FIXATION AND FIXATIVES - (Weekend and Evening)

MLP 309
FIXATION AND
FIXATIVES
CECILIA LEKPOR
10/29/2023 Cecilia Lekpor - Fixation and Fixatives (Weekend Group) 1

LECTURE OBJECTIVE
❑At the end of the lecture students should be able to:
❖ Explain the terms autolysis and putrefaction
❖Explain fixation and know the importance of fixation in the
Histopathology Lab
❖Describe the characteristics of an ideal fixative
❖Classify fixatives
❖Discuss the mechanisms of action of fixation by various types of
fixatives

INTRODUCTION
❑Once a tissue is removed from the body, a process of self-destruction
(or digestion) is initiated soon after cell death by the action of
intracellular enzymes located in lysosomes – This process is known as
AUTOLYSIS
❑This results in the breakdown of protein and final liquefaction of the

cell
❑Autolysis:
• Quickly in tissues rich in enzymes such as the liver, kidney and
brain
• Less in tissues such as fibrous tissue e.g ligaments
INTRODUCTION-CONT
❑Bacterial decomposition (putrefaction) can also produce changes in
tissues that mimic autolysis
❖These changes are produced by bacterial proliferation in the dead

tissue
❖The bacteria may be derived from the body’s normal flora or may be
present in infected tissues at the time of death

FIXATION
❑Fixation is the process by which the constituents of the cells and
tissues are fixed in a physical and partly also in a chemical state, so
that they will withstand subsequent treatment with various reagents
without loss, significant distortion, or decomposition
❑The purpose of fixation is to preserve and harden tissues to retain as

nearly as possible the same relations they had in the living body
❖The cells and extra-cellular materials must be preserved in such a way that
there is little alteration as possible to the structure and chemical composition
of the living tissue
❖ Components of the tissue must be rendered insoluble in all the reagents to

which they will subsequently be exposed
FIXATION-CONT
❖Fixation is the first step in histopathological examination
❖It forms the foundation for subsequent stages in the preparation of

tissues in microscopic examination
❖The chemical and physical agents used in fixation are FIXATIVES
❖Choice of appropriate fixative depends on:

➢the structures and entities to be demonstrated
➢the effects of short and long-term storage
AIMS OF FIXATION
❑The primary function of fixatives is to prevent autolysis (enzymes
attack) as well as putrefaction (bacterial attack) of tissues
❖Preserve cells and tissue constituents in as close to the living state as

possible
❖Allow tissues fixed to undergo further procedures without alteration

in shape or volume
❖Stabilize the tissue and cellular constituents so that they can withstand
subsequent stages of tissue processing and there is no loss of small
molecules
AIMS OF FIXATION-CONT
❖Ensure that antigenic sites for immunohistochemistry are not greatly
altered or lost
❖Leave tissues in a condition which subsequently allows clear staining

of sections

CONSTITUENTS OF TISSUE IN FIXATION
❑The essential tissue constituent during fixation is the protein
component
❑The structure of an animal tissue is determined largely by the structure

of its contained proteins
❑Other tissue components preserved in fixation are:

➢Nucleic acids
➢Carbohydrates (Macromolecules)
➢Lipids-preservation therefore depends largely on avoiding agents
that dissolve them (NB: Most fixatives do not directly affect lipids)
CLASSIFICATION OF FIXATIVES
❖CHEMICAL AGENTS
➢Aldehydes
➢Oxidising agents
➢Protein-denaturing agents
➢Miscellaneous chemical agents
❖ PHYSICAL AGENTS
➢Heat, microwave
➢Freezing

CLASSIFICATION OF FIXATIVES-CONT
❑ CHEMICAL FIXATIVES
❖ Aldehydes and other cross-linking fixatives

➢Formaldehyde, glutaraldehyde, acrolein (acrylic aldehyde), glyoxal,
carbodiimides
❖ Oxidising agents
➢Osmium tetroxide, potassium permanganate, potassium dichromate
❖ Protein-denaturing agents
➢Acetic acid, methyl alcohol (methanol), ethyl alcohol (ethanol)
❖ Miscellaneous chemical agents
➢Mercuric chloride, picric acid
CLASSIFICATION OF FIXATIVES-CONT
❖ PHYSICAL METHOD
➢Heat/microwave irradiation
▪ Application of heat results in coagulation of protein
▪ This results in cellular distortion so that close similarity to the living state
is lost
➢Freezing
▪ Frozen sections-(using cryostat)

