FIXATION AND FIXATIVES  Fixation renders insoluble certain tissue

Histotechnology component that may otherwise leak out

 The art and science performed by the subsequent histologic handling.
histotechnologist to produce a tissue  Two basic mechanisms involved in fixation:
section of good quality that will enable the o Additive fixation
pathologist to diagnose the presence or o Whereby the chemical constituent of
absence of disease. the fixative is taken in and becomes
 The first and most critical step in part of the tissue by forming cross-links
histotechnology involves fixing or or molecular complexes and giving
preserving fresh tissue for examination. The stability to the protein.
process is known as fixation. o Formalin, mercury, and osmium
tetroxide)
FIXATION o Non-additive fixation
 No process of histotechnology is more o The fixing agent is not incorporated
critical to slide preparation than fixation. into the tissue, but alters the tissue
 If the fixation is not adequate, the other composition and stabilizes the tissue
processes will also be inadequate. by removing the bound water attached
 Inadequate or poor fixation will result in a to H bonds of certain groups within the
poorly processed tissue and will make it protein molecule
difficult for the pathologist to render a o Alcoholic fixatives.
proper diagnosis.
 The primary aim of fixation is to preserve
MAIN FACTORS INVOLVED IN FIXATION
the morphologic and chemical integrity of
Hydrogen ion Concentration
the cell
 Satisfactory fixation occurs between ph 6 to
 With fixation, the shape, structure,
8.
intercellular relationship and chemical
 Outside this range, changes may occur that
constituents of tissues are preserved.
are detrimental to ultrastructural
 Fixation prevents degeneration,
preservation of the tissue.
decomposition, putrefaction, and distortion
Temperature
of tissues after removal from the body.
 Room temperature
 The secondary goal of fixation is to harden
 For EM, and some histochemistry , 0-4C
and protect the tissue from the trauma of
 Formalin heated to 60C is sometimes used
further handling, so that it is easier to cut
for the rapid fixation of very urgent biopsy,
during gross examination.
although the risk of tissue distortion is
 Neutral buffered formalin allows a diagnosis
increased.
to be made in most cases.
 Formalin at 100C can be used to fix tissues
 However, certain specialized studies such as
with tuberculosis.
electron microscopy, histochemistry, and
Thickness of the section
immunocytochemistry require specific
 Tissue blocks taken should be either small
fixation procedures.
(1-2 mm for EM and 2cm for light
 The most important reactions for
microscopy) or thin (no more than 0.4cm
maintaining morphology in the fixation of
for light microscopy) or as prescribed by
tissues for routine histopathology are those
tissue processor manufacturer
that stabilize the proteins.
 Large specimen, should be opened or sliced
 Fixatives have the property of forming
thinly.
cross-links between proteins.
 Brain should be suspended whole in
To preserve the tissue
10%buffered formalin for 2-3 weeks to
 Fixation preserves the tissue by stopping all
ensure fixation and some hardening prior to
cellular activities so that the cells can be
sectioning.
viewed under the microscope as if they are
Osmolality
still in their original living state.
 Hypertonic solutions give rise to cell
To prevent breakdown of cellular elements
shrinkage.
 Surgical removal of the tissue from the body
 Isotonic as well as hypotonic fixatives cause
will deprive it of oxygen and nutrition,
cell swelling and poor fixation.
leading to a degradative chemical process
 The best results are usually obtained using
or cell death.
slightly hypertonic solutions ( 400-
 Lysosome contain hydrolytic enzymes that
450mOsM), isotonic solutions (340mosm)
are released when the integrity of the cell is
Concentration
destroyed.
 Formaldehyde is normally used as a 10%
To prevent breakdown of cellular elements
solution and glutaraldehyde is normally
 Fixative prevents autolysis by inactivating
used as 3% solutions
the lysosomal enzymes, or by chemical
 The presence of buffer causes
altering, stabilizing and making the tissue
polymerization of the aldehyde, with
components insoluble.
consequent decrease in its effective
 Fixation also protects the tissue from
concentration.
further decomposition (putrefaction) after
Duration of fixation
death due to bacterial or fungal
 Primary fixation in buffered formalin is
colonization and overgrowth.
usually carried out for 2-6 hours during the
To coagulate or precipitate Protoplasmic substances
day the specimen is obtained, but they
 remain in fixatives over the week end CHARACTERISTICS OF A GOOD FIXATIVES
without much adverse effect.  Cheap
 Prolonged fixation may cause shrinkage and  Stable
hardening of tissue, and may severely  Safe to handle
inhibit enzyme activity and immunological  Kill the cell quickly thereby producing
reactions. minimum distortion of cell constituent
 For EM, it is recommended that diced  Inhibit bacterial decomposition and
tissues be fixed for 3 hours and then placed autolysis
in holding buffer.  Produce minimum shrinkage of tissue
 Harden tissues thereby making the cutting
  of sections easier.
PRACTICAL CONSIDERATION OF FIXATION  Must be isotonic
Speed  Must make cellular components insoluble
 Specimen should be placed in fixatives as to hypotonic solutions and render them
soon as it is removed from the body. This is insensitive to subsequent processing
done to prevent autolysis and putrefaction  Permit subsequent application of many
Penetration staining procedures to facilitate easier and
 1mm/hr profitable examination.
 Slows down as it goes deeper into the tissue So far, no single fixatives has yet been known to
Volume possess all the qualities of an ideal solution. There
 10-25 times the volume of tissue to be fixed are numerous fixatives available, and each has its
 Maximum effectiveness of fixation is noted own advantages and disadvantages.
to be 20 times the tissue volume
Duration of Fixation TYPES OF FIXATIVES ACCORDING TO ACTION AND
 Fibrous organs such as uterus or intestinal COMPOSITION
tract take longer than small or loosely According to COMPOSITION
textured tissues such as biopsies or  Simple fixatives
scrapings.  Aldehydes
 Fixation time can be cut down by using o Formaldehyde
heat, vacuum, agitation or microwave. o glutaraldehyde
 Metallic fixatives
MISCELLANEOUS CONSIDERATION o Mercuric chloride
 The tissue selected for sectioning should be o Chromate fixatives
thin enough to allow penetration by  Potassium
fixatives within reasonable amount of time. dichromate
 To maintain an adequate fixation time of 4-  Chromic acid
6hours, the recommended size of the o Lead fixatives
tissues is 2cm square, and no more than  Picric acid
4mm thick.  Acetic acid
 Most tissues can be cut and trimmed  Acetone
without prior fixation, except for the brain  Alcohol
which is generally soft when unfixed.  Osmium teroxide
 Refrigeration is used to slow down (osmic acid)
decomposition if the tissue needs to be o heat
photographed and cannot be fixed  Compound fixatives
immediately.  made up of two or more fixatives
which have been added together
EFFECTS OF FIXATIVES IN GENERAL to obtain the optimal combined
 They harden soft and friable tissues and effect of their individual actions
make the handling and cutting of sections upon the cells and tissue
easier. Accelerated by the action of alcohol constituent.
during the dehydration process.
