FIXATION

Suresh kumar Mahaseth

S campus
Suresh Kumar Mahaseth
Lecturer(MLT)
[Link]@[Link]
 Introduction:-
 When a tissue is removed from the body it beings to decompose . To preserve the natural state ( as nearly as
possible) the tissue is immediately placed into a solution called fixative & this process is called Fixing or
Fixation .
 It is a process by which the cells or tissues are fixed in chemical and partly physical state so that they can
withstand subsequent treatment with various reagents, with minimal distortion of morphology and no
decomposition.
 fixation is the process of preserving biological tissues by terminating any biochemical reactions
thereby preventing autolysis and putrefaction. It also preserves the integrity and morphology of the
sample by inhibiting bacterial and fungal growth.
 Fixation is a critical step in the preparation of histological sections. If it is not carried out under optimal
conditions or if fixation is delayed, a tissue specimen can be irreversibly damaged.
 Formalin is most commonly used fixative. It is cheap, penetrates rapidly and does not over- harden the
tissues. The primary action of formalin is to form additive compounds with proteins without precipitation.
Formalin brings about fixation by converting the free amine groups to methylene derivatives.
Mode of action of fixative
• Fixation results in denaturation and coagulation of protein in the
tissues. The fixatives have a property of forming cross links between
proteins, thereby forming a gel, keeping everything in their in vivo
relation to each other.
Or
• Fixatives act by denaturing or precipitating
proteins which then form a meshwork due to
cross linking of proteins. This meshwork tends to
hold the other cell constituents in vivo relation to
each other and insoluble proteins provide
mechanical strength for subsequent procedures.
 .
• A fixative is a chemical substance used in biological and medical fields to preserve tissue
samples by stabilizing the structures within cells and preventing decomposition. The mode of
action of a fixative involves several key processes:

[Link]-linking Proteins: Many fixatives, such as formaldehyde, work by cross-linking proteins.

They form covalent bonds between amino acids, especially between the lysine residues in
proteins. This cross-linking stabilizes the structure of proteins and maintains the integrity of the
tissue architecture.

[Link] Enzymes: Fixatives also inactivate enzymes that would otherwise lead to
autolysis (self-digestion) of the tissue. By denaturing these enzymes, fixatives prevent the
degradation of cellular components.

[Link] Lipids and Carbohydrates: While most fixatives are more effective on proteins,
some, like osmium tetroxide, are used specifically to preserve lipids. Fixatives can stabilize lipids
and carbohydrates to some extent, although they may not be as effective as they are with proteins.
4. Preventing Microbial Growth: Fixatives help to prevent bacterial or fungal
growth within the tissue sample, which could otherwise lead to its decomposition.

5. Maintaining Tissue Morphology: By stabilizing proteins and other

macromolecules, fixatives maintain the morphology of the tissue, allowing for
accurate microscopic examination

Examples of Common Fixatives

• Formaldehyde: Works by forming cross-links between proteins.
• Glutaraldehyde: Similar to formaldehyde but forms more extensive
cross-links, making it ideal for electron microscopy.
• Osmium Tetroxide: Used to preserve and stain lipids, often used in
electron microscopy.
• Alcohols (ethanol, methanol): Dehydrate cells and precipitate proteins.
Feature of the ideal fixative :-
 prevent autolysis & putrefaction .
 should not add to remove from the tissue constituent.
 Should penetrate the tissue & cells rapidly evenly & deeply .
 should impart a suitable hardness & texture to allow for easy sectioning .
 Should be non – toxic , non – corrosive & inflammable .
 Should be cheap & easy to prepare .
 Should be stable
 Should allow restoration of some natural colours for museum work & Photography .
 Fixative plays importance role in:-
1. Preservation of cells & tissue constituents
2. Hardening of soft tissues
3. Conversion of semifluid consistency of cells to an irreversible semisolid consistency
4. Alteration of refractive indices to varying degree, which enables unstained components to be more easily seen.

Factors Affecting Fixation process:-

5. Buffer pH: This is necessary for appropriate chemical reaction . pH is adjusted to physiological range 6-8 .
6. Osmolarity : - Best results are obtained by using appropriate osmolarity of the fixative . Hypotonic solutions give rise to
cell swelling & poor fixation . Hypertonic solutions result in cells shrinkage .
7. Temperature :- Increase in temp. will accelerate the fixation process . However , selection of temp. should be done
according to the specification . Routinely tissues are submitted for fixation at room temp. For electron microscopy the
temp. range selected is 0-4 Degree Centigrade .
8. Penetration capacity of fixative : Thickness of the section should be 1 to 4 mm thick . Penetration of fixative reacts with
tissue .
9. Duration : 12 to 24 hours are required for fixation . Prolonged fixation can cause shrinkage & hardening of the tissue.
Prolonged fixation also may affect antigenicity of the specimen .
10. Agitation :- Appropriate agitation improves speed of penetration resulting in better infiltration & impregnation of the
tissue .
11. Concentration : Optimum concentration of fixative is required for satisfactory results.
 Classification of Fixatives:- The fixatives can be divided
into following three main

1. Microanatomical fixatives
2. Cytological Fixatives
3. Histochemical Fixatives

1. Microanatomical Fixatives :-
Microanatomical fixatives are mainly used for the routine use. The fixatives are used to preserve the
anatomy of the tissue , with the correct relationship of tissue layers & large aggregates of cells . Following are various
examples of microanatomical fixatives .

