BIOLOGICAL STAINING

Dr . Samah Kotb Nasr

Eldeen

INTRODUCTION

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.

NATURAL DYES1. 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 .

SYNTHETIC DYES

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.

BASIC, ACID AND NEUTRAL DYES

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.

TERMS USED WHEN STAINING

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.