▪ Freeze-drying: Rapidly freezing of fresh tissue at -160oC and
subsequently removing water molecules by sublimation in a vacuum
▪ Freeze substitution: Rapidly freezing of tissue and the substitution of the
ice removed by a dehydrating solution
MECHANISMS OF FIXATION
❖ REACTION OF FIXATIVES WITH PROTEINS
➢ Most important component in fixation
➢Stabilises proteins by forming cross-links between soluble proteins and

structural proteins, ultimately providing mechanical strength

MECHANISM OF FIXATION-CONT
❖Aldehydes
➢ Cross-links are formed between proteins and aldehyde group of
fixatives
➢Reacts with the basic amino acid residues of proteins
➢Reaction is reversible using water during the first 24 hours of reaction

as the fixative causes relatively little cross-linking under fixation
conditions

ALDEHYDES-CONT
FORMALDEHYDE GLUTALADEHYE
Slow reaction Most effective at cross-linking and the
reaction is rapid
Reversible with excess water Irreversible
Not good morphological picture Good morphological picture
Loss of enzyme and immunological Loss of enzyme and immunological
activity is less. It is ideal for activity is more. It is not ideal for
immunohistochemistry immunohistochemistry
E.g. Acrolein produces more cross-

linking than formaldehyde under optimal
10/29/2023 conditions
Cecilia Lekpor - Fixation and Fixatives (Weekend Group) 15
MECHANISMS OF FIXATION-CONT
❖Oxidizing Agents
➢Reacts with the side chain of proteins, allowing the formation of
crosslinks that stabilize tissue structure
➢Cause denaturation despite preserving fine cell structure
➢Used as secondary fixative
▪ Examples-Osmium tetroxide- used as secondary fixative for electron

microscopy
▪ Potassium permanganate and dichromate are less reactive towards
proteins
MECHANISMS OF FIXATION-CONT
❖Mercuric chloride
➢It reacts with histidine residues in proteins
➢Also, there is H+ ions production making the solution more acidic and
efficient
➢Example-Zenker fixative increases staining brightness and give excellent

nuclear details
➢Penetrate tissue poorly and produces tissue shrinkage
➢Best application is in fixation of haematopoietic and reticuloendotial tissues

REACTION WITH NUCLEIC ACID
❖Fixation brings about change in physical or chemical state of DNA or
RNA in room temperature
➢Few fixatives reacts chemically with nucleic acids
➢ In their normal state within the cell, DNA and RNA do not react to any
extent with formaldehyde or glutaraldehyde
➢Heating at 45°C for RNA and 65°C for DNA, reaction begins to take
place as a results of the uncoiling of nucleic acids
➢During fixation at room temperature, no uncoiling of nucleic acids

occurs and therefore no reaction with the fixative
REACTION WITH NUCLEIC ACID-CONT
➢However, impregnation with molten paraffin wax, results in reaction
with any remaining fixative
➢This lack of initial reaction allows paraffin embedded tissue to be used

for DNA analysis, there is little fixation, thus, retention in tissues
➢Methanol is the best choice for non-cross-linking fixatives for in situ

hybridization and immunohistochemistry
➢For PCR-based detection of DNA or RNA, some denaturing fixatives

like acetone and methanol as well as formaldehyde (“gentle” cross-
linking fixative) are used
REACTION WITH LIPIDS
❖Most lipids are still labile following aldehyde fixation, so lost during
routine processing. Frozen section is best for their demonstration
➢Phospholipids containing amino groups such as phosphatidyl

ethanolamine are fixed by aldehydes
➢The only chemical agents that truly fix lipids are osmium tetroxide
and chromic acid
➢Additives such as tannic acids may be used for demonstration of lipids

with light microscopy
REACTION WITH CARBOHYDRATES
❖Single fixative is not satisfactory for carbohydrate demonstration

