Fixation

The process, by which the components of the cells/tissues are fixed in their physical/chemical state so
that they can withstand the treatment with many reagents with minimal loss in their structure, is called
fixation.

Routine fixative: 10% formalin

Time: 14-16hrs

Volume: fixative: tissue = 10:1

Purpose of fixation: (changes in unfixed-tissue)

A lot of changes occur if a biopsy specimen is not fixed. So, after the removal of tissue, fixation should be
carried out as soon as possible to

● Prevent autolysis (breakdown of tissue due to the action of enzymes from dead cells)
● Prevent putrefaction (decomposition due to the invasion of tissue by bacteria)
● Prevent the tissue from undergoing osmotic shock (burst/lysis after H2O absorption)
● Prevent distortion of the tissue
● Prevent shrinkage of the tissue

Fixatives:

The chemicals used in fixation, are called fixatives.

Properties of an ideal fixative: ( Important )

● Prevents autolysis & putrefaction

● Preserves tissues in their natural state
● Preserves tissue volume
● Fixes all components (protein, carbohydrates, fats)
● Avoids excessive hardness of fixed tissues
● Makes cellular components insoluble to reagents used in tissue processing
● Allows enhanced staining of tissue
● Is non-toxic/non-allergic for the users
● Is cheap/not be very expensive

Principles of fixatives:

The fixative acts to denature proteins by

● Coagulation process (secondary & tertiary proteins → insoluble gels)

● Additive process (crosslinking of end-groups of amino acids)
● Combination of coagulative & additive processes
● Promote the attachment of dyes to particular cell components by opening up of protein side
groups to which dyes may attach
● Remove bound-H2O to increase tissue refractive index to improve optical differentiation
 ● Alter refractive index of tissues to improve contrast for viewing without staining

Precautions:

Sample size = 2-3mm³

When tissue is fixed, it is important to keep the sample size small, as increased thickness will retard
fixative penetration.

Volume-range of fixative = 20-25 times the volume of the tissue.

Removal of extra coatings:

The peritoneum/capsule around the tissue should be removed/pierced. The blood & mucus should be
rinsed off with saline.

Cutting material:

The tissue should be cut with a new, sharp razor blade/scalpel, rather than scissors to avoid tissue
damage by squeezing.

Special handling:

Some tissues/organs (e.g. lung, eye etc.) will require special handling to ensure that the fixative reaches
to all internal components.

Care:

Care should be given to ensure that the specimen has one or more cut sides to guarantee good
penetration of the fixative

Alternative-fixation:

Prolonged fixation may result in the chemical masking of specific protein targets & prevention of Ab-
binding during immunohistochemistry protocols. In such cases, alternative fixation methods may be
used. Therefore, there is no universal fixative, which will serve all requirements.

Types of fixatives
There are 3 main types of fixatives:

● Primary fixatives
● Compound fixatives
● Cytological fixatives
1. Primary fixatives:

Consist of a single fixative in solution. These may be in absolute form.

Examples:

● Absolute ethanol
● 10% formalin
2. Compound fixatives:
Consist of 2/more fixatives in solution.

Examples:

● Zenker’s fluid
● Helly’s fluid
● Bouin’s fluid
3. Cytological fixative:

Preserve cellular structures/inclusions (e.g. mitochondria). These may be alcohol-based.

Examples:

● Ethanol
● Methanol
● Ether

Classification of fixatives
Criteria:

● Action on proteins (coagulant & non-coagulant fixatives)

● Type of fixative
● Use of fixative

Classification:

There are 3 classifications of fixatives

● Tissue fixatives
● Cytological fixatives
● Histochemical fixatives
1. Tissue fixatives:
a) Buffered formal saline
b) Buffered glutaraldehyde
c) Zenker’s formal saline
d) Bouin’s fluid
2. Cytological fixatives:
a) Ethanol
b) Methanol
c) Ether
3. Histochemical fixatives:
a) Formal saline
b) Cold acetone
c) Absolute alcohol

Common fixatives
1. Routine formalin:
Formalin is sold as 40% W/W soln. of formaldehyde gas in H2O.

Use:

It is used as 10-15% V/V soln. in normal saline/CaCl2 soln. called 10-15% formalin/formal saline.

Advantages:

● Is cheapest.
● Most popular fixative.
● Preserves all elements including fats & keeps phospholipid insoluble in fat solvents.
● Does not precipitate protein but combine with –NH2 group of protein to form an insoluble gel.

Disadvantages:

● Formation of acidic crystals “haematin” in the tissue b/c 10% formalin has acidic pH.
● These crystals interfere in staining.
2. Buffered formalin:

Due to acidic pH of routine formalin (10% formal saline), phosphate buffers are added to make it neutral
fixative so that its disadvantages should be minimized.

