FIXATION

This is a process of preserving cells and tissue components in a condition related to life. It is a
critical step in preparation of specimen for various laboratory techniques. It help preserve
cellular structures, prevent decay and maintain the integrity of tissue.
AIMS/EFFECT OF FIXATION
1. To prevent autolysis: it helps maintain the tissue structural integrity/ Autolytic changes
occur due to action of enzyme from the dead cell eg; proteinase, amino peptidase
(proteolytic enzyme).
2. To prevent putrefaction: This action occurs as a result of tissue degradation cause by
bacteria originated from GIT and spread into the surrounding organs (bacteria
decomposition). It helps preserve cellular structures and prevent deterioration.
3. To avoid decay and shrinkage: thus preserve cell morphology (shape and structure) and
also minimize tissue loss of architecture by increasing its strength and rigidity
4. To helps to fortify tissue to withstand subsequent reagent treatment eg; causing tissue to
become firm and hard to facilitate sectioning processes.
5. To co-agulate protein and harden the tissue; Fixation induces denaturation of protein by
crosslinking them thereby rendering diffusible substances insoluble and stabilize
cellular structure (preventing enzymatic degradation)
6. To increase the tissue affinity, optical density and differentiation for stains.; aiding in
visualization of cellular details during subsequent staining procedure.
7. To allow long term storage of the specimen to retain cellular components including
organelles, nuclei and cytoplasm
8. To play critical role in preserving antigens, making them accessible for antibody binding.
METHOD OF FIXATION
Physical Fixation: HEAT/MICROWAVES temperature (air dying) or passing through in a
flame of bursen burner to kill bacteria.
Micro wave fixation: This apply the use of electromagnetic wave frequency 300H Z 300MHZ and
3009HZ with temp of 45-55oC for about 30mins.
Chemical Fixation: This is a process of using chemicals to prevent decay of tissue thereby
maintaining tissue chemistry and morphology in life-like manner it employs: -
 1. Vapourization: Using chemical vapour as a fixative e.g. formaldehyde vapour (cell
smear).
2. Immersion: Insertion in a solution of a volume five times the sizes of specimen, enough
cover the entire surface.
3. Perfusion: It involves the use of host needle or annular to inject chemical inside the
tissues.
Other chemical fixation includes:
Histochemical Fixation
This technique routinely makes use of cold acetone to freeze. It results in less alteration to
antigenic epitopes and loss of enzyme. Use of coolant gas or fluid to a frozen section which may
be air dried, alcohol or formalin fixed - paraffin embedded enzyme southern Blot PCR etc.
Periodiate-Lysine-Paraformaldehyde (PLP) is used for preserving cell morphology and antigen
for immunochemistry.
Secondary Fixation
Wallington, 1955 shown that in some condition, tissue used to be refixed after initial fixation in
10% formal-saline, mercuric chloride and chronic acid. These fixatives tend to form vigorous
precipitant. The effects tend to precipitate the colloidal constituents of a cell as coagulant. It
helps to improve preservation of specific structure, stabilized protein and prepare for special
staining techniquues by treating the fixed tissue with buffered formaldehyde (formalin) or lodide
solution (Mercury chloride) and washing in water for 12-18hrs (for chrome precipitation) before
dehydration.
Post Fixation
Leach 1945, recommended that tissue should be fixed, dehydrate, clear in a fat solvent and then
"Post fixed" in absolute alcohol before re-clearing and wax impregnation. This is to protect the
masked tissue protein which hinder complete penetration of most fixatives and lead to its
subsequent cost.
Post Chromatization/Mordanting
These methods employed to facilitate subsequent preservation and staining of a particular
component eg Mitochondria. It involves treatment of tissue after fixations i.e. before tissue
processing or treatment of section before staining. Mordants are chemical that are used to
intensify or set a stain E.g. the use of a chrome salt and 3% potassium dichromate. Primary stains
may not always bind to all structures uniformly and post mordanting helps to enhance the
staining specificity or intensity.
Factors Affecting Fixation
1. Choice of fixative: This can significantly impact the fixation process. Different fixatives
have varying effects on cellular structure and may be more or less suitable for specific
tissue
2. Buffer and PH: The acidity or basicity of tissue to be fixed should be kept in the
physiological rage between PH 4-9. Formalin can either be buffer formalin (neutral PH).
This is to maintain the stability of cellular component
3. Size of the Specimen/ thickness: This can affect the fixation process; large surgical
dimensional specimen has to be cut to allow easy penetration of the fixative to reach all the
cells. Also, large or thicker samples may require longer fixation period.
4. Duration of Fixation: Size and nature of the specimen depend on the duration thus
