1. Histological techniques: Haematoxylin and Eosin staining
VAN-504, Practical- 03
Histological techniques: Haematoxylin and Eosin staining

2. STAINING

3. GENERAL THEORY OF STAINING
STAINING METHODS:
As there are no ideal methods to demonstrate all tissue material and as sometimes material may need a number of staining methods,
so there are hundreds of stains methods in histology.
Staining has for its first and primary purpose the rendering of outlines and structures more distinct by giving them a color contrast with their surroundings (color image).
A second and more important use is for the differentiation of particular structures or substances which by their selective staining facilitate the histological analysis

4. BIOLOGICAL STAINING 1. Natural dyes: a. Haematoxylin from plant
DYES ARE CLASSIFIED INTO TWO GROUPES:
1. Natural dyes:
a. Haematoxylin from plant
b. Carmine from female cochineal bug
c. Orcein a vegetables dye extract
2. Synthetic: these are derived from
hydrocarbon benzene

5. BASIC DYE, ACID AND NEUTRAL DYES
BASIC DYE: such as methylene blue have the coloring
substance in the basic part of the compound.
ACID DYE: such as the eosin have the coloring substance in
the acid components and the base is colorless.
3. NEUTRAL DYE: such as leishmans stains are obtained by
combining aqueous solutions of basic and acid dyes.
The resultant precipitates are usually insoluble in water but they are soluble in alcohol. They give more colors; the nuclei take the basic dye, cytoplasm takes the acid dye, the granules give the third color. Leucocytes have the blue nuclei, pink or red cytoplasm and purplish granules

6. Classification of Stains
A. According to their chemical composition as
1. Organic; - hematoxylin stains, carmine stains, anilin stains
(coal-tar dyes; benzene derivatives),
2. Inorganic.
B. From another chemical aspect Basic
2. Acid, depending upon the chemical reaction of the staining principle.
3. Neutral
C. Histological stains are:
1. Nuclear (chromatin stains),
2. Plasma or general stains,
3. Special stains,
4. Impregnations.

7. Stains used for demonstrating the general relationship of tissue to each other
Simple stain- when staining solution contains one dye.
Compound stain- the staining solution is composed of more than one or more
dye.
Indirect staining- stained needs a mordant to work.
Direct stains- the stains work without adding a mordant.
A progressive stain- when the different elements in the tissue are colored in sequence an at the correct time differential coloration of tissue are achieved.
A regressive stain- when the tissue is over stained and then differentiated
(washed out) by removing excess stain from the unwanted parts of the
tissue.

8. A selective stain- the staining dyes are more than one substance in the same color; but easy to identify the substance you want to demonstrate either by morphology or by site.
Specific stain- when the stain acts only on a certain constituents of the cell or tissue and has a little or no effect upon other elements.
Impregnation- some elements are demonstrated by methods known as impregnation techniques. The solutions used in the impregnation are not stains ''NOT DYES" but are solutions of metallic salts.
Cytological Stains- which are used to demonstrate of minute structure in the nucleus and the cytoplasm of cells.

9. Routine staining is done to give contrast to the tissue being examined, as without staining it is very difficult to see differences in cell morphology
Hematoxylin and eosin (abbreviated H&E) are the most commonly used stains in histology and histopathology. Hematoxylin colours nuclei blue, eosin colours the cytoplasm pink
To see the tissue under a microscope, the sections are stained with one or more pigments

10. Special Staining
Other compounds used to colour tissue sections include:
safranin
oil red o
congo red
fast green FCF
silver salts
numerous natural and artificial dyes

11. Hematoxylin and Eosin (H & E)
H & E is a charge-based, general purpose stain. Hematoxylin stains acidic molecules shades of blue. Eosin stains basic materials shades of red, pink and orange. H & E stains are universally used for routine histological examination of tissue sections.

12. The Haematoxyline and Eosin
The haematoxyline and eosin stains are probably the most widely used histological stains. Its popularity is based on its comparative simplicity and ability to demonstrate clearly an enormous number of different tissues structure.
The staining procedure:
Removal of wax by xylene.
Removal of xylene by alcohol.
Removal of pigments and removal of colors.
Application of staining solutions.
Dehydration taking through ascending grades of alcohol.
Clear with xylene.
Mounting or cover slipping.
Labeling and examine microscopically.

13. Results Any well fixed tissue.
Fixation
Any well fixed tissue.
Staining Procedure
1. Deparaffinize and hydrate to water
2. If sections are Zenker-fixed, remove the mercuric chloride crystals with iodine and
clear with sodium thiosulphate (hypo)
3. Mayer's hematoxylin for 15 minutes
4. Wash in running tap water for 20 minutes
5. Counterstain with eosin from 15 seconds to 2 minutes depending on the age of the
eosin, and the depth of the counterstain desired. For even staining results dip slides
several times before allowing them to set in the eosin for the desired time
6. Dehydrate in 95% and absolute alcohols, two changes of 2 minutes each or until excess
eosin is removed. Check under microscope
7. Clear in xylene, two changes of 2 minutes each
8. Mount in Permount or Histoclad
Results
Nuclei - blue - with some metachromasia Cytoplasm - various shades of pink-identifying different tissue components

14. Factors which affect staining
Solvents (alcoholic or aqueous solutions).
Low or high temperature during reaction.
Simple or multiple combinations of dyes.
The covering power of the dye.
The time (period) the dye acting.
The type of the tissue and the fixative used.
The type and thickness of the section .
The temperature applied during drying paraffin section on the slides.
The makeup of the dye.

15. Haematoxylin & Eosin Eosin Solutions:
Haematoxylin Solutions
The three main alum haematoxylin solutions employed are:
Ehrlich’s haematoxylin
Harris’s haematoxylin
Mayer’s haematoxylin.
Eosin Solutions:
It can be used to stain cytoplasm, collagen and muscle fibers for examination under the microscope.
Structures that stain readily with eosin are termed eosinophilic. Eosin is most often used as a counter stain to haematoxylin in H&E (Haematoxylin and Eosin) staining. 

16. Mounting TWO TYPES OF MOUNTING MEDIUM AQUEOUS MOUNTING MEDIUM
Generally it is used for temporary mounting media; hours; days; or even weeks. This includes unstained preparations, some fluorescent techniques and most enzymes techniques.
RESINOUS MOUNTING MEDIA
This used for permanent preparations. They are used for routine work except when the substance to stain or the dyes are soluble in the dehydrating, clearing or mounting media.

17. Criteria of a good mounting medium
IT Should-
- be miscible with the last fluid from which you are mounting.
- have the same refractive index of the glass slide used.
- be clear and clean.
- flow freely when cover slipping.
- harden quickly.
- not crack on drying.
- not contract too much when setting or drying.
- not develop granules on drying.
- have the appropriate PH.
- be permanent.
- not colored with age.
- not fluorescence.
- not support life.
- not easy to remove its excess from the cover slip.

18. Staining Machine