Preparation:

To make 10% buffered formal saline, mix

● Pure formalin 10ml

● Sodium dihydrogen phosphate 0.4g
● Disodium hydrogen phosphate 0.65g
● Normal saline up to 100ml

Advantages:

● Tissues can be left in it for longer time period e.g. one year.
● No damage/hardening of tissue.
● Sectioning is easy.
● No formation of haematin crystals.
● A number of staining procedures can be used.

Chemicals used during fixation

1. Ethyl alcohol/ethanol: (C2H5OH)

Use:

Used in 90-100% strength.

Advantages:

● Preserves glycogen.
● Useful for histochemistry (glycogen, uric acid & iron).
Disadvantages:

● Precipitates albumin & globulin but not nucleoproteins.

● Causes shrinkage & hardening of tissue.
● Destroys mitochondria.
● Is a reducing agent and, therefore, cannot be used with chromic acid, chromates & osmium
tetra oxide.
2. Mercuric chloride: (HgCl2)

Use:

Used as 70% saturate/half saturated aqueous soln.

Advantages:

● Valuable for nuclear fixation but rarely used alone.

● Penetrates rapidly, precipitates proteins, fixes chromatin well & enhances its subsequent
staining capability.
● Is a reducing agent and, therefore, cannot be used with chromic acid, chromates & osmium
tetra oxide.
3. Picric acid:

Use:

Used as 1% saturated aqueous soln.

Advantages:

● Not used alone.

● Does not harden the tissue.
● Does not affect the staining.
● Preserves glycogen & nearly all other elements.

Disadvantages:

● Penetrates poorly.
● Causes shrinkage.
4. Chromic acid:

Use:

Used as a pure chemical/mixture of dichrome & acetic acid (e.g. Zenker’s fluid).

Advantages:

● Preserves most elements.

Disadvantages:

● Is an oxidizing agent & therefore incompatible with formalin & alcohol.

5. Potassium dichromate:
Use:

Used as 2-3% aqueous soln.

Advantages:

● Is a good cytoplasmic fixative.

Disadvantages:

● Is a bad nuclear fixative b/c it is a weak oxidizing agent & tend to dissolve chromatin.
● Gives chromaffin reaction.
6. Osmium tetra oxide (osmic acid):

Use:

Used as 2% aqueous soln.

Advantages:

● Preserves fat & gives a black precipitate of osmium dioxide with unsaturated fats.
● Preserves very fine cell details of organelle e.g. golgi apparatus etc.

Disadvantages:

● Is expensive.
● Is unstable.
● Penetrates badly.
● Rapidly coverts to vapors which are irritating.
● Is a powerful oxidizing agent & therefore incompatible with formalin & alcohol.
7. B-5 fixative:

Advantage:

● Used for fixation of lymph nodes.

8. Zenker’s fluid:

Advantage:

● Used for bone marrow trephine biopsy

● Also used for Negri bodies.

Factors affecting fixation

Size & thickness:

Increase in size & thickness of the tissue inversely affect fixation.

Tissues having coatings:

Tissues covered by large amount of mucus/blood fix slowly.

Fatty tissues:
Fatty & lipomatous tissues fix slowly.

Agitation:

Fixation is accelerated by agitation.

Temperature:

Fixation is accelerated by maintaining temp. around 60°C.

Time:

Smaller biopsies fix in few hrs. & larger must be cut, open & then put into fixative for 8-12hrs.

Volume of fixative:

Fixative must be 10-20times the vol. of tissue.

Cost:

Expensive chemicals must be used in low conc.

Penetration of fixative:

Different fixatives have variable penetration e.g.

● Formaldehyde: 0.78mm/h
● Mercuric chloride: 0.5mm/h
● Ethanol: 1.0mm/h
● Potassium dichromate: 1.33mm/h

Fixation containers
Different sized Jars/bottles with screw caps, used for fixation.

1. Large specimens:

These should not be squeezed into small containers. This will result in inadequate fixation & autolysis. If
a specimen is too large, not to fit in container, it should be bought as such to the lab. e.g. Amputated
foot may be wrapped in a rubber sheet.

2. Bulky specimens:

These should be bisected cleanly with a large sharp knife before being placed in fixative e.g. Large
tumors, spleen etc.

3. Solid organs:

Cut slices as big as necessary but not thicker than 5mm.

4. Brain:

For fixing the uncut brain, pass a thick thread under the vessels at the base of the brain. The organ is
gently lowered inti a bucket containing the solution & allowed to float with the help of thread.
 5. Hollow viscera:

Cavity-containing internal organs such as stomach & intestine should be opened at both ends/cut open
along with their length (stomach should be opened at greater curvature.

6. Small biopsies:

To preserve small biopsies in their original orientation, it is better to place them on a piece of filter
paper & then put into fixative.

Transportation of biopsy specimen to the lab.

The specimen should be taken fresh to the lab./wrapped in moist cotton/normal saline to prevent
autolysis.