determines the fixation period. The slice (6mm thickness) and large surgical specimen be
taken into consideration, time for fixation process must be over fixed or under fixed to
avoid tissue hardening and loss of antigenicity.
5. Volume of Fixative: The volume of the fixation should be at least 15-20times greater than
tissue volume. The standard ratio of specimen to the volume of the fixative is 1:50. This is
to avoid zonal fixation
6. Temperature: Fixation should be carried out in a normal room temperature to increases
the speed of fixation. High temperature may damage the specimen.
7. Concentration of Fixative (Osmolality): Alteration in concentration of the fixative may
result in under or over fixation. Hypertonic Solution increase cell shrinkage while
hypotonic give rise to cells swelling and poor fixation. The chemical composition of the
fixation must be maintained.
8. Tissue Composition: Tissue content and density can influence the penetration and
effectiveness of the fixative,
Fixatives
These are chemical agent used to preserve and prevent deterioration of tissue, thereby
maintaining the tissue chemistry and architecture as in life.
Classification of Fixatives
These are various classifications of fixatives based on: -
1. Number of chemicals that constitute the fixative
2. Specific application of the fixatives
3. Action on the amino-acid chain
4. Action on the tissue protein
Classification based on Number of Chemical Constituent
A. Primary/Simple fixatives:
There are fixatives that contain only single item of chemical eg formalin, methanol,
acetone, ethanol, picric acid, acetic acid, mercuric chloride, osmium tetroxide, potassium
dichromate , 10% formalin etc.
B. Secondary/Compound Fixative:
This is combination of two or more constituent. These combinations may be as a result of
advantages of the constituents or act against each other, they may neutralize the effect of
the other consentient to form a better fixation. Eg;
- Zenker formol is combines the advantage of mercury chloride, potassium dichromate
and formalin.
- Bouin's fluid is combination of acetic acid (which swells collagen) with picric acid
(which shrunk tissues).
Other examples of secondary fixatives include for saline sublimate, Carnoy's fluid,
Schaudin's fluid etc.
Classification of fixatives based on action on protein
These fixatives can be classified into 2 groups
1. Coagulating/precipitant fixatives:
These fixatives act by precipitating the collided constituent of a cell to form a coagulant.
Examples: Mercuric chloride, picric acid, ethanol, methanol, acetone, trichloro acetic
acid and chromium trioxide.
2. Non-coagulating/Non -precipitant fixatives:
These fixatives fix protein not by coagulation but by denaturing. Heat also has the same
effect e.g. Fixation of bacteria with the bursen burner. Examples of these fixatives
include - formalin, Osmium tetraoxide potassium di chromate (non-acidified), acetic acid
etc.
Classification of fixatives based on acid on the portion of amino acid chain.
Additives
When the fixative molecules act on side group of the amino-acid chain protein side group.
Non Additive
When they denature protein, split the protein chain act on the exposed inner bond.
Classification of fixatives based on specific application
a. Micro-anatomical Fixatives
These fixatives act to preserve relation of tissue layers and large aggregation of cells to one
another. They deal with preservation of anatomy of the tissue which can be left over a long
period without damage. They are mainly routine formalin fixatives Eg formol-saline
(which has been routine fixative of choice for many years). It can be used as a buffered
formation or formal calcium acetate.
Other micro anatomical fixatives include Bourn's fluid, Zenker's fluid, Heldenhans susa,
Helly's fluid and formol sublimate.
b. Cytological Fixatives
These fixatives preserve the constituent element of the cell to maintain the cytoplasm and
nuclear structure for histochemistry. They deal with preservation of cell and /or tissue
fragments.
There are 2 types of cytological fixatives
1. Nuclear Fixatives
These fixatives penetrative very rapidly to give excellent nuclear preservation. They are
mostly used in the demonstration of RNA and DNA.
Examples: Carnoy', fluid, Fleming fluid with acetic acid, New comer's fluid, Clark's
fluids etc.
2. Cytoplasmic Fixatives
These fixatives penetrate to protect tell morphology details. In most cases it does not
contain any acid Eg. Helly's fluid Shaudin fluid, Fleming's fluid without acetic acid
Champy's fluid etc.
Difference between Micro-anatomical and Cytological Fixative.
S/ Micro-anatomical Fixative Cytological Fixatives
n
1 Common fixative used is formalin (last Alcohol (Ethanol) Fixative is mostly use (few
longer period) days).
2 Deal with preservation of anatomy of Deal with body fluid and soft tissue
tissue
3 Preserves the correct relationship of tissue Preserve free of floaty individual cell
layer & large aggregate of cell. fragment.
4 Involve cutting into translucent slice or Required smearing on the slide using cover
section. slip.
5 Most common stain is H&E Stain commonly used are papaincolaou's stain
may Grunwalds giemsa stain.

