MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION

1st SEMESTER | A.Y. 2022-2023

BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

INTRODUCTION - Fixation should be carried out as soon as possible

after removal of the tissues (in the case of surgical
 Fresh tissues are usually examined when there is pathology) or soon after death (in the case of
an immediate need for evaluation. A better and autopsy) to prevent autolysis.
more effective means, however, of studying tissue, - Fixation terminates any ongoing biochemical
whether normal or abnormal is by examination of reactions, and may also increase the mechanical
strength or stability of the treated tissues.
their sections and smears which have been
- The fixatives employed prevent autolysis by
permanently preserved, stained for demonstration inactivating lysosomal enzymes.
of specific structures, and mounted on glass slides - They stabilize the fine structure, both inside and
with coverslips for permanent keeping. This can be between cells, by making macromolecules
accomplished by preserving and carefully resistant to dissolution by water and other liquids.
processing solid structures and tissues in the - Fixatives also inhibit the growth of bacteria and
molds that give rise to putrefactive changes.
following order:
- The primary goal of fixation is to preserve the
I. Fixation
morphologic and chemical integrity of the cell in as
II. Decalcification (optional) life- like a manner as possible.
III. Dehydration  With fixation, the shape, structure,
IV. Clearing intercellular relationship and chemical
V. Impregnation (Infiltration) constituents of tissues are preserved.
VI. Embedding  Fixation prevents degeneration,
decomposition, putrefaction, and distortion
VII. Trimming
of tissues after removal from the body.
VIII. Section-Cutting (Microtomy)  All vital cellular processes stop when the
IX. Staining tissue is placed in a fixative.
X. Mounting  The weak chemical associations are changed
XI. Labeling into stable complexes that will not break
 Once the tissue has been fixed, it must be down with further histologic handling.
processed into a form in which it can be made into - The second goal of fixation is to harden and
protect the tissue from the trauma of further
thin microscopic sections. Wet fixed tissues (in
handling, so that it is easier to cut and process for
aqueous solutions) cannot be directly infiltrated microscopy.
with paraffin. First, the water from the tissues  Fixation allows the tissue to be more
must be removed by dehydration. This is usually properly oriented in the cassette in
done with increasing concentrations of alcohol preparation for paraffin embedding and
(70% to 95% to 100%). Because water and paraffin microtomy.
NOTE: It is important to know that a fixative will initially
are not miscible, the specimens must be gradually
produce a number of changes to the tissues in
dehydrated to achieve complete replacement of
what is usually an aqueous environment. This will
water with alcohol. Once successfully dehydrated,
include shrinkage, swelling, and hardening of
the next step is called "clearing" and consists of
various components. The tissues will undergo
removing the dehydrating agent (alcohol) with a
further changes during processing when they are
substance that will be miscible with the
placed in a nonaqueous environment. For
embedding medium (paraffin). The commonest
example, fixation in 10% buffered formalin will
clearing agent is xylene. Finally, the tissues are
initially cause slight swelling of tissue specimens.
infiltrated with an embedding agent (almost
During processing, however, the specimen may
always paraffin).
shrink and lose 20% -30% of its volume. The
FIXATION particular fixative employed will also influence the
degree to which individual elements will stain with
- The first and most critical step in histotechnology various histochemical and immuno-histochemical
is FIXATION, a process that preserves tissues from reagents.
decay, thereby preventing autolysis or
putrefaction.  OBJECTIVES OF FIXATION
- If fixation is not adequate, the otherprocesses that
1. To Preserve the Tissue
follow, such as dehydration, clearing, infiltration,
embedding, microtomy and staining, will also be 2. To Prevent Breakdown of Cellular Elements
inadequate.

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

fixation and can result in staining artefacts. One

3. To Coagulate or Precipitate Protoplasmic
way to partially solve the problem is to change the
Substances solution at intervals to avoid exhaustion of the
 METHODS OF FIXATION fixative. Agitation will also enhance fixation of the
- Fixation of tissues can be achieved by specimen. The most common error in
physical or chemical means. Physical methods histotechnology is insufficient ratio of tissue
include heating, microwaving and cryo- volume to fixative volume.
preservation (freeze drying). Chemical fixation is
4. Thickness of section: Tissue blocks should be small
usually achieved by immersing the specimen in (e.g. 1 to 2 mm2 for electron microscopy and 2
the fixative solution (immersion fixation) or, in cm2 wide for light microscopy) and thin (no more
the case of small animals or some whole organs than 0.4 cm for light microscopy).
such as a lung, by perfusing or injecting the
5. Osmolality: The best results are usually obtained
vascular system with fixative (perfusion fixation).
using slightly hypertonic solutions (400-450 mOsm;
For some specialized histochemical procedures, isotonic solutions are 340 mOsm).
fixatives have occasionally been applied in the
vapor form. For example, paraformaldehyde and 6. Concentration: Concentration of fixative should be
osmium tetroxide can be used to vapor-fix adjusted down to the lowest level possible.
freeze-dried tissues. Formaldehyde is normally used as a 10% solution,
and glutaraldehyde is normally used as a 3%
 There are two basic mechanisms involved in solution. Low concentrations of glutaraldehyde
(0.25%) have been found to be an ideal
fixation:
concentration for immuno- electron microscopy.
1. Additive fixation whereby the chemical 7. Duration of fixation: Some tissues take longer to
constituent of the fixative is taken in and fix than others, depending on their structure.
becomes part of the tissue by forming cross- Fixation time can be cut down by using heat,
links or molecular complexes and giving vacuum, agitation or microwave. Prolonged
stability to the protein. (Examples are fixation may cause shrinkage and hardening of
tissue, and may severely inhibit enzyme activity
formalin, mercury, and osmium tetroxide).
and immunological reactions, although washing of
2. Non-additive fixation whereby the fixing the tissue in running water considerably restores
agent is not incorporated into the tissue, but the activity of some enzymes.
alters the tissue composition and stabilizes
the tissue by removing the bound water 8. Speed: Specimens should be transferred to fixative
attached to H-bonds of certain groups within quickly (<1 hour) after surgery as deterioration will
the protein molecule. Upon removal of commence with the loss of blood supply.
water, new cross-links are established within
and among the protein molecules that  PRACTICAL CONSIDERATIONS TO OPTIMIZE FIXATION
stabilize the intercellular components, OF TISSUE
prevent autolysis and make them unsuitable
for bacterial decomposition. (Examples are  Penetration: Formalin diffuses into the tissue at
alcoholic fixatives) the rate of approximately 1mm per hour, and
slows down as it goes deeper into the tissue.
 MAIN FACTORS INVOLVED IN FIXATION  Anatomical barriers to fixation must be
1. Hydrogen Ion Concentration: Satisfactory fixation removed or incised (e.g. fascia, bone, feces,
occurs between pH 6 and 8. thick tissue) and large specimens must be
sectioned or inflated with fixative (e.g. lung)
2. Temperature: Fixation of surgical specimens is or opened and cleaned (gastrointestinal tract)
traditionally carried out at room temperature. to allow penetration.