 They make the cells resistant to damage According to ACTION:
and distortion caused by hypotonic and  Microanatomical fixatives
hypertonic solutions used during tissue  Those that permit the general
processing. microscopic study of tissue
 Inhibit bacterial decomposition structures without altering the
 Increase optical differentiation of cells and structural pattern and normal
tissue components thereby rendering them intercellular relationship of the
more readily visible during examination. tissues in question.
 Act as a mordant or accentuators to  Cytological
promote or hasten staining or they may  Those that preserve specific parts
inhibit certain dyes in favor of another. and particular microscopic
(formaldehyde intensifies while osmium elements of the cell itself
tetroxide inhibits hematoxylin staining)
 Nuclear fixatives
 Reduce the risk of infections during
 Cytoplasmic fixatives
handling and actual processing of tissues.
 Histochemical Fixatives
MICROANATOMICAL FIXATIVES
 10% fomol saline
 10% buffered neutral formalin
 Heidenhains Susa
 Formol sublimate (formol corrosive)
 Zenker’s solution
 Zenker-formol (kelly’s solution)
 Bouin’s solution
 Brasil solution
CYTOLOGICAL FIXATIVES
 Nuclear fixatives
o Preserves the nuclear structures
o Contain glacial acetic acid as their
primary component due to its affinity
for nuclear chromatin.
o Ph of 4.6 or less
 Flemming’s fluid
 Carnoy’s fluid
 Bouin’s fluid
 Newcomer’s fluid
 Heidenhain’s Susa
o Ethanol and methanol, including
Carnoy’s solution, are commonly used
fixatives for nucleic acids.
o Ethanol appears to give the most
valuable usable DNA fragments for
PCR, whereas formaldehyde limits the
size of fragment s which can be
retrieved.
 Cytoplasmic fixatives
o Preserves cytoplasmic structures
o Must never contain glacial acetic acid
which destroys mitochondria and golgi
bodies of the cytoplasm.
o ph is more than 4.6
 Flemming’s fluid without
acetic acid
 Kelly’s fluid
 Formalin with
chroming”
 Regaud’s fluid (Muller fluid)
 Orth’s fluid
o For RNA, the precipitant fixatives
ethanol and acetone give the best
quantitative results using frozen tissues
as the standard.
 Histochemical Fixatives
o Preserves the chemical constituents of
cells and tissues
 Formol saline 10%
 Absolute Ethyl alcohol
 Acetone
 Newcomer’s fluid
LIPID FIXATION
 Cryostat or frozen sections should be used
for demonstrating lipid in tissues, followed
by a general lipid stain.
 Fixatives containing mercuric chloride and
potassium dichromate can be effective for
preservation of lipids in cryostat sections.
 In general, phospholipids, which contain
amino groups, are fixed by aldehydes.
 Baker’s formol-calcium may be used to
preserve phospholipids.
CARBOHYDRATE FIXATION
 Alcoholic fixatives are generally
recommended for glycogen fixation.
Glycogen can be demonstrated
satisfactorily enough for diagnosis, although
losses of glycogen can be high (60-80%) in
aqueous solution.
 Alcoholic formaldehyde is a better fixative
in human skin compared with neutral
buffered formaldehyde.
PROTEIN FIXATION
 Neutral buffered formol saline or
formaldehyde vapor are the most
commonly used fixatives for amino acids.
GLYCOGEN FIXATION
 Most useful fixatives are alcohol based,
such as Rossman’s fluid or cold absolute
alcohol.
 Essential when processing tissues from
patients with glycogen storage disease.
 There is better retention of glycogen if the
section is coated with celloidin.
MIXTURES OF FIXATIVES
 Two aldehyde fixative mixtures have been
particularly useful for Electron cytchemistry.
 The best known is Karnovsky’s
paraformaldehyde-glutaraldehyde solution.
 Acrolein is another aldehyde which has
been introduced as a mixture with
glutaraldehyde or formaldehyde.
 Acrolein penetrates tissues rapidly,
preserves morphology and enzyme activity
at low concentrations, and may be useful
for immersion fixation of surgical biopsies.
ALDEHYDE FIXATIVES
 Formaldehyde (Formalin)
 10% Formol-Saline
 10% Neutral buffered formalin or
“post- phosphate –Buffered formalin
 Formol-Corrosive (Formol Sublimate)
 Alcoholic Formalin (Gendre’s) Fixative
 GLUTARALDEHYDE
Formaldehyde (Formalin)
 Most widely used fixatives is 10% formalin.
 A gas produced by the oxidation of methyl
alcohol and is soluble in water to the extent
of 37-40% weight in volume.
 The commercially available solution of
formaldehyde contains 35-40% gas by
weight.
 Pure stock solution of 40 %
formalin is unsatisfactory
for routine fixation since
high formaldehyde
concentrates tend to over
harden the outer layer of
the tissue and affect
staining adversely
 Commonly used as 4%
solution, giving 10% formalin for tissue
fixation.
 It must be diluted 1:10 to make 10 %
solution.
 Usual fixation time is 24 hours
 buffered to pH 7 with phosphate buffer
Advantages
 o cheap, readily available, easy to prepare,
and relatively stable, especially if stored in
buffered solutions.
o Compatible with many stains
o Does not over harden tissues, even with
prolonged periods of fixation, as long as
solutions are regularly changed.
o Penetrates tissue well.
o Preserves fat and mucin, making them
resistant to subsequent treatment with fat
solvents, thereby allowing them to be
stained for demonstration.
o Preserves glycogen
o It preserves but not precipitate proteins,
thereby allowing tissue enzymes to be
stained.
o Recommended for nervous tissue
preservation.
o Allows natural tissue color to be restored
after fixation by immersing formalin fixed
tissues in 70% alcohol for one hour.