PREPARATION OF FIXATIVES (ADVANTAGES AND DISADVANTAGES)

Formaldehyde
This is a gas made by oxidation of methyl alcohol over a catalyst (sliver) in the form of gauze at
temperature of about 635oC.
Formalin
Formaldehyde gas in dissolved in 60 part of water which is 40% by weight of the gas: This is
commercial formalin or absolute formalin.
This solution polymerizes to form a white perceptual known as paraformaldehyde, addition of
little amount of methanol into can minimize this action.
Over a long period of line formaldehyde undergoes oxidation to from formic acid, placement of
magnesium or calcium carbonate on the bottom of the container will neutralize this effect of
formic acid formation.
Characteristic of Formaldehyde
1. It is a gas made by oxidation of methyl alcohol
2. Soluble in 40% water
3. Forms formic acid
4. Reducing agent (react with protein to bind side groups of protein through formation of
methylene bridges and thus stabilizing protein molecule.
Advantage of formalin
 1. Penetrate Post -Morten tissue excellently
2. Relatively cheap and stable
3. Causes little shrinkage and does not harden tissue excessively
4. Can keep large specimen for indefinite period.
5. Allow application of most staining methods
6. Dose not render tissue brittle
7. Allows natural colour restoration to the specimen
8. Basis for all museum specimen fixation
Disadvantages
1. Has irritants vapour which can cause hazard to nasal mucosa (inflammation of the
sinus).
2. Prolong exposure to skin may cause dermatitis.
3. Formation forms pigment in reaction with haematin from RBC (can be removed by
treatment saturated picric acid in ethanol or fixing in bufffered formalin to prevent it).
4. It is slow in action
5. Causes tissues to shrink during dehydration
6. It causes acid dyes to stain less brightly
Preparation stock: 40% formaldehyde.
Routine formalin
10% formal saline
10% Buffered Neutral formalin
Preparation of 10% Formal-saline
Formalin - 10ml
Sodium chloride (NaCl - 0.85g
Distilled water - 90ml
Application: This has been routine fixative and has been recognized as the most widely used
fixative. Fixation of tissue block not exceeding 5mm in thickness is usually complete in 6-12 hrs
at room temp, 3mm in thickness can be fixed in 1-2hrs at.
10% Buffered Neutral formalin
Formalin - 10ml
Sodium phosphate monohydrate - 0.35g
Anhydrous disodium phosphate - 0.65g
Tap water 90mls
Formal calcium
- Formalin - 10ml
- Calcium acetate/calcium chloride - 2g
- Add H2O to make - 100ml
Buffered formalin or formal calcium have the same advantage and are used in the same manner
as formal saline 7-8 thickness tissues block can be fixed in 12-24hrs.