➢Glycogen and mucins are considered in tissue carbohydrate demonstration
➢In glycogen demonstration:
▪ Removal of bound water molecules from glycogen by the fixative decreases
solubility - equivalent to denaturation
▪ Preservation is due to fixation of its linked proteins rather than glycogen itself or
may be as a result of trapping in a matrix of fixed protein
➢Formaldehye is not a good fixative for carbohydrate but preserves proteins so that
they hold glycogen which is otherwise readily leeched from the cell
REACTION WITH CARBOHYDRATES-CONT
➢Alcoholic or picric fixatives- results in preservation of glycogen which
appears coarse. Eg: Alcoholic formaldehyde
➢Alcohols act partly by forming a meshwork of denatured protein, and

partly by precipitating the carbohydrate moiety
➢Ultra structural studies- Glutaraldehyde is satisfactory
➢Procedures requiring long immersion in aqueous solutions may lead to

some loss of precipitated mucins and glycogen
PHYSICAL METHODS
❖Heat/Microwave irradiation
➢Application of heat results in coagulation of proteins
➢Reacts with polar side chain of protein. This increases the thermal
energy which causes denaturation of protein bringing about tissue
stabilization
➢Water molecules and polar side-chains of proteins throughout the tissue

have their thermal energy increased with consequent heating of proteins
➢ Heat applied externally first acts on the superficial layer, but heat
delivered by microwaves is absorbed at all depths within a specimen
PHYSICAL METHODS-CONT
➢When the specimen is already immersed in a chemical fixative,

microwave radiation:
▪ Promotes diffusion of fixative into the tissue
▪ For formaldehyde-based fixatives, it accelerates the cross-linking of
proteins
➢When microwave radiation is the sole fixative agent, cellular distortion
occurs resulting in loss of close similarity to the living state
➢Current practice is to heat the specimen already immersed in a

chemical fixative
FIXATIVES
❖Fixatives are agents used to preserve and maintain the morphological integrity
(shape, chemical constituents and structure) of tissues and cells
❖It forms cross-links between amino acids and proteins thereby making them
insoluble

FIXATIVES-CONT
❑TYPES OF FIXATIVES
❖Simple (one substance)- Example, Formalin
❖Compound (two or more)-Examples: piric acid, Zenker’s fluid, Bouin’s fluid.
➢Microanatomical- Used to preserve the anatomy of the tissue. Examples; formal saline,
Zenker’s fixative.
➢Cytological-Used to fix intracellular structure. Examples; carnoy’s fluid, clakes’s

Regaud’s fluid.
➢Histochemical-Used to demonstrate the chemical constituents of a cell. Examples;

formal saline, cold acetone, absolute
10/29/2023 Cecilia Lekpor -alcohol.
Fixation and Fixatives (Weekend Group) 26
PROPERTIES OF AN IDEAL FIXATIVE
❑An ideal fixative should:
❖Penetrate a tissue quickly
❖Be rapid in action
❖An ideal fixative is expected to harden (to impact mechanical rigidity
to withstand tissue processing) tissue components and prevent
decomposition, putrefaction, and autolysis
❖An ideal fixative is presumed to transmit mechanical toughness to
tissue so that it resists destruction due to further processing steps
❖Cause a minimum loss and minimal physical and chemical alteration of
the cell and its components
❖ Easy availability, be cheap, stable and safe to handle
TYPES OF FIXATIVES
❖FORMALDEHYDE/FORMALIN
➢It is the most routinely used fixative in histopathology either alone or in
combination with other chemical agents
➢Formaldehyde is a gas soluble in water up to 40% (formalin). For the purposes of

determining the strengths of its diluted solutions, saturated solution is regarded as
100% formalin
➢Formalin fixes tissues by forming cross-link between amino acids of proteins

there by making them insoluble.
➢It is a colourless solution which becomes turbid on long storage through

formation of paraformaldehyde, which does not affect fixation ability.
FORMALDEHYDE-CONT
➢Formalin may become acidic through the formation of formic acid which has
effect on fixation, so neutralization is done by buffers.
➢Acidic formalin results in formalin pigment formation ( a brown pigment similar

