procedure for Hematoxyline and Eosine staining in histopathology detpartment

1. THE STAINING METHODS
PRACTICAL-6

2. 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

3. BIOLOGICAL STAINING
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

4. To determines whether the resulting dye is acid or basic in character:
•BASIC DYE: such as methylene blue have the coloring substance in the basic part of the
compound.
1.ACID DYE: such as the eosin have the coloring substance in the acid components and the
base is colorless.
2.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.
BASIC DYE, ACID AND NEUTRAL DYES:

5. 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
(1) 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.

6. 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.

7. 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.

8. WITHIN THIS COURSE WE ARE GOING TO EXPLAIN AND APPLY FEW OF THEM,
these represent:
General purpose.
Selective methods
Specific methods

9. 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:
1. Removal of wax by xylene.
2. Removal of xylene by alcohol.
3. Removal of pigments and removal of colors.
4. Application of staining solutions.
5. Dehydration taking through ascending grades of alcohol.
6. Clear with xylene.
7. Mounting or cover slipping.
8. Labeling and examine microscopically.

10.  Factors which affect staining:
1.Solvents (alcoholic or aqueous solutions).
2.Low or high temperature during reaction.
3.Simple or multiple combinations of dyes.
4.The covering power of the dye.
5.The time (period) the dye acting.
6.The type of the tissue and the fixative used.
7.The type and thickness of the section .
8.The temperature applied during drying paraffin section on the slides.
9.The makeup of the dye.

11. Haematoxylin & Eosin
 Haematoxylin Solutions
 The three main alum haematoxylin solutions employed are:
1.Ehrlich’s haematoxylin
2.Harris’s haematoxylin
3.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.

12. Mounting
Mounting usually involves attaching the samples to a glass
microscope slide for observation and analysis.
 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.

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