A. 10% (v/v) formalin in 0.9% sodium chloride (normal saline):- This has been the routine fixative of choice for
many years, but this has now been replaced by buffered formal or by formal calcium acetate.
B. Buffered formalin:-
a) Formalin 10ml
b) Acid sodium phosphate - 0.4 gm (monohydrate)
c) Anhydrous disodium - 0.65 gm phosphate
d) Water to 100 ml
- Best overall fixative
gm
c) Water to
100 ml
• Specific features - They have a near neutral pH

- Formalin pigment (acid formaldehyde haematin) is not

formed.
D. Buffered formal sucrose (Holt and Hicks, 1961)
(a) Formalin : 10ml
(b) Sucrose : 7.5 gm
(c) M/15 phosphate to 100 ml
buffer (pH 7.4)
Specific features
- This is an excellent fixative for the preservation of fine structure phospholipids and some enzymes
- It is recommended for combined cytochemistry and electron
E. Alcoholic formalin

Formalin 10 ml
70-95% alcohol 90 ml
F. Formalin ammonium bromide
Formalin 15.0 ml
Distilled water 85.0 ml
Ammonia bromide 2.0 gm
• Specific features
: Preservation of neurological tissues especially
: when gold and silver impregnation is employed
G. Zenker’s fluid

(a) Mercuric chloride 5gm

(b) Potassium dichromate 2.5 gm
(c) Sodium sulphate 1.0 gm
(d) Distilled water to 100 ml
(e) Add immediately before use : Glacial acetic acid : 5 mlSpecific features
- Good routine fixative
- Give fairly rapid and even penetration
- It is not stable after the addition of acetic acid hence acetic acid (or formalin) should be
added just before use 17 .
- Washing of tissue in running water is necessary to remove excess dichromate
H. Zenker formal (Helly's fluid)
(a) Mercuric chloride - 5 gm
(b) Potassium dichromate 2.5 gm
(c) Sodium sulphate 1.0 gm
(d) Distilled water to 100 ml
(e) Add formalin immediately before use 5 ml
Specific features –
- It is excellent microanatomical fixative
- Excellent fixative for bone marrow spleen and blood containing organs
- As with Zenker's fluid it is necessary to remove. excess dichromate and mercuric pigment
I. Bouin's fluid
(a) Saturated aqueous picric acid 75ml
(b) Formalin 25ml
(c) Glacial acetic acid 5 ml

• Specific features
- Penetrates rapidly and evenly and causes little shrinkage
- Excellent fixative for testicular and intestinal biopsies because it gives very good nuclear
details, in testes is used for oligospermia and infertility studies
- Good fixative for glycogen
- It is necessary to remove excess picric acid by alcohol treatment

.
2. Cytological fixatives: - Cytological fixatives are used when the preservation of
intracellular structures or inclusions is very important .
Subdivided into
(A) Nuclear fixatives
(B) Cytoplasmic fixative

A. Nuclear fixatives : As the name suggests it gives good nuclear fixation. This group
includes

a. Carnoy's fluid.
(a) Absolute alcohol 60ml
(b) (b) Chloroform 30ml
(c) Glacial acetic acid 10 ml
• Specific features
- It penetrates very rapidly and gives excellent nuclear fixation. - Good fixative for carbohydrates. - Nissl substance
and glycogen are preserved.
- It causes considerable shrinkage.
- It dissolves most of the cytoplasmic elements. Fixation is usually complete in 1-2 hours. For small pieces 2-3 mm
thick only 15 minutes in needed for fixation.
b. Clarke's fluid
(a) Absolute alcohol 75 ml
(b) Glacial acetic acid 25 ml.
• Specific features
- Rapid, good nuclear fixation and good preservation of cytoplasmic elements.
- It in excellent for smear or cover slip preparation of cell cultures or chromosomal
analysis.
(B) Cytoplasmic Fixatives

(a) Champy's fluid

(b) (a) 3g/dl Potassium dichromate 7ml.
(b) 1% (V/V) chromic acid 7 ml.
(c) 2gm/dl osmium tetraoxide 4 ml.
Specific features
- This fixative cannot be kept hence prepared fresh.
- It preserves the mitochondrial fat and lipids. 21
- Penetration is poor and uneven.
- - Tissue must be washed overnight after fixation.
b. Formal saline and formal Calcium:-
Fixation in formal saline followed by postchromatization gives good
cytoplasmic fixation.
3. Histochemical fixatives:-
The fixatives used for histochemical studies should produce minimum changes in the
elements that is to be demonstrated .Freeze –drying technique is ideal for this purpose . The
various functions of a good histochemical fixative are as follows:
1. Preservation of the tissue- constituents.
2. Preservation of the morphological relationships.
3. Preservation of the specific tissue constituents &
4. No interference with the reagents to be used in the process of visualization .
For a most of the histochemical methods. It is best to use cryostat. Sections are rapidly frozen or
freeze dried. Usually such sections are used unfixed but if delay is inevitable then vapour
fixatives are used.
a. Buffered formalin is the most common fixative used for histochemical purpose . Immersion in
acetone , 0-4 degree cent regrate is widely used for the fixation of tissue in which it is intended to
study enzymes . Fixation of sections cut from freeze dried material may be effect by immersion in
absolute alcohol for 24 Hrs.