3. Volume: The volume of fixative is important.  Fixatives diluted and/or contaminated by bodily
Traditionally, the amount of fixative used has been fluids (e.g. bile, blood, feces) will be reduced in
10-20 times the volume of tissue to be fixed. As concentration and must be replaced to ensure
the fixative molecules bind to the tissue, they are effectiveness.
eventually depleted. This often causes poor  Pinning specimens to a corkboard or inserting a
paper or gauze “wick” into tubular structures can
improve fixation and reduce tissue distortion.

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

 The tissue hardens upon fixation and remains in  TYPES OF FIXATIVES

whatever shape it was fixed in. This should be
considered in those cases where the final - The choice of the fixative is based on tissue and
orientation of the sections is important. anticipated ancillary tests.
 EFFECTS OF FIXATIVES IN GENERAL  According to COMPOSITION
a. They reduce the risk of infection during handling A. Simple Fixatives - are made up of only one
and actual processing of tissues.
component substance.
b. They harden soft and friable tissues and make the
handling and cutting of sections easier. This is
1. Aldehydes
usually accelerated by the action of alcohol during
the dehydration process. 2. Formaldehyde
c. They make the cells resistant to damage and 3. Glutaraldehyde
distortion caused by the hypotonic and hypertonic 4. Metallic Fixatives
solutions used during tissue processing. 5. Mercuric chloride
d. They inhibit bacterial decomposition. 6. Chromate fixatives
e. They increase the optical differentiation of cells 7. Picric acid
and tissue components thereby rendering them
8. Acetic acid
more readily visible during examination.
f. They may act as mordants or accentuators to 9. Acetone
promote and hasten staining, or they may inhibit 10.Alcohol
certain dyes in favor of another (e.g. formaldehyde 11.Osmium Tetroxide
intensifies while osmium tetroxide inhibits
hematoxylin staining). B. Compound Fixatives - are those that are made up
of two or more fixatives which have been added
 CHARACTERISTICS OF A GOOD FIXATIVE
together to obtain the optimal combined effect of
- It must be cheap.
their individual actions upon the cells and tissue
- It must be stable.
constituents.
- It must be safe to handle.
- It must kill the cell quickly thereby producing  According to ACTION:
minimum distortion of cell constituents.
- It must inhibit bacterial decomposition and A. Microanatomical Fixatives -are those that permit
autolysis. the general microscopic study of tissue structures
- It must produce minimum shrinkage of tissue. without altering the structural pattern and normal
- It must permit rapid and even penetration of intercellular relationship of the tissues in question.
tissues.
1. 10% formal saline
- It must harden tissues thereby making the cutting
2. 10% neutral buffered formalin
of sections easier.
3. Heidenhain 's Susa
- It must be isotonic, causing minimal physical and
4. Formal sublimate (formal corrosive)
chemical alteration of the cells and their
5. Zenker 's solution
constituents. This is not, however, a strict rule
6. Zenker-formal (Kelly 's solution)
since there are some hypotonic solutions (i.e.
7. Bouin's solution
glacial acetic acid) producing tissue swelling, which
8. Brasil's solution
are being used in conjunction with hypertonic
solutions (e.g. picric acid) causing shrinkage of B. Cytological Fixatives - are those that preserve
cells, to produce a compound which would give an specific parts and particular microscopic elements
optimal effect on the tissue structure. of the cell itself.
- It must make cellular components insoluble to
hypotonic solutions and render them insensitive to 1. Nuclear Fixatives - are those that preserve
subsequent processing. the nuclear structures (e.g., chromosomes)
- It must permit the subsequent application of many in particular. They usually contain glacial
staining procedures to facilitate easier and more acetic acid as their primary component due
profitable examination. to its affinity for nuclear chromatin. They
have a pH of 4.6 or less.

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

a. Flemming's fluid 1. Formaldehyde and Formalin

b. Carnoy's fluid - The terms “formalin” and “formaldehyde” are
c. Bouin's fluid often used interchangeably, although the
d. Newcomer's fluid chemical composition of each fixative is
e. Heidenhain's Susa different. Formaldehyde is a gas produced by
2. Cytoplasmic Fixatives - are those that preserve the oxidation of methyl alcohol, and is soluble
cytoplasmic structures in particular. They must never in water to the extent of 37-40% weight in
contain glacial acetic acid which destroys volume.
mitochondria and Golgi bodies of the cytoplasm.
They have a pH of more than 4.6. - The commercially available solution of
formaldehyde contains 35-40% gas by weight
a. Flemming's fluid without acetic acid (equivalent to 40% stock or concentrated
b. Kelly's fluid formalin). Pure stock solution of 40% formalin
c. Formalin with "post-chroming" is unsatisfactory for routine fixation since high
d. Regaud 's fluid (Muller 's fluid) concentrations of formaldehyde tend to over-
e. Orth 's fluid harden the outer layer of the tissue and affect
staining adversely.
o For RNA, the precipitant fixatives - ethanol and
acetone - give the best quantitative results using - Formalin is made with formaldehyde but the
frozen tissues as the standard. percentage denotes a different formaldehyde
concentration. For example, 10% neutral-
3. Histochemical Fixatives - are those that preserve the buffered formalin (NBF or simply formalin) is
chemical constituents of cells and tissues. really a 4% formaldehyde solution. This is
because formalin is prepared from commercial-
a. Formal Saline 10%
grade 37 to 40% formaldehyde, by diluting the
b. Absolute Ethyl Alcohol
concentrated formalin 1:10 in phosphate
c. Acetone
buffer. It is recommended that laboratories
d. Newcomer's Fluid
avoid the use of concentrated formalin
solutions by purchasing commercially prepared
There are two major groups of chemical fixatives,
10% formalin.
classified according to their mechanism of action:
2. Buffered Formalin
a. Crosslinking Fixatives (e.g., Aldehydes) that act by
- The most widely used fixative for routine
creating covalent chemical bonds between
histology is 10% neutral buffered formalin
proteins in tissue. This anchors soluble proteins to
(NBF, approximately 4% formaldehyde),
the cytoskeleton, and lends additional rigidity to
buffered to pH 7 with phosphate buffer. This
the tissue.
fixative can effectively prevent autolysis and
b. Precipitating (or denaturing) fixatives (e.g.,
provide excellent preservation of tissue and
alcoholic fixatives) that act by reducing the
cellular morphology. It is considered the
solubility of protein molecules and (often) by
fixative of choice for many other procedures
disrupting the hydrophobic interactions that give
that require paraffin embedding, including
many proteins their tertiary structure. The
immunohistochemistry and interphase
precipitation and aggregation of proteins is a very
Fluorescent In-Situ Hybridization (FISH).
different process from the crosslinking that occurs
with the aldehyde fixatives.  Advantages :
1. It is cheap, readily available, easy to prepare,
 Crosslinking fixatives – Aldehydes
and relatively stable, especially if stored in
- Aldehyde fixatives are satisfactorily for routine
buffered solution.
parasffin sections, for electron microscopy, and
2. It is compatible with many stains, and
when histochemical and enzyme studies are
therefore can be used with various staining
indicated.
techniques depending upon the need of the
tissues.

