L6: Staining Procedure

in Histology Technique

HISTOPATHOLOGY I
HIS 1213


Introduction

Hematoxylin and Eosin stain

Type or classification of dye used for
identification of tissue

Direct and indirect staining technique

Introduction
Tissues and their constituent cells are usually
transparent and colour less when examined
under the light microscope.
Therefore, differentiation between various
structures is not noticeable

 Coloring (dyeing or staining) the sections of

tissues makes it possible to see and study the
physical features and relationships of the
tissues their constituent cells.
Different tissues possess different tissue
components of the cell, hence showing
different affinities for most dyes or stains.
Affinities tendency of a stain to transfer
from solution onto a section

 Hence, no single staining method will

demonstrate all the tissue structures present.
It is often necessary to carry out several
different stains on consecutive sections from a
block of tissue in order to make a diagnosis.

TISSUE STAIN INTERACTION

Van der Waals (eg: intermolecular
attractions)
Occur between all reagents and tissue
substrates .
Hydrogen bonding
It is a dye-tissue attraction when a hydrogen
atom is in between two electronegative atoms
(eg: O, N).
Covalent Bonding

FACTORS INFLUENCING STAINING REACTIONS

Physical factors
Chemical factors

 Physical factors
Osmosis and capillarity
(responsible for the penetration of stains into
porous tissues)
Absorption
(Demonstrated by the action of certain stains on
certain tissues in the presence of mineral
salts)

Selective absorption
(characteristic of certain substances to adsorb
certain ions from a solution more readily
than from others.)

 Chemical factors
 Based on the assumption that certain parts
of biological tissues are acid in nature (eg:
nuclei acid) while other parts are basic (eg:
cytoplasm).
 The colouring matter in basic dyes is
contained in the basic part of the
compound, leaving the acidic part
colourless.
 Vice versa.

Methodology of Staining
1. Removal of paraffin wax (dewaxing)
 Paraffin wax is not permeable to stains, it is
removed by xylene.
2. Removal of xylene
 Xylene is not miscible with watery solutions
and low grade alcohol and therefore it is
needed to be removed by absolute alcohol.

3. Gradual hydration with lower grade alcohol

 By immersion in 90% and 70%
(avoid diffusion currents that cause damages.).
4. Hydration with water
 Sections are rinsed thoroughly in distilled
water..

5. Removal of artifact pigment

 Artifact pigments may be present (due to
fixation).
 Removed by saturated picric acid (removing
formalin pigment)

Hematoxylin and Eosin stain

H&E stain is the most widely used histological
stain.
Advantages:
Simplicity (comparatively to other stainining
method)
Demonstrate clearly an enormous number of
different tissue structures

HEMATOXYLIN
Hematoxylin component stains the cell nuclei
blue black, with good intranuclear detail.
Hematoxylin itself is not a stain.
Instead, the oxidated form of hematoxylin
(Hematein) is a natural dye that is responsible
for the color properties.

 Hematein can be produced from hematoxylin

in 2 ways:
Natural oxidation
(exposure to light and air)
Chemical oxidation
- Chemical oxidizing agents convert
hematoxylin to hematein instantaneously,
hence hematoxylin solutions are ready for use
immediately after preparation

 In the absence of mordants, hematein have

poor affinity for tissue and hence is
inadequate as nuclear stain.
The most useful mordants of hematein are:
 Salts of ammonium
 Iron
 Tungsten
Most mordants are mixed and present in the
hematoxylin staining solutions.

 Type of hematoxylin solutions:

 Alum hematoxylins (Mayers hematoxylin)
(routinely used in H & E stain, produce good
nuclear staining)
 Iron hematoxylins
 Tungsten hematoxylins
 Molybdenum hematoxylins
 Lead hematoxylins
 Hematoxylin without mordant.

EOSIN
Eosins are xanthene dyes.
Eosin component stains cell cytoplasm and
most connective tissue fibers in varying
shades and intensities of pink, orange and
red.
It is most suitable stain to combine with an
alum hematoxylin to demonstrate the general
histological architecture of a tissue.

