FIXATION OF TISSUES.

Fixation is the process of preserving a tissue after death or removal in as life like manner as possible to prevent post mortem changes by use of a fixative.

Postmortem changes are changes that take place on tissue cells after the death of the body or removal from the body.

There are two changes namely;

Putrefaction and autolysis.

Post mortem changes.

Putrefaction.

This is the breakdown of tissue cells after death

due to bacterial action often with formation of

gas.gas.

The bacteria disseminate from the alimentary

tract and the surrounding organs.

During life, these bacteria are usually

commensals.

Contd..

Autolysis.

This is the dissolving of cells due to enzymatic action.

After death, the normal behavior of enzymes is altered. The lysosome rupture releasing the altered. The lysosome rupture releasing the enzymes which dissolve the surrounding cells.

This is especially prominent in specialized organs like the brain, liver, kidney and the alimentary canal.

A lot of times these enzymes break down peptides to individual amino acids.

Signs of Autolysis

During autolysis different parts of the cell undergo different changes;

1. The nucleus.

It first condenses(pyknosis), then fragments (karyorrhesis) and eventually disappears (karyorrhesis) and eventually disappears (karyolysis)

2. The cytoplasm.

It swells, becomes granular and eventually becomes a homogenous mass with the loss of the normal staining reaction.

Contd..

3. The Epithelium.

It desquamates and splits away from the

basement membrane.

4.The glycogen.

It diminishes or diffuses out of the cell, leaving

empty spaces.

Fixation and Fixatives

The chemical substances used to prevent

postmortem changes occurring are called

fixatives.

NB;NB;

Fixation is the foundation for processing of

tissues for diagnosis.

There is no remedy for poorly fixed tissues.

Criteria of a good fixative.

�Must cause sudden death to the tissue cells

�Must be antibacterial

�Must inactivate enzymes

�Must penetrate tissue evenly and rapidly.�Must penetrate tissue evenly and rapidly.

�Must render insoluble substances of the cell.

�Must preserve the cells in as life like manner

as possible

Contd..

�Must harden tissue and enable easy manipulation of

naturally soft tissues of organs such as brain, liver

and spleen.

�Must alter to varying degrees the refractive indices of Must alter to varying degrees the refractive indices of

different cell structures so that they are made more

visible due to differentiation.

�Must fortify the tissue against the harsh effects of

solutions used for processing the tissue.

�Must have good effects on staining.

Contd..

NB;

To obtain good results, the structures to be

demonstrated must be borne in mind when

deciding on fixative to be used.deciding on fixative to be used.

For good results.

1. Tissue must be fixed as soon as it is removed

or after death.

Contd..

1. Amount of fixative should be 15 – 20 times

the bulk of the tissue to be demonstrated.

2. Thin slices of tissue should be made to enable 2. Thin slices of tissue should be made to enable

quick and even penetration of the fixative.

CLASSIFICATION OF FIXATIVES.

They are classified into simple and compound

fixatives;

a. Simple Fixatives.

These consist of only one chemical substance in These consist of only one chemical substance in

solution. Examples include;

1. Formaldehyde.

It is used as 10% formal saline, neutral formal

saline or buffered formal saline.

Formaldehyde Contd..

It is soluble in water up to the extent of 37- 40%

to form formalin.

Advantages.

�A powerful reducing agent which preserves �A powerful reducing agent which preserves

fats and mucin.

�Has beneficial hardening effects on tissues.

Contd..

Disadvantages.

�Gives off an unpleasant vapor that causes

irritation to the eyes and respiratory system.

�Prolonged immersion of hands in this solution �Prolonged immersion of hands in this solution

causes formalin dermatitis.

�When stored for long, formalin often

produces a white precipitate called

paraformaldehyde which is acidic in reaction

due to formation of formic acid.

2. Mercuric Chloride.

Is rarely used alone but incorporated in many compound

fixatives.

Advantages

�Permits brilliant cytoplasmic staining

�Results with metachromatic stains are excellent�Results with metachromatic stains are excellent

Disadvantages

�Hardens and shrinks tissue.

�Does not penetrate tissues quickly.

� Forms mercuric chloride pigments making tissue

radio opaque.

3. Potassium dichromate.

Advantages.

Gives a homogenous fixation of the cytoplasm without

precipitation

Preserves lipids and is the usual fixative employed for Preserves lipids and is the usual fixative employed for

mitochondria.

Disadvantages.

Tissue fixed with potassium dichromate must be

thoroughly washed in water before commencing

dehydration in alcohol to avoid formation of

insoluble oxides.

