Dr. Shrikant Sonune
Dr Ashok Patil,
Dr Shilpa Kandalgaonkar,
Dr Mayur Chaudhary,
Dr Suyog Tupsakhare,
Dr Mahesh Gabhane.
• Function of fixative
• Methods of fixation
• Reaction of the cell(its component) with fixatives
• Simple aqueous fixatives or fixative ingredients
• Factors affecting fixation
• Effect of fixation
Fixation : introduction
• Fixation is the complex series of chemical events and
differs for the different groups of chemical substances
found in tissues.
• It is most essential part of histology. Here where
• Once the tissue is removed from the body it will go
through a process of self-destruction. This process
is known as autolysis.
• If tissue is left without any preservation, then a
bacterial attack will occur, the process is known as
• It is a process by which the constituents of the cells and
therefore of the tissues are fixed in a physical and partly
in a chemical state , so that they will withstand
subsequent treatment with various reagents with
minimum of loss, significant distortion or decomposition.
• The preservation and hardening of a tissue sample
to retain as nearly as possible the same relations
they had in the living body
Aims & objectives of fixation
• To prevent autolysis and putrefaction.
• Rapid and even penetration.
• To preserve cells and tissues in a life like manner as possible.
• Elements that are to be demonstrated must remain in
maximum concentration and precise localization.
• Stabilize labile elements.
• Must be rigid to allow sectioning.
• Must allow staining.
• Optical contrast must be induced for morphological
• Allow long storage of tissues
Acc. to no. of fixatives used:
- Simple fixatives
- Compound fixatives
i) Micro anatomical fixatives
Reaction of fixatives with Protein
Most important reactions which stabilizes proteins
by forming cross links between soluble protein &
structural protein. Ultimately providing some
• Cross links are formed between protein molecules and
Aldehyde group of fixative.
• Aldehydes react with the basic amino acid residues of
proteins & there is an accompanying change in
isoeletric point of proteins.
• This may form the basis for the of the different staining
of tissues after different fixations.
• Process takes places in 2 step
1st step-small polymers are formed
2nd step small polymers cross-link
Formations of cross linkages between Aldehyde and
protein is measured by changes in viscosity, mechanical
strength and molecular size of protein.
• Slow reaction
• Reversible*(in first 24 hr with
excess of water)
• Not good morphological
• Less effective at cross
• Loss of enzyme and
immunological activity is less
• Good morphological
• More effective at cross
• Loss of enzyme and
• React with protein
• Forms cross-links with proteins
• Reflected by rapid increase in viscosity
• After that decrease in viscosity , that phenomenon
is known as secondary liquefaction.
• Osmium tetroxide is more reactive towards protein.
• It reacts with histidine residues in proteins.
• Also there is production of H+ ions making solution
more acidic more efficient.
• But after fixation ultra structural preservation is
• Heat fixation /microwave fixation ------ reacts with
polar side chains of proteins. This increases their
thermal energy which cause denaturation of
proteins. This brings about tissue stabilization.
Reaction of fixative with nucleic acid
• Fixation brings about change in physical or
chemical state of DNA or RNA at room temperature.
• Few fixative react with nucleic acid chemically-
including mercury and chromium salts.
• Heating at 45 and 65 degrees with Aldehyde
fixatives, there is uncoiling of RNA and DNA
• Ethanol, methanol and Carnoy’s fixative are
commonly used. DNA is largely collapsed in
methanol and ethanol.
• Presence of salts is known to be essential for the
maximum precipitation of nucleic acid from alcohol.
Reaction of fixative with lipids
Most of lipids are labile. So lost during routine
processing. To demonstrate them frozen section or
cryostat is used.
Preservation of lipoproteins (fixation of protein
Eg: phospholipids which contain amino group such as
phosphotidyl ethanolamine are fixed by aldehyde.
HgCl₂ react with highly unsaturated compound
which form complex. It also reacts with lipids known
as plasmalogen acetal phosphatides.
Additives such as tannic acid may be used for
demonstration of lipid with light microscopy.
Ultrastructural demonstration---post fixation with
Cholesterol may be fixed with Digitonin for
Reaction with Carbohydrates
Single fixative not satisfactory.
Alcoholic or picric acid fixatives
preservation of glycogen which appear coarse eg:
Alcoholic formaldehyde, Rossman’s solution.
Ultra structural studies gluteraldehyde is
satisfactory while potassium permanganate
increase image contrast.
Tanic acid and cetyl pyrimidium have been found
Additives to vehicle like Alcian blue or ruthenium red
enhance glutaraldehyde fixation of glycogen and
• Powerful reducing agent.