Recommended for colored tissue
photography.
o Allows frozen tissue sections to be
prepared easily.
o Does not require washing out, unless
tissues have stayed in formalin for
excessively long periods of time.
o It is a tolerant fixative, used for mailing
specimen because specimen can be left in
formalin indefinitely.
Disadvantages
o Fumes are irritating to the nose and eyes
and may cause sinusitis, allergic rhinitis or
excessive lacrimation
o Irritating to the skin and may cause allergic
dermatitis.
o Produce considerable shrinkage of tissues
o Soft fixative and does not harden some
cytoplasmic structures adequately enough
for paraffin embedding
o If unbuffered:
 Reduces both basophilic and
eosinophilic staining of cells,
thereby reducing the quality of
routine cytologic staining.
 Forms abundant brown pigment
granules on blood-containing
tissues
o Prolonged fixation may produce:
 Bleaching of the specimen and loss
of natural tissue colors
 Dispersal of fat from the tissues
into the fluid
 Dissolution or loss of glycogen
PRECAUTIONS:
o Prolonged storage of formaldehyde,
especially at very low temperature, may
induce precipitation of white
paraformaldehyde deposits and produce
turbidity although this, in itself, does not
impair the fixing property of the solution.
o Methanol added as a preservatives to
formaldehyde will prevent its
decomposition to formic acid or
precipitation to paraformaldehyde, but it
serves to denature protein, thereby
rendering formalin unsuitable as a fixative
for EM.
o Concentrated solutions of Formaldehyde
must NEVER be neutralized since this might
precipitate violent explosions.
o Room should be properly ventilated with
adequate windows and preferably with
exhaust fan to prevent inhalation of fumes
and consequently injury to the eyes and
nose.
o Dermatitis may be avoided by the use of
rubber gloves when handling specimens
fixed in formalin.
o Bleaching of tissues may be prevented by
changing the fluid fixative every three
months.
o Natural tissue colors may be restored by
immersing tissues in 70% alcohol after
fixation.
o Brown or black crystalline precipitate
formed by the cation of formic acid with
blood can be removed from the sections
prior to staining by treatment with
saturated alcoholic picric acid or a 1%
solution of potassium hydroxide in 80%
alcohol. The use of neutral buffered
formalin will prevent the pigmentation.
o If fatty tissues are to be stored for a long
time, cadmium or cobalt salts are added to
prevent dispersion of fat out into the fluid.
o Acid reaction due to formic acid formation
can be buffered or neutralized by adding
magnesium carbonate or calcium carbonate
to 10-15% formalin. This should be done on
a wide mouth bottle to prevent violent
explosion due to insufficient gas space for
CO2 release.
o To improve staining and produce firmer and
harder consistency, tissues fixed in formalin
for 1-2 hours may be placed again in helly’s
fluid for 4-6 hours or in formol-sublimate
for 4-16 hours (secondary fixation)
o If postfixed in osmic acid, the tissue must
not be washed in demineralized water to
prevent hypotonicity and bleaching.
o Fixation of tissue blocks not exceeding 5mm
in thickness is usually complete in 6-12
hours at room temperature.
o Fixation by formalin is influenced by heat,
vacuum, agitation and microwave
techniques. Fixation can be speeded up by
heating solution at 50C and placing it under
a vacuum or in microwave.
10% Formol-Saline
 Simple microanatomical fixative made up of
saturated formaldehyde (40% weight
volume) diluted to 10% with sodium
chloride.
 Recommended for fixation of CNS tissues
and general post-mortem tissues for
histochemical examination.
 Formula
◦ Formaldehyde, 40%-----------100ml
◦ NaCL __________________9grams
◦ Distilled water___________900 ml
 Fixation time ----24 hours at 35 C (95F)
48 hours at 20-25 C (65-77 F)
Advantages:
o Penetrates and fixes tissues evenly
o Preserves microanatomic and cytologic
details with minimum shrinkage and
distortion
o Large specimens may be fixed for long time
provided that the solution is changed every
three months
o Preserves enzymes and nucleoproteins
o Demonstrates fats and mucin
o Does not over harden tissues, thereby
facilitating dissection of the specimen
o Ideal for most staining techniques, including
silver impregnation
o Allows natural tissue color to be restored
upon immersion in 70%alcohol.
Disadvantages
o Slow fixative
o Tissues tend to shrink during alcohol
dehydration, this may reduced secondary
fixation
o Metachromatic reaction of amyloid is
reduced
o Acid dye stains less briefly than when fixed
with mercuric chloride.
10% Neutral buffered formalin or phosphate –
Buffered formalin
 Recommended for preservation and
storage of surgical, postmortem and
research specimens.
 Formula
◦ Sodium dihydrogen phosphate
(anhydrous) –3.5 grams
◦ Disodium hydrogen phosphate -6.5
grams
◦ Formaldehyde 40% 100ml
◦ Distilled water 900ml
 Fixation time - 4-24 hours
Advantages
o Similar to formol saline with the following
additions
o Prevents precipitation of acid formalin
pigments on postmortem tissues
o Best fixatives for tissues containing iron
pigments and for elastic fibers
o Requires no post treatment after fixation
goes directly into 80% alcohol processing
Disadvantages
o Longer to prepare, time consuming
o Positivity of mucin to PAS is reduced
o May produce gradual loss in basophilic
staining of cells
o Reactivity of myelin to Weigert’s iron
hematoxylin stain is reduced
o Inert towards lipids
Formol-Corrosive (Formol Sublimate)
 Formol mercuric chloride solution is
recommended for routine post mortem
tissue
 Fixation time – 3-24 hours
Advantages
o Penetrates small pieces of tissues rapidly
o Produces minimum shrinkage and
hardening
o Excellent for many staining procedures
including silver reticulum methods
o Brighten cytoplasmic and metachromatic
stains better than with formalin alone.
o Cytological structures and blood cells are
well preserved
o No need for washing out
o Fixes lipid
Disadvantages
o Penetration is slow, sections should not be
more than 1cm thick
o Forms mercuric chloride deposits
o Does not allow frozen section
o Inhibits the determination of the extent of
tissue decalcification.