to malaria pigment.
➢This can be prevented by using buffers such as (Na𝐻2 PO4 and 𝑁𝑎2 HPO4).
➢Addition of MgC𝑂3 or NaOH to neutralized the acidity.
➢Formalin is the widely used fixative in pathology labs worldwide owing to its
convenience in handling, high degree of accuracy and extreme adaptability.
ADVANTAGES OF FORMALIN
➢It is cheap, easy to prepare and relatively stable if buffered
➢It allows subsequent application of many staining techniques.
➢It has a good penetration rate and does not cause excessive hardening of tissues
➢It causes lesser shrinkage compared with other fixatives
➢Staining of some enzymes and fat can easily be carried out on formalin fixed
tissues

DISADVANTAGES
➢Irritant to nostrils, eyes, mucous membrane, causing sinusitis and conjunctivitis to

some workers.
➢In tissue containing blood, dark brown artifact pigments granules are formed
➢It causes dermatitis of the hands of some workers
➢It is a potential carcinogen
➢The use of unbuffered formalin results in the formation of formalin pigments (e.g.
spleen)
GLUTARALDEHYDE
➢ An aqueous solution of glutaraldehyde (glutaric dialdehyde) – is the most
applied fixative in both scanning and transmission electron microscopy (TEM)
➢ In TEM, buffered glutaraldehyde has the reputation of providing the best

ultrastructural preservation in a wide variety of tissues
➢ Solutions kept for long periods at ambient temperatures, leads to formation of

precipitates and aldehyde levels fall, so that purification becomes necessary –
simple shaking with BaCO3

ADVANTGES
➢Glutaraldehyde gives better preservation of cellular and fluid proteins than does
formalin because of formation of more and more stable cross-linkages
➢Causes less shrinkage than formalin and gives better sections of many tissues,
especially of blood clot and brain
➢More pleasant and less irritable to handle because its vapour pressure is very low
➢Wide range of routine and histochemical staining procedures is possible after

glutaraldehyde fixation
DISADVANTAGES
➢More expensive and less stable
➢Penetrate tissue more slowly than formalin thus only small blocks of tissue (1-
2mm) fix well at temperatures of 1-4oC
➢Inferior to formalin for the PAS technique; causes a greater degree of diffuse
background with PAS reaction
➢Mucous membrane and skin irritant, can enter body by inhalation of the vapour
➢Affects the nervous system and significant exposure can result in

unconsciousness
CHROMATE
➢It is prepared and stored as 2% stock solution
➢It is a very strong oxidizing agent and therefore should not combined
with reducing agents

CHROMATE-CONT
❑ADVANTAGES
➢Rapidly fixes small pieces of tissue
➢Stains tissue structures in an additive way; it is reduced and attached as a gray-
black “deposit” to various tissue elements in proportion to their content of reactive
and reducing groups
➢These properties make it useful in electron microscopy
❑DISADVANTAGES
➢Expensive
➢Penetrates tissues slowly
➢Produces great difficulty in counterstaining after its use
➢Easily reduced by light, heat, and dust or other particulate matter and so must be
stored in a cool place and in a cleaned dark glass
➢Produces vapours that are irritating to the eyes and skin

PICRIC ACID FIXATIVE
➢Used in combination with other fixatives
➢Reacts with histones and basic proteins and forms crystalline picrates with amino
acids which are water-soluble until treated with alcohol
➢Penetrates well and leaves tissue soft but causes considerable shrinkage
➢ Causes loss of basophilia (little affinity for basic dyes) unless the tissue is
thoroughly washed following fixation
➢Preserves glycogen well

