Histological techniques for Bio-medical Researchers

Histotechnology

It is the preparation of tissues for microscopic examination. It is an effective diagnostic tool in clinical pathology. Histological  preparations  reveal normal tissue structure, tissue abnormalities  and cancerous conditions.

Branches of histotechnology

—  Histology- the microscopic study of the normal tissues.

—  Histopathology – the microscopic study of  tissues affected by disease.

—  Histochemistry – the techniques provide information on the chemical composition of parts of tissues.

—  Cytochemistry – the techniques provide information on the chemical composition of parts of cells.

Steps in the processing of tissues

1.    Fixation – preservation of tissues in its original condition.

2.    Dehydration – removal of water from tissues.

3.    Clearing – infiltration of paraffin solvent.

4.    Embedding – infiltration of paraffin wax.

5.    Microtomy – preparing thin slices of tissues.

6.    Staining – colouring of tissues.

7.    Mounting – arranging tissues on slides.

     Meaning of fixative 

A fixative is described as a chemical substance which will preserve the shape, structure, relationship and chemical constituents  of tissues and cells after death.

Purpose of fixing agents

1.    To kill and preserve living tissues.

2.    To stabilize the tissue and cell structure for subsequent treatments( wax embedding, sectioning, mounting).

3.    To prepare tissue for staining and optical contrast.

4.    To harden the tissue for section cutting

Requirements of a good fixative

1. Penetrate the tissue and cells rapidly and evenly.
2. Prevent autolysis and bacterial decomposition.
3. Preserve tissues in their natural state and fix all chemical cell components ( proteins, carbohydrates, fats etc.,)
4. Preserve cell volume.
5. Avoid excessive hardness of fixed tissue.
6. Allow enhanced optical differentiation by staining.
7. Make the cellular components insoluble to liquids used in tissue processing.
8. Be nontoxic and non-allergenic
9. Providing iso-osmotic conditions to the tissues.

General principles of fixation

Amount of fixing fluid should be approx. 10 to 20 times more than the volume of tissue held in a container  with a required fixation time. Temperature has an important effect. A lower temperature retard fixation –reduce autolytic reaction. A higher temperature will decrease the required for fixation but will increase autolysis.

Methods  of  Fixation 

—  Fixation by heat  - this denatures and coagulates proteins resulting in some distortion

                              but is useful in fixing smears.

—  Cryostat (freezing) fixing - it does not denatures proteins and minimizes distortion.

                               useful in locating particular chemicals - histochemistry

—  Fixation by chemicals - chemical fixatives are used.

Simple fixatives or primary fixatives or unmixed fixatives

Formalin-  non-coagulant fixative,acidic, cheap,easy to prepare, relatively stable

Mercuric chloride-Coagulant fixative,—  black precipitate in tissues

Glacial acetic acid - Protein precipitant,Colourless solution, Pungent smell

Ethyl alcohol-  Colourless liquid, Reducing agent

Osmium tetroxide- Strong oxidizing agent, Expensive, poor penetration

Potassium dichromate - Strong fixative,Fix lipids

Trichloro acetic acid -  Protein precipitant

Picric acid -  Protein precipitant,—  Used as saturated solutions

Micro-anatomical fixatives

—  10% formal saline

—  10% neutral buffered formalin

—  Heidenhain’s  Susa

—  Formal-sublimate

—  Formal-saline sublimate

—  Zenker’s fluid

—  Helly’s fluid

—  Bouin’s fluid

—  Gendre’s fluid

Physico-chemical features of fixatives

—  Degree of ionization

—  Oxidation-reduction potential

—  Reactions with proteins, lipids, carbohydrates

—  Rate of penetration

—  Shrinkage or swelling

—  Degree of hardening

—  Methods of washed out

—  Effect on staining

—  Compatibility with other fixatives

Paraffin wax technique of tissue blocks

—  Dehydration-the alcohol method

                                  -the acetone method

                                  - the dioxane (diethylene dioxide) method

—  Clearing- de-alcoholisation -clearing agents- xylene, benzene, toluene, chloroform.

—  Embedding- blocking – out in wax-wax impregnation

—  wax-wax impregnation- It can be cold wax infiltration and melted wax infiltration.

—  Complete wax infiltration is essential for the production of good sections.

—  Hard tissues requires a higher melting point wax.

—  Number wax changes and time in each wax change, depend upon the density and size of the tissue.

—  Embedding media – wax, gelatin, celloidin, polyester wax.

Microtomes—

The microtomes cut the tissue at a pre-determined uniform thickness. These instruments are designed for the accurate and serial cutting of thin slices of tissue. Several models are available – sliding, rotary, rocking ultra- thin microtomes.

Processing of paraffin – embedded tissues

Fixing tissues-> Post-fixation treatment->initial dehydration->complete dehydration-->Dealcoholization->wax impregnation->wax embedding->trimming the block->microtomy->drying sections-->de-paraffinization->hydration- down grading->staining->dehydration- up grading->de-alcololization->Mounting-->observing in microscope.

—Staining

Staining is used to obtain contrast between the constituent parts of a tissue section. The depth of colouration is affected by chemical affinity, density, and permeability. Certain stains are metachromatic –i.e. they are capable of imparting one colour to certain constituents and another to others.