b. Vapor fixatives:-
 These are used fix cryostat cut sections of fresh tissue & sections of blocks of frozen dried
tissue .
 These fixatives are used inside an airtight glass container with controlled heat & humidity to
fix cryostat cut section of fresh tissue & sections or block of frozen dried tissue .
Following are the various vapor fixative used –
i. Formaldehyde
ii. acetaldehyde
iii. Glutaraldehyde
iv. Acrolein or Chromyl chloride
Formalin
Mode Of Action
• Formalin acts by polymerizing action, i.e, the
formation of complexes by development of links
(methylene bridges) between protein molecules.
Average thickness of tissue for adequate fixation
– 4mm
Advantages of formalin fixation:
• Cheap & easy to prepare
• Allows the uses of various staining techniques
• Frozen sections can be prepared
• Fat stains can be used as formalin fixed tissues
• Does not cause excessive hardening of tissues
• Natural tissue colour can be restored without difficulty
• It is the best fixative for the nervous system.
Disadvantages of formalin fixation:
• Can lead to dermatitis of the hands & irritation to the nostrils due to
fumes (can be prevented by proper ventilation).
• If excess of blood is present in tissues then formalin leads to formation
of dark brown artifact pigment granules which are doubly retractile.
• Methanol which is added to formalin to prevent the formation of para
formaldehyde causes denaturation of proteins& makes it unsuitable for
electron microscopy.
Different formalin based
fixatives:
• 10% formalin
• 10% formal saline –
• 10% of buffered neutral formalin –
• Formal calcium (calcium acetate formalin) –
Mercuric fixatives

• Mode of Action – Mercuric ions act by combining

with acidic (carboxyl –COOH) groups of proteins &
form especially strong combination with the
sulfer (thiol) radicals
Example: Zenkers
Advantages of mercury
fixatives:
• Better staining of nuclei and connective tissue
• Cytoplasmic staining –enhanced with acidic dyes.
• Nuclear chromatin shown in detail
• Preservation of details for photography.
• Best results with metachromatic stain
• B5 fixative is frequently used for bone marrow,spleen, lymph nodes and other
hematopoetic tissue
• Disadvantages of Hg fixatives:
• Corrodes the metals
• Lysis of RBC & removes much iron from hemosiderin
• Deteriorates rapidly
• Causes Marked shrinkage
• Reduces the amount of demonstrable glycogen
• Slow penetration
• Tissues become hard & brittle
• Formation of Diffuse black granules in tissues
• Radiopaque: preclude use of x-rays to determine and point of calcification
Alcoholic fixatives
• Mode of action Alcohols denaturation and
precipitates protein
• Methylalcohol
• Ethylalcohol
• Carnoy’s alcohol
• Clarke’s fluid
Advantages:

• Carnoy’s fluid is best for small tissue fragments like curetting

• It is a good fixative for glycogen
• Nuclear staining & carbohydrate preservation are good.
• Clarke’s fluid is used as fixative for cell cultures in chromosome
studies
• Disadvantages of alcoholic fixative
• Causes severe shrinkage (unless used at colder temperature)
• Hardens tissue excursively
• Lipids any myelin are dissolved
• At 5 to -20oc it preserves some enzymes like alkaline phosphatase
• Although good fixative for glycogen it leads to “polarization”
because of the streaming of the glycogen granules to one pole of
the cells.
Advantages of fixatives:-
 Its advantages are low cost, simplicity of use and good fixation
traits, which are fast tissue penetration, good preservation of
morphological structures and compatibility with downstream
histological applications.

 It is a good cytoplasmic fixative and gives little shrinkage during

fixation; indeed it is thought to preserve the structure of the living cell
better than any other primary fixative except osmium tetroxide. It is
also inexpensive.
 Fixation of tissue is done for several reasons. One reason is to kill the tissue so
that postmortem decay (autolysis and putrefaction) is prevented.
Fixation preserves biological material (tissue or cells) as close to its natural
state as possible in the process of preparing tissue for examination.
Disadvantages :-
 Prolonged fixation causes hardening and shrinkage of tissue. Tissue penetration: This
process is usually slow with usual fixatives. So, the blocks should be either thin or small .

 Fixation of tissue is done for several reasons. One reason is to kill the tissue so that
postmortem decay (autolysis and putrefaction) is prevented. Fixation preserves biological
material (tissue or cells) as close to its natural state as possible in the process of preparing
tissue for examination.

 Formaldehyde tends to combine strongly with protein, nucleic acids, and unsaturated fatty
acids in a nonenzymatic way. This combination causes cytotoxicity, inflammatory reaction,
necrosis, allergy, and mutagenic effect to be seen by producing denaturation in proteins.