PICRIC ACID FIXATIVE-CONT
❑ADVANTAGES
➢One of the best fixatives for demonstrating glycogen
➢Small fragments of tissue are easily identified as they pick up the yellow colour of
picric acid
➢In a fixative mixture produces bright staining with dyes
❑DISADVANTAGES
➢Lyses red cells
➢Reduces the amount of demonstrable ferric iron
➢Tissues fixed in picric acid solutions crumble when frozen sections are cut
➢Tissues left in fluid for longer than 12 to 24 hours become hard and brittle and
difficult to section
PICRIC ACID FIXATIVE-CONT
❖REMOVING PICRATE SFROM SECTIONS
➢Dewax section and place in a saturated solution of lithium carbonate in 70% ethyl alcohol
for some minutes
➢Dewax section and place in ethyl alcohol followed by 5% sodium thiosulphate. Then wash
in running water

❑MERCURIC CHLORIDE
➢ Usually combined with other fixatives
➢ Mixtures containing mercuric chloride include Zenker’s, Helly’s,
Susa
❖ADVANTAGES
➢ Give better staining of nuclei and connective tissue
➢ Nuclear chromatin is shown in fine detail and trichrome staining is
particularly good
➢ Cytoplasmic staining with acidic dyes is enhanced
➢ Give best results with metachromatic staining

❖DISADVANTAGES
➢Zenker’s solution causes considerable lysis of red cells and removes much iron
from haemosiderin. Helly’s solution is better in these respects
➢Mercurial solutions lead to formation of deposits in the tissues of black

precipitates of mercury, except Susa which must be removed before staining
➢All mercurial fixatives reduce the amount of demonstrable glycogen
➢HgCl2 penetrates very slowly and if tissues are left in fixative for more than 1 to 2
days they become unduly hard and brittle
➢Cutting of frozen sections on tissues fixed in mercurial solutions is extremely

difficult
➢Mercuric
10/29/2023
salts are highly toxic and must not be disposed into sewage systems
Cecilia Lekpor - Fixation and Fixatives (Weekend Group) 41
❖REMOVAL OF MERCURY DEPOSITS
➢Add enough saturated solution of iodine in 96% alcohol in the initial alcohol bath
used in dehydration (brown colour)
➢Removes gross mercuric chloride deposits in tissue blocks prior to sectioning

saving the knife edge
➢When sections are cut, remaining finer precipitates are removed by dewaxing and
placing the sections in 0.5% iodine in 70% alcohol (0.5ml of iodine solution in
100 ml of 70% alcohol) for 5-10 minutes
➢Sections are rinsed water and placed in 5% w./v. sodium thiosulphate for 5
minutes to remove iodine and washed in running water for 5 minutes.
FACTORS AFFECTING FIXATION
➢ Temperature -affects the morphology of the tissue. Fixation times may be
reduced by 25 to 40% if the fixative temperature is raised to 45oC
➢For electron microscopy and some histochemical procedures, the temperature for
fixation is usually 0-4˚C
➢This temperature slows down autolysis and diffusion of various cellular

components in order to obtain a more lifelike appearance
➢ As temperature rises, it increases the rate of penetration, autolysis and diffusion

of cellular components
➢Nucleic acids (DNA and RNA) do not react with fixatives to any extent at room
temperatures
FACTORS AFFECTING FIXATION-CONT
➢SIZE- Ideal size of the tissue should be 3mm. The rate of penetration of the
fixative will inform the maximum size of a block to be fixed by immersion. Large
specimens should be cut into thin slices and placed in the fixative
➢ VOLUME RATIO- Volume of fixative is at least 15 to 20 times greater than

volume of tissue
➢ TIME- Minimum fixation time for 5mm tissue is 12hrs. For electron microscopy,
sliced tissue is preserved for 3 hrs in 3% glutaraldehyde
NOTE: Prolonged fixation in aldehydes can cause shrinkage and hardening of

tissue. This results in severe inhibition of enzyme activity and immunological
reactions
➢ Washing in running water considerably restores the activity of some enzymes

FACTORS AFFECTING FIXATION
➢CHOICE OF FIXATIVES:
➢The method of fixation should be selected immediately once the
specimen is presented
➢Example- For Gout, a fixative of choice is absolute alcohol
➢ Electron Microscopy – the choice is Gluteraldehyde