• Most common fixative for routine fixation of biopsy
• Formalin: 40%formaldehyde gas in water.
• Forms methelene bridges between protein
• Method 4mm block - 8hrs at room temperature
4mm block - 2hrs at 45°C
• MOST COMMONLY IS USED FIXATIVE
10% formalin consist of
• Formalin (40% formaldehyde) 10 ml
• Water 90 ml
• Neutralization is necessary due to formation of formic
acid by addition of buffer to maintain pH of 7.
Buffer added: phosphate buffer
• Protein groups involved in formation of cross links
amino, imino, peptide, hydroxyl, carboxyl and
• Formaldehyde is also obtainable in a stable solid form
composed of high molecular weight polymers known as
Cheap, easy to prepare, relatively stable, staining without
Good preservation of cell morphology
Good penetration properties.
Do not cause excessive hardening.
Best fixative for nervous system
Slow fixation reaction.
Morphological details less accurate than
Dermatitis of hand.
Fumes irritating to nostrils.
In tissue containing blood , dark brown artifact
pigment granules are formed.
Fixative Formula Advantages Disadvantages Uses
•Slow fixative •Hard tissues
10% formalin 40%formaldehyd
•Longer time for
•Used for IHC
Water - 900ml
•Stops formation of
•Fixes tissue rapidly
•Loss of basophillic
staining of the
•Loss of reactivity
of myelin to
Fixative Formula Advantage Disadvantage
Distilled water- 90ml
Formalin - 10ml
•Buffered at pH7 by
•Less hardening or
•Artifacts due to
•Glycogen is better
•RBC are lysed
Formol calcium Formalin- 100ml
Distilled water- 900ml
•Preservation of lipids •Artifacts due to
•Stops formation of
•Fixes tissue rapidly
• Introduced by Sabatini, Bensch and Barrett
• It is a dialdehyde.
• Stable in acid solution: in pH 3 to 5
at 0 ° to 4° C
• Used in electron microscopy with osmium
• Fixation of small tissue: 2.5% solution for 2-4 hrs at
• Fixation of large tissue: 4% solution
for minimum 6-8hrs
fully fixed for 24hrs
Better preservation of cellular and fluid proteins than
More stable cross linkages
More rapid fixing action than formalin.
Less shrinkages than formalin
Give better section of blood clot and brain
Does not corrode metal
More pleasant and less irritating
Penetrates tissue more slowly than formalin
Inferior to formalin for PAS technique.
• Mercuric ions act chiefly by combining with the
acidic group of proteins and strong combination with
sulfur thiol radicles.
Better staining of nuclei and connective tissue.
Give best results with metachromatic staining
Routine fixative of choice for preservation of detail
Corrode all metal except nickel alloy.
Solution deteriorates rapidly.
Reduce amount of demonstrable glycogen.
Penetration is slow.
Long time fixation results in unduly hard and brittle tissue.
Diffuse black granules are seen in tissue fixed with HgCl₂.
Picric acid fixative
• It reacts with histone and basic proteins and forms
crystalline picrates with amino acid.
• It preserves glycogen well.
Considerable shrinkage of tissue.
It dyes the tissue - yellow colour.
• Mechanism of action: alcohol denatures and
precipitate protein, possibly by disrupting hydrogen
and other bonds.
Fixative Type Formula Advantage Disvantage Use
Fixative Formula Type Advantages Use
•Glacial Acetic acid-
• Chromium salts in water form Cr-O-Cr complexes
which have affinity for acidic and hydroxyl group of
proteins so that complexes between adjacent
protein molecules are formed.
• This leads to disruption of internal salt linkages of
protein ,thereby increasing the reactive basic
groups and enhancing acidophilia in staining.
Fixative Formula Type Advantage and use Disadvantage
•Solution darken on
tend to bleach all
of PAS reaction.
• It is highly reactive substance , being easily
• It gels protein probably by a process of bridge
formation between compounds.
• With lipid it forms mono and diester linkages which
are then rendered insoluble and non extractable by
fat solvent such as alcohol and xylene.
• Rapid fixing agent
• Stains tissue structure in a additive way as a grey
Fixative Formula Type Advantages Disadvantage Use
Acetic acid -1ml
•It is expensive.