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

3. It does not over-harden tissues, even with 6. Prolonged fixation may produce:
prolonged periods of fixation, as long as a. Bleaching of the specimen and loss of
solutions are regularly changed. natural tissue colors.
4. It penetrates tissues well. b. Dispersal of fat from the tissue into the
5. It preserves fat and mucin, making them fluid.
resistant to subsequent treatment with fat c. Dissolution or loss of glycogen, and urate
solvents, and allowing them to be stained for crystals
demonstration.
6. It preserves glycogen.  FACTORS THAT INFLUENCE FORMALIN FIXATION
7. It preserves but does not precipitate
proteins, thereby allowing tissue enzymes to a. Post-Mortem or Post-Surgical Interval .
be studied. It does not make tissues brittle, - Rapid fixation by formalin is essential because
and is therefore recommended for nervous autolysis begins immediately following disruption
tissue preservation. of blood and can influence the cellular morphology
8. It allows natural tissue colors to be restored and staining. The time interval between death and
after fixation by immersing formalin-fixed fixation should be minimized and controlled
tissues in 70% alcohol for one hour, and is whenever possible. If there is a necessary delay in
therefore recommended for colored tissue fixation, the tissue should be immersed in cold
photography. phosphate-buffered saline (PBS). Tissues should
9. It allows frozen tissue sections to be not be allowed to dry before (or after) fixation. For
prepared easily. certain studies, vascular perfusion with fixative
10. It does not require washing out, unless may be recommended.
tissues have stayed in formalin for
b. Composition of Fixative.
excessively long periods of time.
- Formaldehyde fixation is usually optimal near
 Disadvantages: physiological pH and ionic strength. Unfortunately,
formalin is not stable and will gradually acidify to
1. Fumes are irritating to the nose and eyes and form more complex polymers which can have
may cause sinusitis, allergic rhinitis, or adverse effects on the tissue. This can be
excessive lacrimation. minimized by appropriate buffering and addition
2. The solution is irritating to the skin and may of small amounts of methanol. Commercial
cause allergic dermatitis on prolonged preparations in proper specimen containers are
contact. strongly recommended, but should not be used
3. It may produce considerable shrinkage of beyond the expiration date (usually 2 years),
tissues. because of its short life span.
4. It is a soft fixative and does not harden some
cytoplasmic structures adequately enough c. Volume of Fixative.
for paraffin embedding. - Tissues should be immersed in a 10-20 fold volume
5. If unbuffered: of fixative. Large amounts of blood or protein-rich
fluid can decrease the effective fixative
a. Formalin reduces both basophilic and
concentration. If necessary, this problem can be
eosinophilic staining of cells, thereby
minimized by briefly washing the tissue with PBS
reducing the quality of routine cytologic
prior to fixation. If the fixative becomes cloudy or
staining. Acidity of formic acid may,
bloody after tissue is immersed, the fixative should
however, be used to an advantage when
be changed.
applying the silver impregnation
technique of staining. d. Fixation Time.
b. It forms abundant brown pigment - Fixation with 10% NBF at room temperature for a
granules on blood-containing tissues, e.g., minimum of 24 hours at room temperature is
spleen, due to blackening of hemoglobin. recommended in most instances. However, some
bloody, fatty or fetal tissues may require
significantly longer fixation, e.g., up to 48- 72

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

hours. If the fixed tissue shows internal areas with of incompletely fixed tissue to alcohol precludes
normal coloration upon staining, the tissue should be normal tissue processing.
left in fixation for a longer time period. Small surgical
biopsies are sometimes fixed for only 6 hours, but 12  MIXTURE OF FIXATIVES
or more hours will often provide a better result. - Two aldehyde fixative mixtures have been
Inadequate- fixation can cause artefacts following particularly useful for electron cytochemistry. The
exposure to alcohol during the subsequent tissue best known mixture of fixative is Karnovsky's
processing, or it can causedamage during paraformaldehyde-glutaraldehyde solution.
decalcification. Too much-fixation can impair staining, Acrolein is another aldehyde which has been
and complicate sectioning. introduced as a mixture with glutaraldehyde or
formaldehyde. It penetrates tissues rapidly,
e. Temperature. preserves morphology and enzyme activity at low
- An increase in temperature can increase the rate concentrations, and may be used for immersion
of fixation but can also increase the rate of fixation of surgical biopsies.
autolysis. Primary fixation in the cold can slow
autolysis, but it also slows the process of fixation,  Precautions:
and premature cooling of a specimen in fixative
1. Prolonged storage of formaldehyde, especially at
can lead to inadequate fixation.
very low temperature, may induce precipitation of
f. Tissue Thickness. white para-formaldehyde deposits and produce
- Formalin fixation is dependent on diffusion of the turbidity although this, in itself, does not impair
fixative into the tissue. The thicker the tissue, the the fixing property of the solution. Precipitates
more time is required to obtain complete may be removed by filtration or by addition of I0%
penetration and crosslinking of tissue proteins. If methanol.
the specimen is too thick, the center of the tissue 2. Methanol added as a preservative to
can suffer from autolysis or incomplete fixation. formaldehyde will prevent its decomposition to
For this reason, tissues should be no thicker than formic acid or precipitation to paraformaldehyde,
3-5 mm. Careful slicing of tissues and solid organs but it serves to denature protein, thereby
before transferring them to fixative can greatly rendering formalin unsuitable as a fixative for
influence the efficiency of fixation by increasing electron microscopy.
exposed surface area and decreasing total 3. Concentrated solutions of formaldehyde must
thickness. This is particularly important for fatty, NEVER be neutralized since this might cause
bloody organs and encapsulated tissues. When violent explosions.
slicing tissues, it is important to use a sharp, clean 4. Room should be properly ventilated with adequate
blade to minimize compression artifacts. For very windows and preferably with an exhaust fan to
soft tissues, it is sometimes helpful to stiffen the prevent inhalation of fumes and consequent injury
tissue by brief fixation prior to cutting of thinner to eyes and nose.
sections and additional fixation. Tubular organs 5. Dermatitis may be avoided by the use of rubber
can be cross-sectioned at small intervals to gloves when handling specimens fixed in formalin.
enhance exposure of the lumen to the fixative. 6. The bleaching of tissues may be prevented by
Any luminal contents (like feces and mucus) should changing the fluid fixative every three months.
also be removed. 7. Natural tissue colors may be restored by
immersing tissues in 70% alcohol after fixation.
g. Post-Fixation Storage. 8. Brown or black crystalline precipitate formed by
- Non-coagulant fixatives such as formalin continue the action of formic acid with blood can be
to crosslink proteins as long as they are in contact removed from the sections prior to staining by
with the tissue. If there is to be a delay in treatment with saturated alcoholic picric acid or a
processing after complete fixation (usually 24 1% solution of potassium hydroxide in 80%
hours or more), the fixed tissue can be stored for alcohol. The use of neutral (phosphate) buffered
up to 3 days in the cold in 70% ethanol. However, formalin will prevent the pigmentation.
it is essential that the tissue is completely fixed
prior to transfer to the alcohol. Premature transfer