Alcoholic Formalin (Gendre’s) Fixative
 Formula
◦ 95% ethyl alcohol saturated with
picric acid -80ml
◦ Strong formaldehyde solution 15ml
◦ Glacial acetic acid 5ml
 Postfixation with phenol-formalin for 6
hours or more can enhance
immunoperoxidase studies on the tissues,
and in some cases, EM, if it is necessary at a
later time to establish a diagnosis
Advantages
o Fixation is faster
o Used for rapid diagnosis
o Good for the preservation of glycogen and
for micro-incineration technique
o Used to fixed sputum
Disadvantages
o Produce gross hardening of tissues
o Causes partial lysis of RBC
o Preservation of iron containing pigments is
poor
o Formaldehyde does not give as good a
morphological picture as glutaraldehyde
o Causes little cross-linking
Glutaraldehyde
 Made up to two formaldehyde
residues, linked by three carbon
chains
 Buffered glutaraldehyde, followed
by secondary fixation in osmium
tetroxide is satisfactory for EM
 Fixation time varies from half hour
to two hours.
Advantages
o It has more stable effect on tissues, giving a
firmer texture with better tissue sections,
especially of CNS.
o Preserves plasma proteins better
o Produces less tissue shrinkage
o Preserves cellular structures better,
recommended for enzyme histochemistry
and EM
o More pleasant and less irritating to the nose
o Does not cause dermatitis
Disadvantages
o More expensive
 o Less stable
o Penetrates tissues more slowly
o Tends to make tissue brittle
o Reduces PAS positivity of reactive mucin
PRECAUTION:
o Specimen vial must be kept refrigerated
during the fixation process
o Solution may be changed several times
during fixation by swirling the vials to make
sure that the specimen is in contact with
fresh solution all the time.
METALLIC FIXATIVES
 MERCURIC CHLORIDE
◦ Zenker’s fluid
◦ Zenker-formol (helley’s solution)
◦ Heidenhain’s Susa solution
◦ B5 fixative
 CHROMATE FIXATIVES
◦ Chromic acid
◦ Potassium dichromate
◦ Regaud’s (Muller) Fluid
◦ Orth’s fluid
 LEAD FIXATIVES
MERCURIC CHLORIDE
 Mercuric chloride is the most common
metallic fixative, frequently used in
saturated aqueous solutions of 5-7%
 Widely used as secondary fixative reacting
with a number of amino acid residues and
accompanied by spectroscopic changes,
probably due to reaction with histidine
residues.
 Penetrates poorly
and produces
shrinkage of tissues
 Tissues fixed with
mixtures containing
mercuric chloride
(except susa) contain
black precipitates of
mercury.
Advantages
o Penetrates and hardens tissues rapidly and
well
o Nuclear components are shown in fine
detail
o Precipitates all protein
o Greater affinity to acid dyes
o Trichrome staining is excellent
o Routine fixative of choice for preservation
of cell detail in tissue photography
o Permits brilliant metachromatic staining of
cells
o Recommended for renal tissues, fibrin,
connective tissues and muscles
Disadvantages
o Causes marked shrinkage of cells
o Rapidly hardens the outer layer of the
tissue with incomplete fixation of the center
o Penetration beyond the first 2-3 mm is`
slow, should not used more than 5mm
o If left in fixative for more than 1-2 days, the
tissue becomes unduly hard and brittle
o Prevents adequate freezing of fatty tissues
and makes cutting of frozen tissues difficult
o Causes lysis of RBC and removes iron from
hemosiderin
o Inert to fats and lipids
o Leads to formation of black granular
deposits in the tissues.
o Reduces the amount of demonstrable
glycogen
o Compound solutions containing mercuric
chloride deteriorate rapidly upon addition
of glacial acetic acid to formalin
o Extremely corrosive to metals
PRECAUTIONS
o Black deposits may be removed by adding
saturated iodine solution in 96% alcohol,
the iodine being decolorized with absolute
alcohol in the subsequent dehydration .
o Compound solutions must always be freshly
prepared.
o The use of metallic forceps and of metal
caps to cover the bottles containing the
fixative should be avoided.
o Contact of mercuric fixatives with personal
jewelries should be avoided
 Mercuric chloride stock solution formula
◦ Mercuric chloride 5grams
◦ potassium dichromate 2.5grams
◦ sodium sulfate 1 gram
◦ Distilled water 100ml
 Just before use, glacial acetic acid may be
added to form Zenker’s solution or strong
formaldehyde solution may be added to
form Zenker-Formol solution (Kelly’s)
Zenker’s fluid
 Made up of mercuric chloride stock solution
to which glacial acetic acid has been added
just before its use to prevent turbidity and
formation of a dark precipitate.
 Recommended for fixing small pieces of
liver, spleen, connective tissue fibers and
nuclei.
 Fixation time – 12-24hours
 Formula
◦ Mercuric stock solution 95ml
◦ Glacial acetic acid 5ml
Advantages
o Produces a fairly rapid and even fixation of
tissues
o Stock solution keep well without
disintegration
o Recommended for trichrome staining
o Permits brilliant staining of nuclear and
connective tissue fibers
o Compatible with most stains
o May act as a mordant to make certain
special staining reactions possible
Disadvantages
o Penetration is poor
o Not stable after addition of Acetic acid
o Prolonged fixation will make tissues brittle
and hard
o It causes lysis of red blood cells and
removes iron from hemosiderin
o Does not permit cutting of frozen sections
o Tendency to form mercuric pigment
deposits or precipitates
o Tissue must be washed in running water for
several hours before processing.
Insufficient washing may inhibit or interfere
with good cellular staining

Precautions and practical considerations
o Do not left tissues stay in solution for more
than 24 hours
o Solutions must always be freshly prepared
o Tissues must be washed out thoroughly in
running water to permit good staining.
o Mercuric deposits may be removed by
immersing tissues in alcoholic iodine
solution prior to staining, through a process
known as de-zenkerization
o Tissues should be cut thin (2-3mm)and
hollow organs should be opened to
promote complete penetration and fixation.
