Dr. Samah Kotb Nasr Eldeen

Biological stains are prepared from dyes which have been manufactured to rigid specifications; to ensure that they are suitable for specialized purpose for which they are made.

Dyes classified into two main groups: Natural dyes. Synthetic dyes.

1. Haematoxylin – from plant. Is extracted from the heartwood of the tree Haematoxylum. 2. Carmine – derived from female cochineal bug. 3. Orcein – a vegetable dye extract.

Are derived from hydrocarbon benzene. Simple benzene compounds have absorption bands in the U.V range of the spectrum.

Chromophores added to the benzene compound to move the absorption bands into visible portion of the spectrum giving the visible color. Benzene compounds containing chromophores are known as CHROMAGENS.

Chromagen is a colored substance; but its not a dye. That because it can easily washed out or removed. Another radical known as AUXOCHROME is added to the chromagen to make a dye not easily washed fast ex: Methylene blue, Eosin and others.

Benzene derivative + a chromophore + an auxochrome = Synthetic dye.

The nature of auxochrome determines whether the resulting dye is acid or base in character. Basic dye such as Methylene blue is coloring substance in the basic part of the compound; the acid radical remains colorless. And so on for the acid dye such as Eosin.

Neutral dyes as leishmans’ stain are obtained by combining aqueous solutions of basic and acid dyes; And they give more than two colors; the nuclei take the color of the basic dye while the cytoplasm stained by the acidic dye and the granules take the third color.

1. Micro-anatomical stains: used for demonstrating the general relationship of tissues to each other. 2. Cytological stains: used for demonstration minute structures in the nucleus and cytoplasm of cells. 3. Indirect staining: stain need a mordant; so the stain can work.

4. Direct staining : no need to add a mordant to the stain. 5. Simple stain : stain contains only one dye. 6. Compound stain : stain contains more than one stain. 7. Progressive stain: when the different elements in the tissues are colored in sequence and at the correct time differential coloration of tissues are achieved. It does not need differentiation.

8. Regressive stain: when tissues are over-stained and then destained or differentiated (washed out) by removing excess stain from the unwanted parts of the tissues. 9. Selective stain: stains more than one element in the same color, but its easy to identify the element you want to demonstrate either by morphology or site or by both.

10. Specific stain: when the stain acts only on a specific constituent or element of the cell or tissue and has no effect upon other elements.

11. Vital stain:  The living cells may be stained after removal from the organism and in this case the staining called SUPRAVITAL stain.  If the stain take place while the elements are still part of the living organism; the staining called INTRAVITAL stain. Intravital staining is done by injecting or introducing the stain into the body.

12. Negative staining: when the organism do not take the stain, but these unstained organisms are sharply contrasted against a stained background. 13. Fluorescent staining: staining by flurochrome dyes. there are two types: a) Primary or Auto-fluorescence. B) Secondary or induced fluorescence.

14. Mordant: are metallic substances (Aluminum, Iron & others) which act as a link between the stain and the tissue to be stained. Mainly used for indirect staining. These mordants are used in 3 ways: 1) Before the application of the dye “pre-mordanating”. 2) In conjunction with the stain. 3) After the application of the stain “post-mordanating”.

15. Accentuators: used for indirect stains. They differ from mordants. They are not essential for chemical union of the dye. But they increase the intensity and selectivity of the staining. 16. Accelerators: increase the staining power. They used with the metallic impregnation (not with the dyes).

17. Differentiation: it’s a de-staining or differentiation of an over stained tissues in a regressive staining techniques. Mordants and some dyes also act as differentiating agents. Washing in water or alcohol is a common means of differentiation. 18. Using controls in histology: when you apply a new staining solution you have to apply it side by side with your old working staining solution on sections from the same tissues.