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

Recommended Application:
9. If fatty tissues are to be stored for a long time, This is a simple microanatomical fixative made up of
cadmium or cobalt salts can be added to prevent saturated formaldehyde (40%, by weight volume)
dispersion of fat out into the fluid. diluted to 10% with sodium chloride. This mixture of
10. After use, formalin should be collected, sealed in formaldehyde in isotonic saline was widely used for
appropriately labelled containers and disposed of routine histopathology prior to the introduction of
by a commercial waste service. phosphate buffered formalin. It is recommended for
11. All empty containers should be washed thoroughly fixation of central nervous tissues and general post-
with water. mortem tissues for histochemical examination. It is
12. Formalin should not be released into soil, drains also recommended for the preservation of lipids,
and waterways. especially phospholipids. The buffer tends to prevent
13. Acid reaction due to formic acid formation can be the formation of formalin pigment. Many epitopes
buffered or neutralized by adding magnesium require antigen retrieval for successful
carbonate or calcium in a wide-mouth bottle to immunohistochemical (IHC) staining procedure
prevent violent explosion due to insufficient gas following its use. Most pathologists feel comfortable
space for CO2 release. interpreting the morphology produced with this type of
14. Calcium acetate may be used to buffer formalin fixative. It often produces formalin pigment.
but it leaves a calcium deposit. The greatest
amount of calcium deposit appears wherever the Advantages:
tissue is most exposed to the fixative (i.e., 1. It penetrates and fixes tissues evenly.
periphery of the block, within and around the 2. It preserves microanatomic and cytologic details
walls of blood vessels, or in the proximity of with minimum shrinkage and distortion.
hollow structures). 3. Large specimens may be fixed for a long time
15. To improve staining and produce firmer and provided that the solution is changed every three
harder consistency, tissues fixed in formalin for 1 - months.
2 hours may be placed again in Helly's fluid for 4-6 4. It preserves enzymes and nucleoproteins.
hours or in formol-sublimate for 4-16 hours 5. It demonstrates fats and mucin.
secondary fixation). 6. It does not over-harden tissues, thereby facilitating
16. Formic acid develops over time producing an acidic dissection of the specimen.
solution, requiring a buffer to remain neutral pH 7. It is ideal for most staining techniques, including
(7.0). silver impregnation.
17. Acidic formalin causes hematin pigment deposition 8. It allows natural tissue color to be restored upon
in tissues particularly in hematogenous tissue after immersion in 70% alcohol.
storage for extended periods of time. Methods are
available to remove hematin but it is better to Disadvantages are similar to formaldehyde with the
prevent its deposition by maintaining a neutral pH. following additions:
18. If post-fixed in osmic acid, the tissue must not be 1. It is a slow fixative. The period of fixation is
washed in demineralized water to prevent required to be 24 hours or longer.
hypotonicity and bleaching. 2. Formal-saline fixed tissues tend to shrink during
19. Fixation of tissue blocks not exceeding 5 mm. in alcohol dehydration; this may be reduced by
thickness is usually complete in 6-12 hours at secondary fixation.
room temperature. 3. Metachromatic reaction of amyloid is reduced.
4. Acid dye stains less brightly than when fixed with
1.1 10% Formal-Saline
mercuric chloride.
FORMULA:
40% formaldehyde: 100 ml
1.2 10% Neutral-Buffered Formalin
Distilled water: 900 ml
FORMULA:
Sodium dihydrogen phosphate monohydrate:4 gm
Mix together:
Disodium hydrogen phosphate anhydrous 6.5 gm
Sodium Dihydrogen Phosphate (Na2HPO4), anhydrous,
The solution should have a pH of 6.8
6.5 gm
Fixation time: 12 – 24 hours

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
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BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

Sodium Dihydrogen Phosphate (NaH2PO4•H20) 4 gm 4. It brightens cytoplasmic and metachromatic

