Cell staining.pptx

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Cell / Tissue
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
GENERAL THEORY OF STAINING

Staining Methods
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.

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.

or Accentuator is A substance (e.g., aniline) the presence of
which allows a combination between a tissue and a stain that
might otherwise be impossible.

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

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:

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.

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.

Step by Step Guide to Performing an H&E Stain
The hematoxylin and eosin stain (H&E) is the most widely used stain in histology
and histopathology laboratories.
When it is properly performed it has the ability to demonstrate a wide range of
normal and abnormal cell and tissue components and yet it is a relatively simple
stain to carry out on paraffin or frozen sections. In histopathology, a high
proportion of cases can be diagnosed by an experienced pathologist using an H&E
stain alone.
Small numbers of slides can be effectively stained manually, while in laboratories
that have a high throughput, staining can be performed successfully and
consistently by using automated slide stainers.
There are a number of different hematoxylin and eosin formulations in popular use,
each with various advantages and disadvantages. Some laboratories prefer to
prepare their own solutions whilst others choose ready-to-use commercial
products.

Introduction to H & E
The H&E stain provides a comprehensive picture of the microanatomy of organs
and tissues.
Hematoxylin precisely stains nuclear components, including heterochromatin and
nucleoli, while eosin stains cytoplasmic components including collagen and elastic
fibers, muscle fibers and red blood cells.
In a high-quality H&E stain, there are subtle differences in the shades of color
produced by the stains, particularly eosin, and this aids in the detection and
interpretation of morphological changes associated with disease.
It is important that people performing and assessing H&E stains for quality are
aware of the subtleties of the stain, know what can be achieved when the stain is
properly performed with high-quality reagents, and know what to look for
microscopically.
The maintenance of consistent, high-quality H&E stains is a fundamental
requirement in histopathology laboratories.
In the following sections, the basic steps in performing an H&E stain are outlined.

H & E Procedure
Remove the Wax
Following the preparation of a paraffin section, all the elements are infiltrated with
and surrounded by paraffin wax which is hydrophobic and impervious to aqueous
reagents. The majority of cell and tissue components have no natural color and
are not visible. The first step in performing an H&E stain is to dissolve all the wax
away with xylene (a hydrocarbon solvent).
Hydrate the Section
After thorough de-waxing, the slide is passed through several changes of alcohol
to remove the xylene, then thoroughly rinsed in water. The section is now hydrated
so that aqueous reagents will readily penetrate the cells and tissue elements.

H & E Procedure cont..
Apply the Hematoxylin Nuclear Stain
The slide is now stained with a nuclear stain such as Harris hematoxylin, which
consists of a dye (oxidized hematoxylin or hematein) and a mordant or binding
agent (an aluminum salt) in the solution. Initially this stains the nuclei and some
other elements a reddish-purple color.
Complete the Nuclear Stain by “Blueing”
After rinsing in tap water, the section is “blued” by treatment with a weakly alkaline
solution. This step converts the hematoxylin to a dark blue color. The section can
now be rinsed and checked to see if the nuclei are properly stained, showing
adequate contrast and to assess the level of background stain.

Remove Excess Background Stain (Differentiate)
On most occasions when Harris hematoxylin is employed, a differentiation
(destaining) step is required to remove non-specific background staining and to
improve contrast. A weak acid alcohol is used. After this treatment, blueing and
thorough rinsing is again required. Staining methods that include a destaining or
differentiation step are referred to as “regressive” stains.
Apply the Eosin Counterstain
The section is now stained with an aqueous or alcoholic solution of eosin
(depending on personal preference). This colors many nonnuclear elements in
different shades of pink.
slide will reveal all the important microscopic components and be stable for many
years.

Rinse, Dehydrate, Clear and Mount (Apply Cover Glass)
Following the eosin stain, the slide is passed through several changes of alcohol to
remove all traces of water, then rinsed in several baths of xylene which “clears” the
tissue and renders it completely transparent.
A thin layer of polystyrene mountant is applied, followed by a glass coverslip. If the
stain and all the subsequent steps have been properly performed, the

 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.

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.

• 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.
 Criteria of a good mounting medium:

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