4. Osmium Tetroxide.

It’s a pale yellow powder.

Advantages.

It is used in electron microscopy for good

preservation of nuclear detail preservation of nuclear detail

it demonstrates lipids by fixing and staining

them black

Contd..

Disadvantages

It penetrates poorly and therefore only fixes thin

tissue sections.

It is poisonous and may be deposited in the eye

causing conjunctivitis. It should be therefore

used in a fume cupboard.

5. Picric Acid.

Picric acid is a peculiar chemical since it has several

applications such as;

As a stain

As a fixativeAs a fixative

As a differentiator

Advantages.

Because of the yellow coloration it imparts to

tissues it is useful in locating minute tissue

fragments that otherwise be overlooked.

Advantages contd..

It gives good results with trichrome stains.

Disadvantages.

Its highly explosive and must be kept damp by

storing under water.storing under water.

It forms water soluble picrates.

NB; it precipitates proteins and combines with them

to form water soluble picrates. These must be

made insoluble by treating them with alcohol.

6. Chromic Acid

Advantages.

It precipitates all proteins and perhaps is the only

fixative that preserves carbohydrates rendering

them insoluble in water.them insoluble in water.

Disadvantages.

Tissues fixed in chromic acid must just as in

potassium dichromate be washed well in running

tap water to avoid the formation of an insoluble

oxide.

7. Trichloroacetic Acid (TCA)

It causes swelling in tissues and thus is used in

compound fixatives like heiden hain susa to

counteract the shrinkage effect.

It is at times used as a weak decalcifying It is at times used as a weak decalcifying

solution.

It’s a general protein precipitant.

8.Glacial acetic acid.

Is a colorless solution with a pungent smell.

Its called glacial because it solidifies at 17 o Celsius.

It swells the collagen fibers and because of this it is

used in many compound fixatives to counteract used in many compound fixatives to counteract

the shrinkage effects of other reagents.

It does not fix proteins but nucleoproteins are

precipitated and therefore used in fixatives for

chromosomes.

9. Absolute alcohol.

Ins mainly used in dehydration of tissues.

Occasionally it is employed as a fixative in fluid

particularly used for enzymes.

Also used in the fixing smears.Also used in the fixing smears.

It has a slow penetration except when used in

carnoy’s fluid.

COMPOUND FIXTIVES

These are made of two or more chemical fixing

agents.

They are divided into;

�Micro anatomical fixatives�Micro anatomical fixatives

�Cytological fixative

a) Nuclear fixatives

b) Cytoplasmic fixatives

Microanatomical Fixatives.

These preserve the microanatomical structure of

the tissue with correct tissue layers and cells.

They preserve various layers of tissue and cells

in relationship to one another and allow the in relationship to one another and allow the

study of the general tissue structure.

1. Formal sublimate.

Formula;

Saturated acq. Mercuric chloride – 900ml

Formaldehyde – 100mlFormaldehyde – 100ml

Duration; 12 – 24 hours.

Advantage; Is good when secondary fixative.

Gives brilliant staining reactions with

acidic dyes and metachromatic

reactions.

2. Zenker’s formal

Formula

Mercuric chloride – 5g

Potassium dichromate – 2.5g

Sodium sulphate – 1gSodium sulphate – 1g

Distilled water – 100ml

Acetic acid( ethanoic acid) 5ml, added just before

use.

Duration; 3 – 18 hours.

Advantages

Is very good for cytoplasmic and connective

fiber stains.

DisadvantagesDisadvantages

Forms mercuric chloride pigments in the tissues.

Tissues fixed in zenker’s fluid must be washed in

water to remove the potassium dichromate.

3. Helly’s fluid

It has same ingredients as those of zenker’s

fluid except that instead of acetic acid, 5ml of

formaldehyde is added just before use.

Duration of fixation; 6 – 24 hours.Duration of fixation; 6 – 24 hours.

It is used as a secondary fixative and must be

washed in running tap water thereafter.

4. Bouin’s fluid.

Formula

Saturated acq. Picric acid – 75 ml

Formaldehyde – 25mlFormaldehyde – 25ml

Acetic acid (ethanoic acid) – 5 ml.

Duration of fixation ; 6 – 24 hours

Advantages

Preserves glycogen well.

Is a good fixative for chromosomes.

Disadvantage

Causes complete lysis of the red blood cells.Causes complete lysis of the red blood cells.

It makes the collagen fibers to swell.

It imparts a yellow color, however the tissue is removed

by treating the tissue with alcohol followed by 2.5%

sodium thiosulphate to avoid the formation of a

precipitate with aniline dyes.

