SECTIONING & STAINING

SECTIONING

Paraffin blocks of tissues are sectioned by means of a Microtome. The sections which are

produced are capable of transmitting light and are at micron level thickness. Black

bottomed water both is used in sectioning because there is no interference of light.

The sectioning procedure includes following steps:

1. After embedding paraffin blocks are kept on a cold plate with their surfaces upside

down. Just before sectioning blocks are transferred to ice cubes.

2. A suitable knife is inserted in microtome and knife edge is kept slightly away from

the block holder by using feedback mechanism.

3. Then paraffin block is fixed in the block holders.

4. Knife edge is brought towards the block surface by using feedback mechanism when

block just touches the knife edge, microns are set for trimming of the block.

5. Trimming is done at 10-15u but in case of tiny biopsies trimming is rarely done.

6. After trimming fine sectioning is done at 3-4u and ribbon of sections is floated on

the surface of cold tap water in a kidney tray and wrinkles are removed by using

forceps to flatten the ribbon of sections.

7. Then 2 or 3 sections from the ribbon are transferred to warm water in a water bath

having temperature 5-10o

C less than the melting point of paraffin wax. Here sections

are completely flattened due to expanding of paraffin wax.

8. Then sections are picked on slides already smeared with adhesive. (Mayer¶s albumin

glycerol adhesive).

9. The slide is then properly numbered and kept on a hot plate having temperature of

about 5-10o

C above than the melting point of the wax. This step is meant for drying

of sections and melting of paraffin wax surrounding the sections.

Adhesive

Normally Mayer's Albumin glycerol adhesive is used. No adhesive is used when tissues are

of uniform thickness. It is used when:

i. Tissues are not of uniform thickness.

ii. Tough tissues material is received in laboratory

iii. There are prolonged staining techniques, e.g in immune-histochemistry.

Preparation:

1. Egg white is highly viscous.

2. Glycerol is a form of alcohol so termed as glycerin.

3. Glycerin is also viscous so 50% of it is added to 50% egg white to form Adhesive.

4. Fungus may grow in the adhesive so preservatives like thymol can be added.

STAINING

When an unstained section is observed under a microscope, different cellular components

transmit the white light almost equally. In such a case, no sharp contrast is present and we

can observe very little details of the cells. Staining is preformed to colour the cellular

components differently so producing a contrast or differentiation. By this way different

cellular components become more prominent for microscopic studies. The colour is infact a

perception of eye and determined by the specific wavelength that is perceived by a normal

eye.

Staining is helpful to produce a contrast because different cellular components possess

affinities for different stains, e.g. nuclei show a high affinity for basic dyes like

Haemotoxylin whereas cytoplasm components shown a high affinity for acidic dyes like

Eosin.

Factors Affecting Staining

In staining, different physical and chemical factors are involved. The most important factors

are:

ƒ Acidic and Basic Reaction

Cellular components are acidic or basic in nature, so as the stains. Very clearly therefore,

acidic components of cell are stained with basic dyes and vice versa. 

ƒ Adsorption

In adsorption certain small molecules of dyes are attached and fixed on the surface of larger

molecules of the tissues components e.g. staining of dextrons with iodine.

ƒ Differential Solubility

In differential solubility, a stain or dye bears different solubilities into different solvents,

e.g. in lipids staining the dyes like Sudan Black B or Oil Red O are alcoholic solutions.

When these solutions are applied to lipids, due to higher solubility in lipids the dyes

gradually leave the original solvent (alcohol) and enter the lipids and so staining is brought

about.

Classification of Stains/Dyes

Stains are coloured materials and are such chemicals which react with different components

like tissue components with different affinities to give them different colours. Stains are

classified by two ways:

1st Classification

Stains are classified as natural stains and synthetic stains.

i. Natural Stains:

Natural stains are extracted from a natural source and they constitute a small group of dyes.

Examples are haemotoxylin, Orcein, Litmus, Saffron and Carmine.

ii. Synthetic Stains:

because these are synthesized from substances obtained from coal tar. Examples are Eosin,

basic fuchsin, toluidine etc.

2nd Classification

Stains are classified as acidic stains, basic stains and neutral stains.

i. Acidic Stains:

In acidic stains, e.g. eosin, the colouring substance is contained in the acid component while

basic component is colourless. These stain the basic components of cell. The base is usually

sodium. 

ii. Basic Stains:

In basic stains, such as haemotoxylin, the colouring substance is contained in the basic part

of compound. The acidic radical is colourless. These stain the acidic parts of the tissues. 

iii. Neutral Stains:

These are obtained by combining aqueous solutions of basic and acidic dyes. The resulting

dye is insoluble in water but soluble in alcohol.

nuclei (acidic part) and cytoplasm (Basic part).

3rd Classification

i. Microanatomical Stains:

These stains are used for demonstrating the general relationship of tissues to each other. 

Nuclei and cytoplasm are differentiated but their included structures are not necessarily

differentiated.

ii. Cytological Stains:

Cytological stains demonstrate the minute structures in the nucleus and cytoplasm of cells

without the general differentiation of various tissue types.

Simple Benzene and Derivatives

Benzene C6H6 and its other simple derivatives are colourless in visible spectrum of light

because their adsorption band lies in the UV region. There are certain atomic groups which

when added to colourless benzene molecules, the adsorption is shifted from UV region to

A chromogen is a coloured product but it differs from a stain or dye because any colour

which it imparts to a tissue is easily washed off and is not retained by the tissue.

A dye or stain bears acidic and basic parts and so it shows salt forming properties. The

retention of a dye by any tissues component is its basic feature. So a synthetic dye may be

portrayed as a benzene subordinate, to which a chromophore and an auxochrome have been 

added.