Mercuric Chloride
This is a white needle-like crystalline substance.
Characteristic
1. Soluble to the extent of 7% in water and 33% in alcohol at room temperature.
2. Precipitates protein and does not combine well with it.
3. Used in saturated aqueous solution form
Advantage
1. It is good antiseptic and disinfectant
2. Can undergoes hydrolysis to produce hydronium ions
3. Coagulates proteins and proteolytic enzyme e.g. pepsin.
4. Penetrate and harden tissue quickly.
5. Cause staining of particulars of cytoplasm brighter and more differentiating.
Disadvantages
1. Gives a black deposit in tissue which can be removed with lugolis iodine followed by
5% sodium bisulphate.
2. It is a poisonous and corrosive substances
3. Because of its radio-opaque nature, its presences in decalcified tissue presides the use
of X-ray to test the end point.
4. Its penetration is slow and cannot fix large pieces of tissue.
Preparation
Stock: saturated aqueous solution.
Osmium tetra oxide (Osmotic acid)
- Sold in glass sealed tubes contain 0.5g - 1g pale yellow crystal substance soluble in H20 up
to 6% at 20oC.
Characteristics
1. Strong oxidizing agent
2. Soluble to 6% in water at 20oC
3. Stop at 2% solution
Aqueous solution is easily reduced by light, it is stored at dark
Advantages
1. Used for fixing tiny pieces of tissue for electron microscopy
2. Little pieces of tissue can best be fixed through vapour penetration.
3. Fixes lipids and preserves cytoplasmic structure
Disadvantage
1. It is expensive/costly
2. Tissue must be washed in running water before processing
3. Prolonged exposure to the vapour may cause deposit collection in cornea resulting in
blindness.
4. Must not be used in conjugation with fixative which are reducing agent.
5. It las a poor penetration power and cannot fix large pieces of tissue.
Preparation
Stock: -2% aqueous, solution used as 0.5 to 1% solution in H20.
Potassium Dichromate (K2 Cr2 O7 )
- It an enrage crystalline substance
Characteristic
1. Strong oxidizing agent
2. PH 3.4-3.8
3. Act like chronic acid by precipitating both nucleus and cytoplasm
Advantage
1. Cytoplasm and mitochondria are homogenously fixed
2. Preserve phosphatide
3. Fixation used in mitochondria & myelinated nerve fiber study
Preparation of Potassium dichromate
Dissolve in 2.5% solution in H20
Stock: 5% aqueous solution.

Chromic acid (CrO3 )

- Dark red crystal solution from chromium oxide
characteristic
- A strong oxidizing agent
- Covalently stored as a 2% stock solution.
Advantages
1. Used in fixing of carbohydrate
Disadvantage
1. Tissue must be washed in running water before treatment with alcohol to avoid
precipitation.
2. A strong protein precipitant
Preparation
Dissolving in hydride chromic oxide (CrO3) in distilled water.
Stock: 2% aqueous solution.

Picric Acid (C H2 (No2 )3 OH

- Bright yellow crystalline substance
Characteristic
1. It has explosive properties and must be kept damp preferably water.
2. It is about 1% soluble in water at Room temperature) but nearly 5% in alcohol and 10%
benzene can combine with protein to form picrates
Advantage
1. Useful constituent fixative for glycogen
2. They are soluble to water
Disadvantage
1. Precipitates nucleoproteins
2. Causes shrinkage but little hardening
 3. It precipitates proteins
4. Not ideal for kidney tissue
Preparation
Stock: Saturated aqueous solution

Acetic Acid (CH3COOH)

- Colourless liquid
CHARACTERITICS
1. It solidified at about 17oC have called Glacial
2. It has a pungent smell/ odour
Advantages
1. Used in studying chromosomes
2. The chromatin precipitate makes it useful in nuclear study
3. It is mostly used to counteract the shrinking effect of the fixatives
Disadvantage
1. Destroys mitochondria and Golgi elements.
2. Causes excessive swelling of collagen fibers.
3. It is never used alone
Preparation
Stock: Keep as glacial acetic acid
Ethyl alcohol (Ethanol)C2H5 OH)
- Colorless, miscible substance
Characteristics
1. It is a reducing agent dehydrating agent.
2. It is an inflammable liquid with boiling at 78%
Advantage
A simple fixative used at concentration 70-100%
Disadvantage
1. Causes shrinkage and hardening
2. It coagulates proteins
3. And precipitate glycogen
Preparation
Stock solution - absolute ethyl alcohol
Acetone
Used in demonstration of enzyme eg; phosphates and lipase
Disadvantage
Glycogen is not well preserve