Distilled water 900 ml stains better than with formalin alone.
Adjust pH to 7.4, then add: 40% formaldehyde 100 ml 5. Cytological structures and blood cells are well
preserved. There is no need for "washingout".
Advantages are similar to formal-saline with the following Tissues can be transferred directly from
additions: fixative to alcohol.
1. It prevents precipitation of acid formalin pigments 6. It fixes lipids, especially neutral fats and
on post mortem tissue. phospholipids.
2. It is the best fixative for tissues containing iron Disadvantages:
pigments and for elastic fibers which do not stain 1. Penetration is slow; hence, tissue sections
well after Susa, Zenker’s or chromate fixation. should not be more than 1 cm thick.
3. It requires no post-treatment after fixation and
2. It forms mercuric chloride deposits.
goes directly into 80% alcohol for processing.
3. It does not allow frozen tissue sections to be
Disadvantages:
made.
1. It is longer to prepare; hence, is time-consuming.
2. Positivity of mucin to PAS is reduced.
4. It inhibits the determination of the extent of
tissue decalcification.
3. It may produce gradual loss in basophilic staining
of cells.
1.5 Paraformaldehyde
4. Reactivity of myelin to Weigert's iron hematoxylin
Paraformaldehyde is a polymerized form of
stain is reduced.
formaldehyde, usually obtained as a fine white
5. It is inert towards lipids, especially neutral fats and
powder, which depolymerizes back to formalin when
phospholipids
heated. It is suitable for paraffin embedding and
sectioning, and also for immunocytochemical analysis.
1.3 Zinc formalin (unbuffered)
Paraformaldehyde fixed samples can also be stained
FORMULA:
for general histology but the degree of fixation is less
Zinc sulphate 1 gm
vigorous than Bouin’s so the quality of the morphology
Deionized water 900 ml
obtained will be less. This fixative allows for
Stir until dissolved, then add – 40% formaldehyde 100
subsequent immuno-detection of certain antigens and
ml
should therefore be used when the objective is to
Recommended Applications:
study morphology and protein expression
Zinc formalin solutions were devised as alternatives to
simultaneously. Its effects are reversible by excess
mercuric chloride formulations. They are said to give
water and it avoids formalin pigmentation. Other
improved results with immunohistochemistry. There
benefits include: Long term storage and good tissue
are a number of alternative formulas available some of
penetration.
which contain zinc chloride which is thought to be
slightly more corrosive than zinc sulphate
1.6 Karnovsky’s Fixative (4% Paraformaldehyde-1%
Glutaraldehyde in 0.1M Phosphate Buffer)
1.4 Formol-Corrosive (Formol-Sublimate)
Karnovsky’s fixative is a mixture of paraformaldehyde
FORMULA:
and glutaral-dehyde. It is suitable for use when
Sat. Aq. Mercuric chloride 90 ml.
preparing samples for light microscopy in resin
Formaldehyde 40% 10 ml.
embedding and sectioning, and for electron
Fixation time: 3-24 hours
microscopy. This fixative should always be prepared
Formol-mercuric chloride solution is recommended for
fresh.
routine post-mortem tissues.
To prepare 8 % Paraformaldehyde (PFA):
Advantages:
1. It penetrates small pieces of tissues rapidly. 1. Working in a fume cupboard wearing appropriate
PPE, heat up 90 ml of water to 65 °C on a hotplate
2. It produces minimum shrinkage and
stirrer.
hardening.
2. Add 8 g of paraformaldehyde (PFA) powder to the
3. It is excellent for many staining procedures
warm water. Use a magnetic stirrer and hotplate
including silver reticulum methods.
to dissolve PFA (approximately 15-20 minutes).

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
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BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

3. Slowly add drops of 1 M sodium hydroxide treated with inert amine-containing molecules prior to the
solution until the solution is clear. Make up to 100 immunoassay. Free, unsaturated aldehyde groups are
ml with additional water. available to covalently link amine-containing moieties such
4. Filter the fixative and adjust pH to 7.3-7.4. as antibodies. The most efficient aldehyde blockers are
5. Allow fixative to cool to room temperature. Store ethanolamine and lysine.
at −20 °C in aliquots. Frozen aliquots are stable for
up to 6 months. A standard fixative (2% buffered glutaraldehyde)
6. Dispense 25 ml for use in fixative. followed by secondary fixation in osmium tetroxide, is
satisfactory for electron microscopy. The glutaraldehyde
must be cold and buffered and not more than 3 months
To prepare 0.2 M phosphate buffer:
old. The tissue must be as fresh as possible and preferably
1. Solution X: Weigh out 35.61 g of disodium
sectioned and fixed in glutaraldehyde at a thickness of no
monohydrogen phosphate (Na2HPO4.2H2O) ±
more than 1 mm to enhance fixation. It penetrates very
make up to 1000 ml with distilled H2O, stir until
poorly, but gives best overall cytoplasmic and nuclear
dissolved. 1 year shelf life at room temperature.
detail. A 2.5% solution is used for small tissue fragments
2. Solution Y: Weigh out 27.6 g of sodium dihydrogen
and needle biopsies fixed in 2-4 hours at room
phosphate (NaH2PO4.H2O) ± make up to 1000 ml
temperature. A 4% solution is recommended for larger
with distilled H2O, stir until dissolved. 1 year shelf
tissues less than 4 mm thick, fixed in 6-8 hours up to 24
life at room temperature.
hours. Some fixation protocols call for a combination of
3. Add 40.5 ml of Solution X to 9.5 ml of Solution Y to formaldehyde and glutaraldehyde so that their respective
give 50 ml 0.2 M phosphate buffer. strengths complement one another.
4. Adjust to pH 7.4. This is to be used fresh
Advantages of Glutaraldehyde over Formalin:
To prepare Karnovsky’s Fixative (for 100 ml add the 1. It has a more stable effect on tissues, giving a
following together) firmer texture with better tissue sections,
FORMULA: especially of central nervous tissues.
8 % paraformaldehyde 25 ml
2. It preserves plasma proteins better.
25 % glutaraldehyde 10 ml
3. It produces less tissue shrinkage.
0.2 M phosphate buffer 50 ml.
4. It preserves cellular structures better; hence, is
Make up to 100 ml with distilled water. This is to be
recommended for electron microscopy.
used fresh.
5. It is more pleasant and less irritating to the nose.
GLUTARALDEHYDE 6. It does not cause dermatitis
Disadvantages of Glutaraldehyde over Formaldehyde:
Another popular aldehyde for fixation is 1. It is more expensive.
glutaraldehyde, made up of two formaldehyde residues, 2. It is less stable.
linked by a three carbon chain. Glutaraldehyde is a larger
3. It penetrates tissues more slowly.
molecule than formaldehyde, and so its rate of diffusion
4. It tends to make tissue (i.e. renal biopsy) more
across membranes is slower than formaldehyde.
brittle
Consequently, glutaraldehyde fixation on thicker tissue
5. It reduces PAS positivity of reactive mucin. This
samples may be hampered, but this problem can be
may be prevented by immersing
overcome by reducing the size of the tissue sample.
glutaraldehydefixed tissues in a mixture of
concentrated glacial acetic acid and aniline oil.
One of the advantages of glutaraldehyde fixation is
that it causes rapid and irreversible changes, fixes quickly, is
Precautions:
well suited for electron microscopy, it fixes well at 4oC, and
it gives best overall cytoplasmic and nuclear detail. 1. The specimen vial must be kept refrigerated during
However, like formaldehyde, glutaraldehyde causes the fixation process.
deformation of alpha-helix structure in proteins so it is not 2. Solution may be changed several times during
good for immunohistochemical staining. Tissues that have fixation by swirling the vials to make sure that the
been fixed with a glutaraldehydebased fixative must be specimen is in contact with fresh solution all the
time.

