L6: Staining Procedure

in Histology Technique

HISTOPATHOLOGY I

HIS 1213

Introduction

Hematoxylin and Eosin stain

Type or classification of dye used for 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.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 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 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 .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 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.)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 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 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)formalin pigment)

Hematoxylin and Eosin stain

H&E stain is the most widely used histological

stain.

Advantages: 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 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 andshades 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 celltypes 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 BEosin 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 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 Section thickness

Excessive hot plate temperature

Type of dye used for identification

of tissue

CONNECTIVE TISSUE STAINS

Trichrome stain 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 striationsThe 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 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 red-

purple 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.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)(DPX or Kirkpatrick and Lendrum, Clarite)