Trichloracetic and (CCL3CoCH3)

Advantages
1. Used to counteract the shrinkage effect of certain fixative
2. Has decalcifying effect
Disadvantage
1. It is never used alone
2. Generally precipitate protein
Preparation
Gendre's solution
Glacial Acetic acid - 5ml
Saturate Solution Picric Acid (95% alcohol 80ml )
40% concentration formaldehyde solution - 15ml
Advantages
1. It is macroaromotic fixative widely used
2. It is similar to Bouin's fluid in action
3. Has superior over the preservation of glycogen because of presence alcohol & picric acid.
Disadvantages
Tissue should be washed in several changes of alcohol
Preparation of Compound fixation
Mercuric chloride - formalin (Formal sublimate)
Susa fixation (Heidenhain's susa)
- Mercuric chloride - 1.5g
- Sodium chloride - 0.5g
- Trichloroacetic acid - 4ml
 - Formalin - 20ml
- Distilled water - 80ml
Fixation of tissue blocks 3-24hrs
Advantages
1. Widely used for routine surgical tissue and fibrous tumors.
2. Cause minimal shrinkage & hardening
3. Permit many staining procedure eg; Sliver impregnation, Wright .
Disadvantages
1. Poor differentiation of RBC
2. Cause excess hardening after 24hrs.
Zenker's fluid
Composition:
- Mercuric Chloride- 5g
- Potassium dichromate - 2.5g
- Sodium sulphate - 1g
- Distilled water- 100ml
- Acetic acid - 5ml (add immediately before use)
Fixation duration 3-18hrs
Advantage
1. General fixative highly recommended for small pieces of liver and spleen
2. Good for cytoplasmic & fiber stains preservation
3. Gives excellent cytoplasm details
4. Good for fixing of decalcified tissue (15-24hrs
Disadvantage
1. It bright staining of nuclei & connective tissue
2. Rbcs are not well preserved.
3. Penetration is poor and tissue should not exceed 0.5cm in thickness
4. Form precipitates (mercury) and should be treated with iodine.
Helly's Fluid (Zenker - Formal)
Preparation
Acetic acid 5ml a replaced with formalin before use
Tissue block fixation - 6-24hrs
Advantages
1. Recommended for fixation of pituitary tissues
2. Suitable for bone marrow spleen, lymph, glands, pancreases etc.
3. Fixation is within 24hrs
4. Preserve accurately both cytoplasm and nuclei
Bouin's Fluid
Composition:
- Saturated aqueous picric acid - 75ml
- Formalin - 75ml
- Acetic acid - 5ml
Tissue block fixation - 6-24hrs
Advantages
1. Causes little shrinkage
2. Does not cause excessive hardening
3. Give brilliant staining with cytoplasmic stains
4. Preserve glycogen
5. It is good for demonstration of chromosomes
Disadvantage
1. Causes partial or complete lysis of RBC
2. Collagen fibers may be swollen
3. Does not preserves kidney & some cytoplasm granules