5. Hedenhain’s Susa

Formula

Mercuric chloride – 4.5 g

Sodium chloride – 0.5 g

TCA – 2g

Acetic acid (ethanoic acid) – 4mlAcetic acid (ethanoic acid) – 4ml

Formaldehyde – 20 ml

Distilled water – 100ml.

Duration; 12 – 24 hours.

It penetrates tissues evenly and it gives brilliant staining

results.

5. 10% formal saline.

Formula

Formaldehyde – 100 ml

Sodium chloride – 8.5 g

Tsp water – 900 mlTsp water – 900 ml

Duration; 6 – 48 hours.

Disadvantages

It forms acid formaldehyde which can be

prevented by adding buffer or by storing it on

a layer of marble chips ( calcium carbonate) a layer of marble chips ( calcium carbonate)

which neutralizes the acid.

It forms formalin pigment in the tissue.

7. Buffered 10% formalin ph 7.0

Formula

Formaldehyde – 100 ml

Tap water – 900 ml

Sodium dihydrogen phosphate – 4gSodium dihydrogen phosphate – 4g

Anhydrous sodium phosphate – 5 g

Duration ; 24 – 48 hours.

Advantages

Imparts uniform consistency to the tissue.

Restores color of tissue

Cytological fixatives.

These generally preserve the intracellular

structure and inclusions of the cell.

i) Nuclear fixatives; these preserve the nuclei

and its inclusions.and its inclusions.

Some common nuclear fixatives include;

carnoy’s fluid, Fleming's fluid, and Clarkes's

fluid.

1. carnoy’s fluid.

Formula

Absolute ethanol; 60 ml

Chloroform; 30ml

Acetic acid(ethanoic acid); 10mlAcetic acid(ethanoic acid); 10ml

Duration; 10ml

It is used for very urgent biopsies.

It penetrates the tissue quickly and evenly

2. Fleming's fluid.

Formula

1% chromic acid;15 ml

2% osmium tetroxide ; 4ml

Acetic acid(ethanoic acid); 1mlAcetic acid(ethanoic acid); 1ml

Duration; 2 -3 days.

It penetrates poorly

Causes blackness to the tissue

Does not do well with alum hematoxylin

3. Clarkes fluid

Formula

Absolute ethanol; 75 ml

Acetic acid ; 25 ml

Duration ; 15 minutes.Duration ; 15 minutes.

Properties.

Penetrates tissue rapidly

Is good for chromosomal studies.

Cytoplasmic fixatives.

These preserve the cell cytoplasm and cytoplasmic

inclusions.

Common cytoplasmic fixatives include Muller’s fluid,

Orth’s fluid, and Schaudin’s fluid.

1. Muller’s fluid.

Formula

Potassium dichromate

Sodium sulphate

Distilled water.

Properties.

It takes 24 – 48 hours.

Has good mordanting effects on tissue.

2. Orth’s fluid.

Formula

To 100 ml of Muller's fluid add 10 ml of

formaldehyde just before use.

Properties.

Takes 24 – 48 hours to fix a tissue.

Is the best fixative for the demonstration of

chromaffin tissue in cases of chromaffin tissue in cases of

pheochromocytoma ( a tumor of the adrenal

gland)

3. Schaudinn’s fluid.

Formula ; saturated acq.mercuric chloride 2

parts and 1 part of absolute ethanol.

PropertiesProperties

Takes 15 minutes to fix smears.

Is a good fixative for wet smears.

Forms mercuric chloride pigments if fixation is

prolonged.

Special fixatives.

These include histochemical, vapor and fixatives

for exfoliative cytology.

Special fixatives.

Histochemical fixatives are used to preserve Histochemical fixatives are used to preserve

lipids and enzymes in the tissue.

The commonly used fixatives are buffered

formal saline, cold acetone and absolute

alcohol.

1. Buffered formal saline.

The tissue must be thoroughly fixed and the

tissue must be washed well in tap water

before cryostat sections are cut.before cryostat sections are cut.

2. Cold acetone

This fixative gives very good results particularly

in cases for the demonstration of phophates.

3. Absolute ethanol.

This fixative is good for sections cut from freeze

dried tissues.

Note;Note;

A good histochemical tissue should preserve the

constituents to be demonstrated.

It should not react with the reagents to be used

in the process of visualization.

Vapor fixatives.

These are fixatives that are used to fix

�cut sections of fresh tissues

�Thin blocks of freeze dried tissues

�Cytological smears�Cytological smears

The three common vapor fixatives include;

�Formaldehyde

�Acetaldehyde

�Glutaradehyde.