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

PRECIPITATING (ALCOHOLIC) FIXATIVES a. Methyl Alcohol 100%

Advantages:
Alcohols are protein denaturants and are not used 1. It is excellent for fixing dry and wet smears,
routinely for tissues because they cause too much blood smears and bone marrow tissues.
brittleness and hardness. The protein denaturants - 2. t fixes and dehydrates at the same time.
methanol, ethanol and acetone - are rarely used alone for Disadvantage:
fixing blocks unless studying nucleic acids. They are also 1. Penetration is slow
very good for cytologic smears because they act quickly and 2. If left in fixative for more than 48 hours, t
give good nuclear detail. Spray cans of alcohol fixatives are issues may be over hardened and difficult to
marketed to physicians doing PAP smears. Alcohol alone cut.
(preferably methanol) is suitable for fixing thin layer b. Isopropyl Alcohol 95%
preparations such as blood films or cell cultures. Solid It is used for fixing touch preparations , although
specimens taken from patients with gout are usually fixed some touch preparations are air dried and not fixed,
in 95% ethanol for subsequent histochemical detection of for certain special staining procedures such as Wright-
sodium urate crystals, which can be dissolved out of the Giemsa.
tissue by water. Ethanol appears to give the most usable c. Ethyl Alcohol
DNA fragments for polymerase chain reaction (PCR), It is used at concentrations of 70-100%. If the
whereas formaldehyde limits the size of fragments which lower concentrations are used, the RBC's become
can be retrieved. hemolyzed and WBC's are inadequately preserved. It
may be used as a simple fixative. It is, however, more
Alcohol rapidly denatures and precipitates proteins by frequently incorporated into compound fixatives for
destroying hydrogen and other bonds. It must be used in better results.
concentrations ranging from 70 to 100% because less Fixation Time: 18-24 hours
concentrated solutions will produce lysis of cells. Ethanol Advantages:
(95%) is fast and cheap. Since smears are only a cell or so 1. It preserves but does not fix glycogen.
thick, there is no great problem from shrinkage, and since
2. It fixes blood, tissue films and smears.
smears are not sectioned, there is no problem from
3. It preserves nucleoproteins and nucleic acids,
induced brittleness. They are not good for electron
hence, is used for histochemistry, especially
microscopy, though, because they cause tissue shrinkage .
for enzyme studies.
Absolute alcohol can be used to fix and preserve glycogen,
pigments, blood, tissue films and smears. The color of the 4. It fixes tissue pigments fairly well.
specimen can be preserved for photographic work using 5. It is ideal for small tissue fragments.
80% alcohol. Sometimes the specimen is taken out from 6. It may be used both as a fixative and
fixative and placed in 80% alcohol to bring out some of the dehydrating agent.
original color. Glycerin is also used in combination with Disadvantages:
alcohol for this purpose. 1. Hemosiderin preservation is less than in
buffered formaldehyde.
Alcohol (ethanol or methanol) alone instantly 2. It is a strong reducing agent; hence, should
coagulates proteins but causes considerable distortion of not be mixed with chromic acid, potassium
the tissue micro-anatomy. These unwanted changes are dichromate and osmium tetroxide which are
opposed by dilution of the alcohol with chloroform strong oxidizing agents.
(immiscible with water), water, and/or acetic acid (which 3. Lower concentrations (70-80%) will cause RBC
coagulates chromatin and opposes shrinkage) being hemolysis and inadequately preserve
miscible with water, alcohol and hydrocarbons. There are leukocytes.
also many mixtures containing an alcohol (usually ethanol), 4. It dissolves fats and lipids, as a general rule.
formalin, acetic acid and 10% to 70% water, where the Alcohol-containing fixatives are
chemical reactions of formaldehyde with proteins are not contraindicated when lipids are to be studied.
retarded by buffering to a near-neutral pH. All these 5. It causes glycogen granules to move towards
alcoholic fixatives contain acetic acid, which produces the poles or ends of the cells (polarization).
characteristic patterns of coagulated nuclear chromatin, 6. Tissue left in alcohol too long will shrink,
facilitating the recognition of cell types. making it difficult or impossible to cut.

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

7. It causes polarization of glycogen granules. Recommended Applications:

8. It produces considerable hardening and Clarke’s solution has been used on frozen sections
shrinkage of tissues and smears. It can produce fair results after
conventional processing if fixation time is kept
d. Carnoy’s Fixative very short. It preserves nucleic acids but extracts
FORMULA: lipids. Tissues can be transferred directly into 95%
Absolute alcohol 60 ml. ethanol.
Chloroform 30 ml.
Glacial acetic acid 10 ml. f. Alcoholic formalin
Fixation Time: 1-3 hours FORMULA:
Advantages: 40% Formaldehyde: 100 ml
1. It is considered to be the most rapid fixative 95% Ethanol: 900 ml
and may be used for urgent biopsy specimens 0.5 g calcium acetate can be added to ensure
for paraffin processing within 5 hours. neutrality
2. It fixes and dehydrates at the same time. Fixation time: 12 – 24 hours
3. It permits good nuclear staining and
differentiation. Recommended Applications:
4. It preserves Nissl granules and cytoplasmic Alcoholic-formalin combines a denaturing fixative
granules well. with the additive and cross-linking effects of
5. It preserves nucleoproteins and nucleic acids. formalin. It is sometimes used during processing to
6. It is an excellent fixative for glycogen since complete fixation following incomplete primary
aqueous solutions are avoided. formalin fixation. It can be used for fixation or
7. It is very suitable for small tissue fragments post-fixation of large fatty specimens (particularly
such as curettings and biopsy materials. breast), because it will allow lymph nodes to be
8. Following fixation for one hour, tissues may be more easily detected as it clears and extracts
transferred directly to absolute alcohol- lipids. If used for primary fixation specimens, it can
chloroform mixture, thereby shortening be placed directly into 95% ethanol for processing.
processing time.
9. It is also used to fix brain tissue for the g. Formol-acetic alcohol
diagnosis of rabies. FORMULA:
Ethanol absolute: 85 ml
Disadvantages: 40% formaldehyde: 10 ml
1. It produces RBC hemolysis, dissolves lipids and Acetic acid glacial: 5 ml
can produce excessive hardening and Fixation time: 1 - 6 hours
shrinkage.
2. It causes considerable tissue shrinkage. Recommended Applications:
3. It is suitable only for small pieces of tissues Formol-acetic acid alcohol is a faster acting agent
due to slow penetration. than alcoholic formalin due to the presence of
4. It tends to harden tissues excessively and acetic acid that can also produce formalin
distorts tissue morphology. pigment. It is sometimes used to fix diagnostic
5. It dissolves fat, lipids, and myelin. cryostat sections. If used for primary fixation, the
6. It leads to polarization unless very cold specimens can be placed directly into 95% ethanol
temperatures (-70°C) are used. for processing.
7. It dissolves acid-soluble cell granules and
pigments. h. Gendre's Fixative
FORMULA:
e. Clarke’s solution 95% Ethyl alcohol saturated with picric acid 80 ml.
FORMULA: Strong formaldehyde solution 15 ml.
Ethanol (absolute) 75 ml Glacial acetic acid 5 ml.
Acetic acid glacial 25 ml Post-fixation with phenol-formalin for 6 hours or
Fixation time: 3 - 4 hours more can enhance immunoperoxidase studies on