CYTOLOGICAL FIXATIVES
Carnoy's Fluid
Preparation
- Absolute alcohol- 60ml
- Chloroform- 30ml
- Acetic acid - 10ml
Fixation (about 5m thickness) 30-90 mins
Advantages
 1. Quick and rapid penetrating fixative
2. Used in preservation of chromosomes for study
3. Glycogen is preserved
4. Good for both micro anatomical & cytological specimens
Disadvantages
1. Lyses RBC
2. Dissolve cytoplasm
Clarke's fluid
Preparation
- absolute alcohol 75ml
- glacial acetic acid 25ml
Advantages
1. penetrate rapidly and gives good nuclear fixation
2. used for smear & coverslip in cell culture for chromosomal analysis
3. preserve cytoplasm (cytoplasmic fixatives)
Flemming fluid
Preparation
- 1% Chronic acid -15ml
- 2% Osuim tetricoxide -4ml
- Acetic acid -1ml or less
Fixation: 12-48hrs
Advantages
1. Preserve fat permanently
2. Preserve nuclear structures especially chromosome
3. Useful in cytoplasmic fixation with omission of acetic acid
Disadvantage
1. It is costly
2. Has low penetration power
3. Use for small tissue
New Comer’s fluid
Preparation
 - Isopropanol- 60mls
- Propionic acid- 30mls
- Petroleum ether- 10mls
- Acetone -10mls
- Dioxane- 10mls
Advantages
1. It penetrates rapidly
2. It is used for chromosomal analysis
3. Fixation is 2-3hour

PRATICAL GUIDE TO CHOICE OF FIXATIVES

Choice of fixatives depend on;
1. Nature of element to be demonstrated
2. Type and freshness of the specimen
3. Size of specimen
4. Duration
5. Stains to be employed

Type of specimen Size Fixatives Duration Treatment after

fixation
Autopsy tissue 6mm thickness Formol saline 70% alcohol
block (trim & refix in
fresh fomalin)
Cellular biopsy Surgical samples 80-90%
Small Formol Mercury alcohol
Large Formal saline
(select in theatre
& refix in formol
Mercury)
Cytological Smear Ethanol 30mins -1 week 95% or
specimen Palpable absolute
(make a smear)
Frozen section Fresh frozen
Rapid section unfixed
(Formal-saline
Nervous tissue Small pieces or formalin)
Bone marrow, Tissue block Helly/Zenker 6-12hrs Wash in
spleen, lymph, 6mm thickness formal running H2O
pancreas, glands
Urine and other Add equal Ethyl alcohol 30mins- 1weeks
fluids volume of
fixatives

PROPERTIES OF GOOD FIXATION

1. It had excellent penetration ability to reach all cells within the tissue, thereby ensuring
uniform and thorough fixation.
2. It should facilitate rapid fixation to minimize the risk of tissue autolysis or change in
cellular morphology.
3. It is stable and has ability to harden tissue without damage thus maintaining integrity of
cellular component.
4. It should be easily affordable ie inexpensive. Its cost effectiveness should balance the
need for efficient fixation with economic consideration
5. It should have little shrinkage effect
6. It should be available (not scarce) and easily handle in the laboratory setting, allowing
straightforward application and storage

REMOVAL OF FIXATION PIGMENT AND PRECIPITATE

Formalin pigment
Formalin shows artifact pigment which is similar to malaria parasite presenting a dark-brown
granular material. This is caused by action of acid formalin with hemoglobin. It is more
commonly seen in post mortem tissue.
Method of Removal
Bayley (1949) described the artifacts as pink disease and advocates the use of 20% acetic acid in
10% formalin as a means of avoiding its occurrence or when present treatment of dewaxed
section with 1% HCL acid in absolute alcohol for 1hr immediately before staining.
Treatment with saturates alcohol is for 20mins.
Mercury Pigment
This occur as a result of corrosive crystal substance maturation with extracellular cells to form a
green black pigment.
Removal of mercury pigment
Treatment with Iodine
Iodine react with mercury to give mercury iodate (HgI) which is soluble in alcohol so no pigment
is formed.
Method of Deposit Removal
Dehydrate tissue block by addition of 0.25 to 0.5% iodine is 70-80% Alcohol.
Before staining treat section as follows;
1. Treat with xylene and wash in absolute alcohol
2. Treat in 0.5 iodine in 70% alcohol (i.e. 99.5ml of 70% alcohol + 0.5ml iodine) wash in
water
3. Treat in 25% Sodium thiosulphate wash in water before staining
Chromic Deposit
It occurs as a brown dark granule
- Removal is washing the tissue block in running tap water
NB: read more on fixation pigment formation and removal