1% Chromic acid -
fat, yolk, lipids
•Needs to be
EFFECT OF FIXATION
RULE #1 IS THAT FIXATIVES DENATURE
MACROMOLECULES; FIXATION CHANGES THE
SHAPE OF LARGE MOLECULES. THIS RULE IS
THE BASIS FOR THE VARIED FUNCTIONS OF
FIXATION AND WHY FIXED SPECIMENS LOOK
THE WAY THEY DO UNDER THE MICROSCOPE.
• RULE #2 IS THAT DIFFERENT FIXATIVES
PRODUCE THEIR OWN MORPHOLOGICAL
PATTERNS. THAT IS AN OBJECTIVE FACT THAT
DOES NOT IMPLY GOOD OR BAD. WHETHER WE
LIKE WHAT WE SEE IS A SUBJECTIVE MATTER
PREDOMINANTLY BASED ON OUR INDIVIDUAL
TRAINING. MANY CHEMICALS ACT AS
FIXATIVES IN THAT THEY DENATURE
MACROMOLECULES, BUT FEW PRODUCE
RULE #3 IS THAT FIXATION IS A CHEMICAL
REACTION THAT IS NOT INSTANTANEOUS. ITS
RATE IS DEPENDENT UPON THE CHEMICAL
NATURE OF THE FIXATIVE SOLUTION AND ITS
Freida L. Carson
Factors affecting fixation.
Hydrogen ion concentration
Substances added to vehicle
Hydrogen ion concentration
• Satisfactory fixation occurs between pH 6 to 8.
• Stabilization of tertiary and quaternary structure of
• By addition of acids pH decreases
destruction of proteins and cause precipitation.
• Hence, fixatives must be neutralized by adding buffer.
Commonly used buffer system are : Phosphate, s-
collidine, veronal acetate, Tris and cacodylate.
•pH chosen must be as near the biochemical optimum
•For electron microscopy , tissue must be fixed with a
gradually increasing pH
Rapid fixation reactions favors fixation.
Fixation should be carried out at gradually increasing
Disadvantages : 1. Risk of tissue distortion
2. Deleterious effect on
Use : 1.Rapid fixation of urgent biopsy
2.To fix tissue with tuberculosis formaldehyde at
100°C is used.
Low temp. Slows down Autolysis more accurate
Ultra structure and enzyme histochemistry and electron
microscopy , temp. range of 0 – 4 degrees is required.
• Fixation depends on diffusion of fixative into the
• Penetration of fixatives is a slow process.
• Size of specimen is important to ensure complete
penetration of fixatives.
• Small or thin slices of blocks - satisfactory fixation
• Large blocks of specimen - slow fixation 63
• Slow rate of diffusion and reaction give rise to various
zones of tissue fixed to different degrees.
• d=k √t (d-depth penetrated , t-time , k-coefficient of
• Fixed tissue acts as a barrier to subsequent inward
diffusion of fixatives.
• Hypertonic solutions - cell shrinkage.
• Isotonic and hypotonic solutions - cell swelling
• In general fixatives that act mainly on protein
precipitants cause shrinkage irrespective of what
the osmotic pressure is and for non protein
precipitants, reverse is true.
• By varying the Osmolality, structure of membrane system
within various cells can be altered.
Thus , additives to fixatives can alter extracellular space in
• Sucrose is commonly added to osmium tetroxide for ultra
• Fixative solutions must be preferably isotonic, thus
cell swelling is compensated by processing and wax
• Low concentration of fixative with neutral pH favors
• Glutaraldehyde solution is used as 3% solution but it is
effective even at concentration as low as 0.05% with
correct pH of fixative.
• Presence of buffer causes polymerization of Aldehyde
with a consequent decrease in effective concentration.
• Staining of tissue is altered with the concentration of
• Long duration
- In Aldehyde :
- a) inhibit enzyme activity and immunological reactions
- b) shrinkage and tissue hardening.
• longer duration of fixation
• effective polymer formation
- In oxidizing fixatives : degrade the tissue by oxidative
cleavage of proteins and loss of peptides.
i) Changes in volume-
Ideally, changes in processing and fixation cancel each other
giving no net change.
Formalin fixed tissues along with paraffin embedding causes
33% shrinkage .
ii) Substances added to vehicle:
fixative agent + Buffer + water.
Salts added have denaturing and stabilizing effect on
Eg : NaCl2 + HgCl2 , Tannic acid, Alcian blue for
A technique or process used to preserve
bodies or body parts, tissue. The water and fat
are replaced by certain plastics(or resin),
yielding specimens that can be touched, do
not smell or decay, and even retain most
properties(especially morphology) of the
John D. Bancroft: Theory & Practice Of
Culling’s: Histological Techniques
Fixation and Processing-Freida L. Carson