Zenker-formol (helley’s solution)
 Fixation time 12-24 hours
Advantages
o Excellent microanatomic fixative for
pituitary gland, bone marrow and blood
containing organs such as spleen and liver
o Penetrates and fixes tissues well
o Nuclear fixation and staining is better than
zenker’s
o Preserves cytoplasmic granules well
Disadvantages
o Similar with Zenker’s except that brown
pigments are produced if tissues are
allowed to stay in the fixative for more than
24 hours due to RBC lysis.
o This may be removed by immersing the
tissue in saturated alcoholic picric acid or
sodium hydroxide
Heidenhain’s Susa solution
 Recommended for skin biopsies
 Excellent cytologic fixative
 Formula
◦ Mercuric chloride 45grams
◦ Sodium chloride 5grams
◦ Trichloroacetic acid 20grams
◦ Glacial acetic 40ml
◦ Formaldehyde, 40 % 200ml
◦ Distilled water 800ml
 Fixation time 3-12 hours
Advantages
o Penetrates and fixes tissues rapidly and
evenly
o Produces minimum shrinkage and
hardening of tissues due to counter-balance
of the swelling effects of acids and the
shrinkage effect of mercury.
o Permits most staining procedures to be
done, including silver impregnation,
producing brilliant results with sharp
nuclear and cytoplasmic details
o Permits easier sectioning of large blocks of
fibrous connective tissues
o Susa-fixed tissues may be transferred
directly to 95% alcohol or absolute alcohol,
thereby reducing processing time.
Disadvantages
o Prolonged fixation of thick material may
produce considerable shrinkage, hardening
and bleaching.
o RBC preservation is poor
o Some cytoplasmic granules are dissolved
o Mercuric chloride deposits tend to form on
tissues
o Weigert’s method of staining elastic fibers is
not possible in susa-fixed tissues
Precaution
o After using Heidenhain’s Susa fixative, the
tissue should be transferred directly to a
high grade alcohol, to avoid undue swelling
of tissues caused by treatment with low
grade alcohol or water.
B5 fixative
 Commonly used for Bone marrow biopsies
 Formula
◦ Distilled water 90ml
◦ Mercuric chloride 6grams
◦ Sodium acetate 1.25 grams
 Just prior to use, add 1cc of formaldehyde
(40%) for 10 cc of B5.
Advantages
o Rapid fixation can be achieved in ½ to 2
hours
o Good fixative for cytology of bone marrow
biopsies
Precautions and practical considerations
o Overfixation hardens the tissue and make
cutting difficult
o Two working solutions are kept separate ,
since the mixture is unstable. Mix just prior
to use.
o Mercury pigments can be removed by de-
zerkerization.
CHROMATE FIXATIVES
Chromic acid
 Used in 1-2 % aqueous solution, usually as a
constituent of a compound fixative
 Precipitates all proteins and adequately
preserves carbohydrates
 Strong oxidizing agent, hence a strong
reducing agent must be added to chrome
containing fixatives before use in order to
prevent counteracting effects and
consequent decomposition of solution upon
prolonged standing.
Potassium dichromate
 Used in 3% aqueous solution
 Fixes but not precipitate cytoplasmic
structures
 Preserves lipids
 Preserves mitochondria(if used in pH 4.5-
5.2)
Regaud’s (Muller) Fluid
 Formula
◦ Potassium dichromate 3%---------80ml
◦ Strong formaldehyde 40%---------20ml
 To be added just before use
◦ Fixation time -------12-48hours
Advantages
o Penetrate tissues well
o Harden tissues better and more rapidly than
orth’s fluid
o Recommended for demonstration of
chromatin, mitochondria, mitotic figures,
Golgi bodies, RBC and colloid containing
tissues.
Disadvantages
 o Deteriorates and darkens on standing due o Excellent fixative for preserving soft and
to acidity, hence the must always be freshly delicate structures
prepared. o Penetrates rapidly and evenly, causes little
o Penetration is slow shrinkage
o Chromate fixed tissues tend to produce  Yellow stain is useful when handling
precipitates of sub oxide, hence should be fragmentary biopsies
thoroughly washed in running water prior o Permits brilliant staining of tissues
to dehydration. o Preferred fixative for tissues to be stained
o Prolonged fixation blackens tissue by Masson’s trichrome for collagen, elastic
pigments or connective tissue
o Glycogen penetration is poor o Preserves glycogen
o Does not preserves fats o Does not need “washing out”
o Preserves hemosiderin less than buffered
formation Disadvantages
o Intensity of PAS reaction is reduced o Penetrates large tissues poorly, hence, its
use is limited to small fragments of tissue
Orth’s fluid o Picrates are soluble in water, hence, tissues
 Formula should not be washed in running water but
◦ Potassium dichromate 2.5% -----100ml rather, transferred directly from fixative to
◦ Sodium sulfate (optional)---------1gram 70% alcohol
◦ Strong formaldehyde 4----------10ml o Not suitable for fixing kidney structures,
to be added just before use lipid and mucus.
 Fixation time -36 to 72 hours o Destroys cytoplasmic structures,
mitochondria
Advantages o Produces RBC hemolysis and removes
o Recommended for the study of early demonstrable ferric iron from blood
degenerative processes and tissue necrosis pigments.
o Demonstrate rickettsiae and other bacteria o Reduces or abolishes Feulgen reaction due
o Preserves myelin better that buffered to hydrolysis of nucleoproteins.
formalin
Disadvantages Brasil’s alcoholic Picroformol Fixative
o same as regaud’s fluid  Formula
◦ Formaldehyde 37%---------------2040ml
LEAD FIXATIVES ◦ Picric acid ---------------------------80grm
 Used in 4% aqueous solution of basic lead ◦ Ethanol or isopropyl alcohol---6000ml
acetate ◦ Trichloroacetic acid --------------65grm
Advantages
o Recommended for acid Brasil’s solution
mucopolysaccharides Advantages
o Fixes connective tissue mucin o Better and less messy than bouin’s solution
Disadvantages o Excellent fixative for glycogen
o Takes up CO2 to form insoluble lead
carbonate especially on prolonged standing.  Overnight tissue fixation by automatic
This may be removed by filtration or by processing technique may utilize 3-4
adding acetic acid drop to lower the ph and changes of Brasil’s fixative at ½ to 2 hours
dissolve the residue. each, succeeded directly by absolute
alcohol.