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

the tissues, and in some cases, electron METALIC FIXATIVES

microscopy, if it is necessary at a later time to
establish a diagnosis. Mercurials fix tissues through an unknown mechanism that
Advantages: increases staining brightness and gives excellent nuclear
detail. However, mercurial penetrate poorly and produce
1. Fixation is faster (fixation time is reduced to one-
tissue shrinkage. Their best application is for fixation of
half).
hematopoietic and reticuloendothelial tissues.
2. It can be used for rapid diagnosis because it fixes
and dehydrates at the same time, e.g., in the
1. Mercuric Chloride
frozen section room.
Mercuric chloride is the most common metallic
3. It is good for preservation of glycogen and for
fixative, frequently used in saturated aqueous solutions of
micro-incineration technique (the burning of a
5-7%. Mercuric chloride is widely used as a secondary
minute tissue specimen for identification of
fixative reacting with a number of amino acid residues and
mineral elements from the ashes).
accompanied by spectroscopic changes, probably due to
4. It is used to fix sputum, since it coagulates mucus. reaction with histidine residues. Mercuric chloride-based
Disadvantages: fixatives are used as an alternative to formaldehyde-based
1. It produces gross hardening of tissues. fixatives to overcome poor cytological preservation and
2. It causes partial lysis of RBC. include such well-known fixatives as B-5 and Zenker's
3. Preservation of iron-containing pigments is poor. solution. They penetrate relatively poorly and cause some
4. Formaldehyde does not give as good a tissue hardness, but give excellent nuclear detail. These
morphological picture as glutaraldehyde. fixatives work by additive and coagulative means.
5. Formaldehyde causes little cross-linking under
usual fixation conditions where low concentrations The major advantages of using these fixatives include
of proteins are used, while glutaraldehyde is most good penetration resulting in more intense immunostaining
effective at cross-linking. and the preservation of cytological detail that allow for
easier morphological interpretation. These fixatives often
Post-fixation with phenol-formalin for 6 hours or more contain neutral salt to maintain tonicity and can be mixed
can enhance immunoperoxidase studies on the tissues, with other fixatives to provide a balanced solution. Their
and in some cases, electron microscopy, if it is best application is for fixation of hematopoietic and
necessary at a later time to establish a diagnosis. reticuloendothelial tissues.

i. Newcomer's Fluid Mercuric chloride penetrates poorly and produces

FORMULA: shrinkage of tissues, so it is usually combined with other
Isopropyl alcohol 60 ml. fixative agents. Tissues fixed with mixtures containing
Propionic acid 30 ml. mercuric chloride (except Susa) contain black precipitates
Petroleum 30 ml. of mercury. Mercury deposits are removed from
Ether 10 ml. deparaffinized sections before staining, by treating the
Acetone 10 ml. section with 0.5% iodine solution in 70% ethanol for 5- 10
Dioxane 10 ml. minutes. Sections are rinsed in water, decolorized for 5
Fixation time: 12-18 hours at 3°C minutes in 5% sodium thiosulfate and washed in running
water.
Advantages:
1. It is recommended for fixing Following mercuric chloride post-fixation, the
mucopolysaccharides and nuclear proteins. ultrastructural preservation is poor, but trichrome staining
2. It produces better reaction in Feulgen stain methods also work well and many immunoperoxidase
than Carnoy's fluid. techniques are satisfactory.
3. It acts both as a nuclear and histochemical
fixative If the glacial acetic acid is replaced by 5 ml of formalin
(37–40% formaldehyde), the resulting solution is Helly's
fixative, sometimes also called "Formol-Zenker". Helly’s
fixative is stable for only a few hours because the

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
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BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES

formaldehyde and dichromate components react, 4. Contact of mercuric fixatives with personal jewelries
producing formic acid and chromic ions; the orange should be avoided.
solution becomes greenish. 5. Mercury-containing solutions (Zenker's or should
always be discarded into proper containers. Mercury, if
Advantages: poured down a drain, will form amalgams with the
1. It penetrates and hardens tissues rapidly and well. metal that build up and cannot be removed
2. Nuclear components are shown in fine detail.
3. It precipitates all proteins. 1.1 Zenker's Solution
4. It has a greater affinity to acid dyes and is preferred in FORMULA: Mercuric chloride 5 gm Potassium dichromate
lieu of formaldehyde for cytoplasmic staining. 2.5 gm Distilled water 100 mlAcetic acid, glacial 5 ml (to be
5. Trichrome staining is excellent. added just before use)
6. It is the routine fixative of choice for preservation of Heat, cool, filter in brown bottle
cell detail in tissue photography. Wash sample for 24 hours with distilled water after fixation.
7. It permits brilliant metachromatic staining of cells. Fixation time: 12-24 hours
8. It is recommended for renal tissue, fibrin, connective
tissue and muscle. Advantages:
1. It produces a fairly rapid and even fixation of tissues.
Disadvantages: 2. Stock solutions keep well without disintegration.
1. It causes marked shrinkage of cells (this may be 3. It is recommended for trichrome staining.
counteracted by addition of acid). 4. It permits brilliant staining of nuclear and connective
2. It rapidly hardens the outer layer of the tissue with tissue fibers.
incomplete fixation of the center; therefore, thin 5. It is recommended for congested specimens (such as
sections should be made. lung, heart and blood vessels) and gives good results with
3. Penetration beyond the first 2-3 millimeters is slow; PTAH and trichrome staining.
hence, not more than 5 mm. thickness of tissues 6. It is compatible with most stains.
should be used. 7. It may act as a mordant to make certain special staining
4. If left in fixative for more than 1-2 days, the tissue reactions possible.
becomes unduly hard and brittle. 8. It is a stable fixative that can be stored for many years.
5. It prevents adequate freezing of fatty tissues and
makes cutting of frozen tissues difficult. Disadvantage:
6. It causes considerable lysis of red blood cells and 1. Penetration is poor
removes much iron from hemosiderin. 2. It is not stable after addition of acetic acid.
7. It is inert to fats and lipids. 3. Prolonged fixation (for more than 24 hours) will make
8. It leads to the formation of black granular deposits in tissues brittle and hard.
the tissues. 4. It causes lysis of red blood cells and removes iron from
9. It reduces the amount of demonstrable glycogen. hemosiderin
10. Compound solutions containing mercuric chloride 5. It does not permit cutting of frozen sections.
deteriorate rapidly upon addition of glacial acetic 6. It has the tendency to form mercuric pigment deposits
acid to formalin. or precipitates.
11. It is extremely corrosive to metals. 7. Tissue must be washed in running water for several
hours (or overnight) before processing. Insufficient
Precautions: washing may inhibit or interfere with good cellular
1. Sections must be cleared of mercury deposits before staining.
immunostaining. Black deposits may be removed by
adding saturated iodine solution in 95% alcohol, the Precautions:
iodine being decolorized with absolute alcohol in the 1. Do not let tissues stay in solution for more than 24
subsequent stages of dehydration. hours.
2. Compound solutions must always be freshly prepared. 2. Solutions must always be freshly prepared.
3. The use of metallic forceps and of metal caps to cover
the bottles containing the fixative should be avoided.

