These decolorized dyes are known as Leuco compound.

* They are used as an indicator of oxidation & reduction reactions.

* Examples - leucofuchsin used in periodic acid - schiff (PAS) stain.

    Natural Stain 

 * These are very few in numbers.

* Examples :

1) Hematoxylin (Natural Black 1) , a naturally occurring flavonoid compound derived from the logwood tree, Haematoxylon campechianum.

2) Carmine (Carmine red) a natural red dye extracted from the dried females of the insect Dactylopius coccus var. Costa (cochineal).

 Fluorochrome  

* A fluorophore (or fluorochrome) is a flurorescent chemical compound that car re-emit light upon light excitation.

* Fluorochrome typically contain several combined aromatic groups or planar or cyclic molecules  with several  π bonds.

* They are notably used to stain tissues, cells or materials in a variety of analytical methods, i.e., fluorescent imaging and sperctroscopy.

* Examples : Fluorescein, Acridine orange, Acridine yellow.

Decolorizing agent

* They are used to remove dye from a stained microscopic specimen.

* Decolorizing agents can be as simple as water or ethanol.

* Example: Ethanol or an ethanol and acetone solution in Gram's staining; water in endospore staining