PICRIC ACID FIXATIVES
 Bouin’s solution GLACIAL ACETIC ACID
 Brasil’s alcoholic Picroformol Fixative  Acetic acid is normally used in conjunction
with other fixatives to form a compound
Bouin’s solution solution. Solidifies at 17C, hence, the name
 Recommended for fixation of embryos and glacial acetic acid.
pituitary biopsies Advantages
 Formula o Fixes and precipitates nucleoproteins
◦ Saturated solution of picric acid o Precipitates chromosomes and chromatin
75 ml materials, hence, is very useful in the study
◦ Strong formaldehyde 40% of nuclear components of the cell.
25ml o Causes tissues to swell, to counteract the
◦ Glacial acetic acid shrinkage produced by other products
5ml Disadvantages
 Fixation time -------------6-24 hours o When combine with
Advantages Potassium Dichromate, the
o Produces minimal distortion lipid-fixing property of the
of micro-anatomial later is destroyed (zenker’s
structures and can be used fluid).
for general and special stains o Contraindicated for
cytoplasmic fixation since it
 destroys mitochondria and golgi elements
of the cells.
ALCOHOLIC FIXATIVES
◦ Methyl alcohol 100%
◦ Isopropyl Alcohol 95%
◦ Ethyl alcohol
◦ Carnoy’s fluid
◦ Newcomer’s Fluid
 Alcohol rapidly denatures and precipitates
proteins by destroying hydrogen and other
bonds.
 It must be used in concentrations ranging
from 70 to 100% because less concentrated
solutions will produce lysis of cells.
 Absolute alcohol can be used to fix and
preserve glycogen, pigments, blood, tissue
films and smears.
 The color of the specimen can be preserved
for photographic work using 80% alcohol.
Glycerin is also used in combination with
alcohol for this purpose.
Advantages
o Ideal for small tissue fragment
o Used both as fixative and dehydrating agent
o Excellent for glycogen preservation
o Preserves nuclear stain
Disadvantages
o Lower concentration (70-80%) will cause
RBC hemolysis and inadequately preserve
leukocytes.
o Dissolves fats and lipids, as a general rule.
ALCOHOL CONTAINING FIXATIVES ARE
CONTRAINDICATED WHEN LIPIDS ARE TO
BE STUDIED.
o Causes glycogen granules to move towards
the poles or ends of the cells (polarization)
o Tissue left in alcohol too long will shrink,
making it difficult or impossible to cut.
Methyl alcohol 100%
Advantages
o Excellent for fixing dry and wet smears,
blood smears and bone marrow tissues.
o Fixes and dehydrates at the same time.
Disadvantages
o Penetration is slow
o If left in fixative for more than 48hours,
tissues may be overhardened and difficult
to cut
Isopropyl Alcohol 95%
 Used for fixing touch preparation, although
some touch preparations are air dried and
not fixed, for certain special staining
procedures such as wright -Giemsa.
Ethyl alcohol
 Used at concentration of
70-100%.
 If lower concentrations
are used, the RBC’s
become hemolysed and
WBC’s are inadequately
preserved.
 Fixation time
--------------18-24 hours
Advantages
o Preserves but not fix glycogen
o Fixes blood, tissue films and smears
o Preserves nucleoproteins and nucleic acids,
hence used for histochemistry especially
for enzyme studies.
o Fixes tissue pigments fairly well.
Disadvantages
o Causes polarization of glycogen granules
o Produces considerable hardening and
shrinkage of tissues.
o Hemosiderin preservations is less than in
buffered formaldehyde
o Strong reducing agent, hence, should not be
mixed with chromic acid, potassium
dichromate and osmium tetroxide which
are strong oxidizing agents.
Carnoy’s fluid
 Recommended for fixing chromosomes,
lymph glands and urgent biopsies.
 Rapid in action and may be used for urgent
biopsy specimens for paraffin processing
within 5 hours.
 Tissues fixed with carnoy’s fixative for 1
hour can be transferred directly to absolute
alcohol or an alcohol-chloroform mixture
(1:1).
 It is also used to fix brain tissue for
diagnosis of rabies
 Formula
◦ Absolute alcohol------------------60ml
◦ Chloroform----------------------30ml
◦ Glacial acetic acid ----------------10ml
 Fixation time 1-3 hours
Advantages
o Most rapid fixative
o Fixes and dehydrates at the same time
o Permits good nuclear staining and
differentiation
o Preserves Nissl granules and cytoplasmic
granules well
o Preserves nucleoproteins and nucleic acids
o Excellent fixative for glycogen since
aqueous solutions are avoided.
o Very suitable for small tissue fragments
such as curettings and biopsy materials
o Following fixation, tissue may be
transferred directly to absolute alcohol,
thereby shortening processing time.
Disadvantages
o Produces RBC hemolysis
o Causes considerable tissue shrinkage
o Suitable only for small pieces of tissues due
to slow penetration.
o Tends to harden tissues excessively and
distorts tissue morphology.
o Dissolves fat, lipids, and myelin
o Leads to polarization unless very cold
temperature (-70C) are used
o Dissolves acid-soluble cell granules and
pigments
Newcomer’s Fluid
 Formula
◦ Isopropyl alcohol ------------------60ml
 ◦ Propionic acid---------------------30ml
◦ Petroleum ether -------------------10ml
◦ Acetone--------------------------10ml
◦ Dioxane--------------------------10ml
 Fixation time -----------------12-18hours
Advantages
o Recommended for fixing
mucopolysaccharides and nuclear proteins
o Produces better reaction in Feulgen stain
than Carnoy’s fluid
o Acts both as nuclear and histochemical
fixative.
OSMIUM TETROXIDE (OSMIC ACID)
 Pale yellow powder which dissolves in
water (up to 6% at 20C) to form a strong
oxidizing solution.
 Causes complete denaturation of protein
 Potassium permanganate and potassium
dichromate are also oxidizing agents but are
less reactive towards proteins than in
osmium tetroxide
Advantages
o Fixes conjugated fats and lipids
permanently by making them insoluble
during subsequent treatment with alcohol
and xylene
o Preserves cytoplasmic structures well
o Fixes myelin and peripheral nerves
o Produces brilliant nuclear staining with
safranin
o Adequately fixes material for ultrathin
sectioning in EM, since it rapidly fixes small
pieces of tissues and aids in their staining.
o Precipitates and gels proteins
o Shows uniformly granular nuclei with clear
cytoplasmic background
o Some tissues (adrenal gland) are better
fixed in vapor form of osmium tetroxide.