THANK YOU
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MLP 309

10/29/2023 Cecilia Lekpor - Fixation and Fixatives (Weekend Group) 1

10/29/2023 Cecilia Lekpor - Fixation and Fixatives (Weekend Group) 2

❑This results in the breakdown of protein and final liquefaction of the

❖These changes are produced by bacterial proliferation in the dead

10/29/2023 Cecilia Lekpor - Fixation and Fixatives (Weekend Group) 4

❑The purpose of fixation is to preserve and harden tissues to retain as

❖ Components of the tissue must be rendered insoluble in all the reagents to

❖It forms the foundation for subsequent stages in the preparation of

❖The chemical and physical agents used in fixation are FIXATIVES

❖Choice of appropriate fixative depends on:

❖Preserve cells and tissue constituents in as close to the living state as

❖Allow tissues fixed to undergo further procedures without alteration

❖Leave tissues in a condition which subsequently allows clear staining

10/29/2023 Cecilia Lekpor - Fixation and Fixatives (Weekend Group) 8

❑The structure of an animal tissue is determined largely by the structure

❑Other tissue components preserved in fixation are:

10/29/2023 Cecilia Lekpor - Fixation and Fixatives (Weekend Group) 10

❖ Aldehydes and other cross-linking fixatives

▪ Frozen sections-(using cryostat)

➢Stabilises proteins by forming cross-links between soluble proteins and

10/29/2023 Cecilia Lekpor - Fixation and Fixatives (Weekend Group) 13

➢Reacts with the basic amino acid residues of proteins

➢Reaction is reversible using water during the first 24 hours of reaction

10/29/2023 Cecilia Lekpor - Fixation and Fixatives (Weekend Group) 14

E.g. Acrolein produces more cross-

➢Cause denaturation despite preserving fine cell structure

➢Used as secondary fixative

▪ Examples-Osmium tetroxide- used as secondary fixative for electron

➢Example-Zenker fixative increases staining brightness and give excellent

➢Penetrate tissue poorly and produces tissue shrinkage

➢Best application is in fixation of haematopoietic and reticuloendotial tissues

➢Few fixatives reacts chemically with nucleic acids

➢During fixation at room temperature, no uncoiling of nucleic acids

➢This lack of initial reaction allows paraffin embedded tissue to be used

➢Methanol is the best choice for non-cross-linking fixatives for in situ

➢For PCR-based detection of DNA or RNA, some denaturing fixatives

➢Phospholipids containing amino groups such as phosphatidyl

➢Additives such as tannic acids may be used for demonstration of lipids

❖Single fixative is not satisfactory for carbohydrate demonstration

➢Alcohols act partly by forming a meshwork of denatured protein, and

➢Ultra structural studies- Glutaraldehyde is satisfactory

➢Procedures requiring long immersion in aqueous solutions may lead to

➢Water molecules and polar side-chains of proteins throughout the tissue

➢When the specimen is already immersed in a chemical fixative,

➢Current practice is to heat the specimen already immersed in a

10/29/2023 Cecilia Lekpor - Fixation and Fixatives (Weekend Group) 25

❖Simple (one substance)- Example, Formalin

❖Compound (two or more)-Examples: piric acid, Zenker’s fluid, Bouin’s fluid.

➢Cytological-Used to fix intracellular structure. Examples; carnoy’s fluid, clakes’s

➢Histochemical-Used to demonstrate the chemical constituents of a cell. Examples;

➢Formaldehyde is a gas soluble in water up to 40% (formalin). For the purposes of

➢Formalin fixes tissues by forming cross-link between amino acids of proteins

➢It is a colourless solution which becomes turbid on long storage through

➢Acidic formalin results in formalin pigment formation ( a brown pigment similar

➢Addition of MgC𝑂3 or NaOH to neutralized the acidity.

➢It allows subsequent application of many staining techniques.

➢It causes lesser shrinkage compared with other fixatives

10/29/2023 Cecilia Lekpor - Fixation and Fixatives (Weekend Group) 30

➢Irritant to nostrils, eyes, mucous membrane, causing sinusitis and conjunctivitis to

➢It causes dermatitis of the hands of some workers

➢It is a potential carcinogen

➢ In TEM, buffered glutaraldehyde has the reputation of providing the best

➢ Solutions kept for long periods at ambient temperatures, leads to formation of