THEORY OF STAINING

Dr . Samah Kotb Nasr Eldeen

INTRODUCTION

INTRODUCTION Several types of staining processes

are used to color tissues for

microscopical examination.

Staining methods depend on the

metallic impregnation or the production

of a colored substance from colorless

reagents.

The mechanism of how some of these

techniques work is not yet known.

There are physical and chemical

theories for staining mechanism.

Theories for staining mechanism

A.Physical theory: this depends on:

1. Osmosis.

2. Solubility.

3. Absorption.

4. Adsorption.

5. Permeability of the tissues.

B.The chemical theory: this is depends

on the pH.

1. basic.

2. Acidic.

3. Neutral.

4. Amphoteric “ acts as a base in acidic

solution and as an acid in a basic

solution .

Main Types Of Staining Processes Use In Histology

1) Production of colored chemical

substances:

Divided into:

(a) stable method

ex: Pearls and iron pigments “ferric salts” .

(b) Unstable method:

This is applied in enzyme histochemistry

In both methods we use colorless chemical

substances and end up after the reaction

with colored production; this is a pure

chemical mean “chemical theory”.

2) Metallic impregnation:

Silver technique and other like osmium

tetroxide and mercuric chloride. The metallic

impregnation with silver techniques divided into:

(a)Argentophilic: “direct”

This is when dealing with or demonstrating

substances which do not need an outside

reducer i.e. direct impregnation for

substances like melanin.

(b) Argyrophilic: “indirect”

This is when using outside reducer as light

(sun or artificial light), photographic developer

or hydroquinone to demonstrate substances

like fibrils and calcium.

Factors affect staining

Factors affect staining

1. The mode of action of dyes.

2. Solvents ex: alcoholic or aqueous solutions.

3. Low or high temperature during the reaction.

4. Simple or multiple combination of dyes.

5. Strong or weak dye solutions.

6. The covering power of the dye.

7. The time.

8.The type of the tissue.

9.The fixative used.

10.Type of section.

11.The thickness of section.

12.The temperature during drying paraffin sections.

13. The make up of the dye (in the factory).

Preparation of the stains

Preparation of the stains

1. Clean dry glassware.

2. The correct solvent.

3. Flasks and pipettes for silver solution

should marked and kept separately.

4. Silver and osmic tetroxide solutions should

be kept in dark bottles in a cool place.

5. Dilute ammonia for staining should be

freshly prepared and the stock kept in the

fridge.

6. Constituents of stains should be dissolved

in the order giving in the formulae.

7. Alcoholic solutions of stain should be kept

in glass-stoppered bottles or containers to

avoid evaporation which cause

precipitation of the stain.

8. All dye solution used to demonstrate

bacteria should be filtered immediately

before used.

9. Appropriate batch of dyes should be

used.

10. Controls of stains and test slides should

be applied.

Solvents

1. Water: tap or distilled water.

2. Ethyl alcohol (ethanol).

3. Methyl alcohol (methanol).

4. Acetone.

5. Phenol.

6. Buffer solutions.

7. Aniline water.

Staining process Equipment

1. Coplin jars.

2. Staining dishes.

3. Staining racks + staining

sets.

4. Staining machines.

Staining procedures

1. Removal of wax by xylene.

2. Removal of xylene by alcohol; taking by

descending grades of alcohol.

3. Removal of pigments.

4. Application of staining solution.

5. Dehydration; taking through ascending

grades of alcohol.

6. Clearing with xylene.

7. Mounting or cover slipping.

8. Labelling.

9. Examine under the microscope.

Thank you