Disadvantages
o very expensive
o poor penetrating agent, suitable only for
small pieces of tissues (2-3mm thick)
o readily reduced by contact with organic
matter and exposure to sunlight , forming a
black precipitate which settles at the
bottom of the container.
o Prolonged exposure to acid vapor can
irritate the eye, producing conjunctivitis or
cause the deposition of black osmic oxide in
the cornea , producing blindness.
o Inhibits hematoxylin and makes
counterstaining difficult
o Extremely volatile
Precautions
o Eyes and skin may be protected by using a
fume hood or wearing protective plastic
masks or gloves.
o Should be kept in dark colored, chemically
clean bottle to prevent evaporation and
reduction by sunlight or organic matter.
o Should be placed in cool place or
refrigerated to prevent deterioration
o Addition of saturated aqueous mercuric
chloride solution will prevent its reduction
with formation of black deposits
o Black osmic oxide crystals may be dissolved
in cold water.
o To prevent contact with black precipitate
formed in the bottom of the jar, the tissues
may be wrapped in cotton gauze and
suspended in the fluid by means of a
thread.
o Osmic acid-fixed tissues must be washed in
running water for at least 24 hour to
prevent formation of artefacts.
Flemming’s Solution
 Most common chrome-osmium acetic acid
fixative used, recommended for nuclear
preparation of such sections.
 Formula
◦ Aqueous chromic acid 1%----15ml.
◦ Aqueous osmium tetroxide 2% --4ml.
◦ Glacial acetic acid ---------------1ml.
 Fixation Time ------24-48 hours
Advantages
o Excellent fixative for nuclear structures
o Permanently fixes fat
o Relatively less amount of solution is
required for fixation (less than 10x the
volume of the tissues to be fixed)
Disadvantages
o Poor penetrating agent
o Solution deteriorates rapidly and must be
prepared immediately before use
o Chromic-osmic aid combination depress the
staining power of hematoxylin
o Has tendency to form artifect pigments
o Very expensive
 
Flemming’s Solution without acetic acid
 Made up only of chromic and osmic acid,
recommended for cytoplasmic structures
particularly the mitochondria.
 The removal of acetic acid from the formula
serves to improve the cytoplasmic detail of
the cell.
 Fixation time - - 24-48hours
TRICHLOROACETIC ACID
 Sometimes incorporated into compound
fixatives
Advantages
o Precipitates proteins
o Its marked swelling effect on tissues serves
to counteract shrinkage produced by other
fixatives.
o May be used as a weak decalcifying agent
o Softening effect on dense fibrous tissues
facilitates preparation of such sections.
Disadvantages
o Poor penetrating agent
 
ACETONE
 Used at ice cold temperature ranging from
-5C to 4C
Advantages
o Recommended for the study of water
diffusible enzymes especially phosphatases
and lipases
o Used in fixing brain tissues for diagnosis of
rabies
 o Used as a solvent for certain metallic salts
to be used in freeze substitution techniques
for tissue blocks
Disadvantages
o Produce inevitable shrinkage and distortion
o Dissolves fat
o Preserves glycogen poorly.
o Evaporates rapidly.
HEAT FIXATION
 Procedure involves thermal coagulation of
tissue proteins for rapid diagnosis, usually
employed for frozen tissue sections and
preparation of bacteriologic smears.
Advantages
o Fixation is better
o Preserve nuclear and cytoplasmic detail
o Suitable for frozen tissue preparation
Disadvantages
o Produces considerable tissue shrinkage and
distortion
o Destroys RBC
o Dissolves starch and glycogen
SECONDARY FIXATION
 The process of placing an already fixed
tissue in a second fixative in order:
◦ To facilitate and improve the
demonstration of particular
substances
◦ To make special staining
techniques possible (with
secondary fixative acting
mordant)
◦ To ensure further and complete
hardening and preservation of
tissues
 This may be done before dehydration and
on deparaffinized sections before staining,
usually with 10% formol saline as primary
fixative. The tissue may be placed in a
primary fixative for storage or may require
further fixation for special staining.
POST-CHROMATIZATION
 Form of secondary fixation whereby a
primarily fixed tissue is placed in aqueous
solution of 2.5 -3% potassium dichromate
for 24 hours to act as mordant for better
staining effects and to aid in cytologic
preservation of tissues.
 
WASHING OUT
 The process of removing excess fixative
from the tissue after fixation in order to
improve staining and remove artefacts from
the tissues.
 Several solutions may be used:
◦ Tap water is used to remove:
 Excess chromates from
tissues fixed in kelly’s,
zenker’s and flemming’s
solutions
 Excess formalin
 Excess osmic acid
◦ 50-70% alcohol- used to wash out
excess amount of picric acid
(Bouin’s solution)
◦ Alcoholic iodine – used to remove
excessive mercuric fixatives
FACTORS THAT AFFECT FIXATION OF TISSUES:
 Retarded by:
◦ Size and thickness of the tissue
specimen-larger tissues
◦ Presence of mucus – prevents
complete penetration of fixative
◦ Presence of fat
◦ Presence of blood
◦ Cold temperature – inactivate
enzymes
 Enhanced by:
◦ Size and thickness- smaller tissues
◦ Agitation – fixation is accelerated
when automatic or mechanical
tissue processing is used
 Moderate heat (37-56C)
accelerates fixation but
hastens autolytic changes
and enzymes destruction.
PRINCIPLES AND PRECAUTIONS IN HANDLING AND
FIXATION OF SPECIMENS IN GENERAL
 Autopsy material should be fixed as soon
after death as possible to prevent
decomposition and autolysis due to
deprived oxygen metabolism
 Surgical specimens should be fixed as soon
as possible after removal or refrigerated if
fixation is to be delayed, to prevent drying
of surface layers and ultimate tissue
as
distortion.
 If placed in NSS during the operation,
autolysis may occur before fixation is
carried on.
 All tissue specimens must be properly
labeled and identified
 If tissues are refrigerated, slow freezing of
unfixed tissues near 0C must be avoided
since this may promote formation of ice
crystal artefacts.
 Tissue slices should be taken at right angles
to the surface of the organ and should be
sufficiently deep to show the normal
anatomic components.
 Tissues should not be more than 5mm thick
except in lung edema, with minimum
squeezing and handling.
 Thin sections allow complete penetration
by fixative in a short time.