 Eosin is able to differentiate and distinguish

between:
 Cytoplasm of different types of cell
 Connective tissue fibres between different
types of cell
Acetic acid may be added to sharpen the
staining.

 The commercially available types of Eosin:

Eosin Y (most widely used)
Ethyl eosin
Eosin B

 The need for consistency of staining is vital to

avoid difficult histological interpretation.
Hence, it is important to ensure the quality of
staining.
In general, automated staining machines is
able to produce accurate and consistent
staining.
However, problems might arise with the
hematoxylin staining.

 Factors affecting the quality of staining:

 Fixation
 Variation in processing schedules
 Section thickness
 Excessive hot plate temperature

Type of dye used for identification

of tissue
CONNECTIVE TISSUE STAINS
Trichrome stain
general term for a number techniques for the
selective demonstration of muscle, collagen
fibers, fibrin and erythrocytes.

 Example:
The demonstration of fibrin
(Techniques : Gram Weigert; phosphotungistic
acid-hematoxylin; trichrome methods)
The demonstration of muscle striations
( H & E; trichrome methods.)

Periodic acid-Schiff (PAS) technique

Is the most versatile and widely used
technique for the demonstration of
carbohydrates and glycoconjugates.
With PAS, we can assess the thickness of
basement membrane
(increase in basement membrane can be a
pathological conditions eg: kidney.)

 The PAS technique is based upon the

reactivity between free aldehyde groups
within carbohydrates and the Schiff reagents.
In PAS technique, it involves the oxidation of
1,2 glycols within carbohydrates to form
adjacent aldehydes.

 Oxidizing agents commonly used is periodic

acid (HIO4) (0.5-1.0%, 5-10 mins).
Other oxidizing agents are: potassium
permanganate, chromic acid, etc.
The reaction between the aldehyde and the
Schiff reagent results in the formation of a
bright red magenta end product.

 The intensity of the color that develops

following the reaction with Schiff reagent is
dependent upon the concentrations of glycol
structures in the tissue.

FAT STAINS
The most common stains used to demonstrate
fats/lipids are oil soluble dyes.
This group of dyes includes Sudan III, Sudan
IV, oil red O and Sudan black B
Sudan black B is the most sensitive of them.

 In order to penetrate fats, the Sudans must

dissolved in organic solvents (solvent cehicle).
The solvents should be sufficiently diluted to
avoid extracting the lipids itself.
Example of solvents used:
Ethanol
Isopropanol
 Triethyl phosphate
 Propylene

 For general use, 70% of ethanol is an

adequate solvent for Oil red O and Sudan
black.
Besides lipids, Sudan black B stains
phospholipids and neutral fats.
The stained lipids will appear in orange-red.

FEULGEN REACTION
It is the standard technique for demonstrating
deoxyribose.
It involves mild acid hydrolysis (employing 1
M HCL at 60oc) which is the critical part of the
method.
It causes the purine-deoxyribose bond to
break, resulting the exposure of aldehydes
which can be demonstrated by the use of
Schiffs reagent.

 Elements containing DNA are stained at redpurple color, whereas RNA is not
demonstrated.

METHYL GREEN-PYRONIN
Demonstrating both DNA and RNA.
Staining DNA in green and cytoplasmic RNA in
red.
Methyl green is impure dye containing methyl
violet which can be removed by washing with
chloroform.
Pure methyl green appears to be specific for
DNA.

 Pyronin on the hand, binds with RNA.

The pH and concentration of the staining
solution is critical.
Dehydration after staining is important as well
as it will give a greener nuclear staining effect.

Mounting stained Specimens

In order to obtain best result with stained
sections, the slide is mounted in a
transparent medium with refractive index
close to that of the glass slides.
Mounting medium is desired to protect the
stained section from physical damage.
It can also avoid fading of the stain due to
heat or oxidation.

 Example of mounting media:

Canada Balsam
Gum Damar
Synthetic Resins
(DPX or Kirkpatrick and Lendrum, Clarite)