 Purulent materials, exudates or exudates
should be marked and kept for possible
cultures, smears and other bacteriologic
examination.
 The amount of fixative must be adequate
(20times the volume of specimen) except
for osmium tetroxide which is very
expensive, requiring only 5-10 times that of
tissue volume for fixation.
 For prolonged fixation (museum
preparation) the volume of fixing fluid
should not be less than 50-100 times that of
the tissue.
 Contamination of fixed tissue with
precipitates should be avoided.
 In most instances, fixed tissues must be
washed thoroughly to remove salts and or
pigments before staining.
 Low temperature retards fixation but
prevents autolysis.
 The required period of fixation should not
be exceeded since this may cause excessive
hardening or brittleness of tissues.
 There must always be an adequate supply
of fixatives at all times.
 Drying should be avoided to prevent
shrinkage and distortion of tissue with loss
of cellular detail. Small tissue biopsies may
be placed in a petri dish with moistened
filter paper to prevent drying.
 Hollow organs should be packed with
cotton soaked in fixative or completely
opened before being immersed in adequate
fixing solution.
 Air filled lungs may float on fixative. To
avoid this, the organ may be covered with
several layers of gauze to maintain it under
surface.
 Human brains may be suspended by a cord
tied under the circle of willis to prevent
flattening, and may undergo intravascular
perfusion.
 Eyes must be dissected before they are
fixed since this may lead to immediate
tissue collapse and wrinkling to the escape
of vitreous humor.
 Frozen sections may lead to formation of
ice crystal artifacts.
 To avoid rigor contraction and staining of
artifacts when fixing muscle, the fresh
biopsy material may be stretch for by
sutures on each end, or laid flat in a moist
filter paper before suspending in fixative.
 Water should not be used for glycogen
containing tissues because glycogen is
soluble in water.
 To assure rapid access of the fixative to all
parts of the tissue, the tissue may be
minced, that is, small pieces of specimen
may be divided into small fragments and
transferred to the vial of a fixative by
means of a toothpick.
 Hard tissues may be washed out in running
water overnight and immersed in 4%
aqueous phenol solution for 1-3 days
(Lendrum’ method). This will soften the
tissue and allow easier sectioning without
producing any marked distortion of the cell
structures.
 Good cutting and staining of section is
greatly dependent upon proper fixation.
  Proper tissue processing should start with
proper fixation and preservation since a
badly preserved tissue will later on yield a
badly processed specimen and may prove
to be unsuitable for. The choice of fixatives
and mode of processing must therefore
vary depending upon the following factors:
◦ Need for immediate examination
◦ Tissue structure or component to
be studied
◦ Type of tissue to be processed
◦ Staining technique to be applied
◦ Type of section to be made,
whether serial or individual.
SOME OF THE DIFFICULTIES ENCOUNTERED
BECAUSE OF IMPROPER FIXATION ARE:
FIXATION ARTEFACTS
 Formalin pigment is a well known artifact
that may be produced under acid
conditions.
 The pigment may be eliminated or reduced
by fixation in phenol formalin.
 “Crush artifact” may be found in surgical
specimens particularly in liver biopsies,
associated with intense eosinophilic
staining at the center of the tissue. This
may be due to partial coagulation of
partially fixed protein by ethanol or by
incomplete wax impregnation during
subsequent histological processing.
MICROWAVE TECHNIQUE
 Works as a physical agent similar in
mechanism to vacuum, oven (heat) and
agitation to increase the movement of
molecules and accelerate fixation.
 Used to accelerate staining , decalcification,
immunohistochemistry, and electron
microscopy.
 Interacts with dipolar molecules, causing
oscillation at a frequency of 2450 mHz
 Water molecules and polar side chains of
proteins increase their thermal energy, with
subsequent heating of the proteins.
 Allows light microscopic techniques used in
routine histopathology to be performed
adequately.
Advantages
 Chief advantage of microwave fixation is
that the tissue is heated right through the
block in a very short time, thereby
potentially allowing the study of cellular
processes that proceed very rapidly
 Non-chemical technique that is useful in
preserving neurochemical substances in
brain, such acetylcholine.
 Used for rapid fixation of routine surgical
specimens.
  Significantly reduces the time taken for
immunohistochemistry and in-situ
hybridization
Disadvantages
 Microwaves generated by commercial
ovens only penetrate tissue to a thickness
of 10-15mm.
 No significant cross-linking of protein
molecules, and subsequent chemical
fixation may be needed.
 Viable spores and other pathogens may
remain in tissues processes with alcohol-
based fixatives or microwaving alone.
 
 Fixation: procedure
◦ Fix tissue------------------------4hours
◦ Soak blocks in water at room temp
--------1 minute
◦ Place blocks in 100ml of formalin---------
Place in microwave , 450watts,55C------
1.5 to 4mins
◦ Remove blocks and slice tissue to 2mm
thick
◦ Place directly in alcohol
 Dehydration can be achieved in one step
(instead of 6) in microwave, using ethyl or
methyl alcohol.
 Clearing agents – ethyl alcohol (dehydrant),
isopropanol (, best), histoclear,xylene,
chloroform, methyl alcohol
◦ Isopropanol absorbs microwave
better than other reagents , less
toxic than xylene and can
effectively boil out at paraffin
stage due to boiling point.
◦ Cheaper than ethanol and help
dehydrate further.
◦ It need only one change in paraffin
bath, or 2 changes if dense tissue is
being processed.

IMMUNOFLOURESCENCE AND
IMMUNOPEROXIDASE TECHNIQUES
 Immunoflourescence technique are
commonly used in pathology for the
demonstration of antibodies.

Enzyme histochemistry
 The general aim of fixation is to preserve
the maximum enzyme activity at its original
localization, while also preserving structural
integrity.
 The tissue for study may be fixed in 4 %
formaldehyde or formol saline overnight, or
fresh frozen cryostat sections may be fixed
in acetone or formaldehyde and washed in
distilled water prior to enzyme testing.

Electron microscopy
 The most useful fixative for EM are osmium
tetroxide, glutaraldehyde and
paraformaldehyde, with the whole
procedure performed at 4C.
 For routine studies, glutaraldehyde or
osmium are adequate.
 For electron histochemistry and electron
immunocytochemistry karnovsky
paraformaldehyde-glutaraldehyde is useful.