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
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BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES
3. Nuclear fixation and staining with Helly’s solution is
3. Tissues should be cut thin (2-3 mm.) and hollow better with Zenker’s
organs should be opened to promote complete 4. It preserves cytoplasmic granules well
penetration and fixation.
4. Tissues must be washed out thoroughly in running Disadvantages:
water to permit good staining. The disadvantages of Helly's solution are similar to Zenker's
5. It produces mercury pigment which should be except that brown pigments are produced if tissues
removed from sections prior to staining and can (especially blood containing organs) are allowed to stay in
produce chrome pigment if tissue is not washed in the fixative for more than 24 hours due to RBC lysis. This
water prior to processing. After water washing, tissue may be removed by immersing the tissue in saturated
should be stored in 70% ethanol. alcoholic picric acid or sodium hydroxide.
6. Mercuric deposits may be removed by immersing
tissues in alcoholic iodine solution prior to staining, b. Lillie’s B-5 Fixative
through a process known as dezenkerization. Lillie’s B5 fixative is 4% aqueous formaldehyde with
0.22M mercuric chloride and 0.22M acetic acid. This
Chemically, de-zenkerization is done by oxidation with mixture enhances nuclear detail, which is important for
iodine to form mercuric iodide, which can be subsequently identifying normal and abnormal cell types in bone marrow
removed by treatment with sodium thiosulfate, using the (hematopoietic tissue) specimens. The coagulation of
following procedure: nuclear chromatin is an effect of the acetic acid. The
1. Bring slides to water. mercuric chloride ensures rapid structural stabilization and
2. Immerse in Lugol's iodine (5 minutes). also facilitates bright staining by many of the dyes used in
3. Wash in running water (5 minutes). microtechnique. The reasons for this effect on stainability
4. Immerse in sodium thiosulfate 5% (5 minutes). are not understood.
5. Wash in running water (5 minutes). A dirty looking brown crystalline precipitate, probably
6. Proceed with required water soluble stain. mercurous chloride (Hg2Cl2) forms in all parts of tissues
fixed in mixtures containing HgCl2. It is called mercury
a. Zenker-Formol (Helly’s) Solution pigment and before staining must be removed by
FORMULA: sequential treatments with iodine and sodium thiosulfate
Mercuric chloride, 5 gm Potassium dichromate, 2.5 gm solutions. Because it contains mercury, B-5 is subject to
Distilled water, 100 ml 40% formaldehyde 5 ml (to be toxic waste disposal regulations, which apply to the fixative
added immediately before use) solution and every solution thereafter that has been
Heat, cool, filter in brown bottle. contaminated with mercury.
Wash sample for 24 hours with distilled water after fixation.
Fixation time: 4 – 24 hours FORMULA:
B-5 Stock solution
Practical Applications: Mercuric chloride: 12 g
Zenker’s solution is an excellent fixative for bone marrow, Sodium acetate anhydrous: 2.5 g
extramedullary hematopoiesis and intercalated discs of Distilled water: 200 ml
cardiac muscle. However, it produces mercury pigment Working solution: (prepare immediately before use)
which should be removed from sections prior to staining B-5 stock solution: 20 ml 40%
and it can produce chrome pigment if tissue is not washed formaldehyde: 2 ml
in water prior to processing. After water washing, fixed Fixation time: 4 – 8 hours
tissue should be stored in 70% ethanol. Because of the low
pH of this fixative, formalin pigment may also occur. Never Practical Applications:
use metal forceps to handle tissue. 1. Despite its mercuric chloride content and consequent
problems with disposal, this solution is popular for fixation
Advantages: of hematopoietic, bone marrow biopsies and lymphoid
1. It is an excellent microanatomic fixative for pituitary tissue.
gland, bone marrow and blood containing organs such 2. Rapid fixation can be achieved in 1 1/2 - 2 hours.
as spleen and liver.
2. It penetrates and fixes tissues well.

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
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BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES
3. It produces excellent nuclear detail, provides good results 5. Weigert's method of staining elastic fibers is not
with many special stains, and is recommended for possible in Susa fixed tissues.
immunohisto-chemical staining.
4. Sections will require the removal of mercury pigment prior Precautions:
to staining. After using Heidenhain's Susa fixative, the tissue should be
5. Tissue should not be stored in this fixative but placed in transferred directly to a high-grade alcohol, e.g. 96% or
70% ethanol instead. absolute alcohol, to avoid undue swelling of tissues caused
6. Over-fixation hardens the tissue and makes cutting by treatment with low- grade alcohol or water.
difficult.
7. The two working solutions are kept separate, since the OXIDIZING AGENTS
mixture is unstable. Mix just prior to use. Oxidizing agents include permanganate fixatives
8. Some B-5 solutions will form precipitate on standing, but (potassium permanganate), potassium dichromate, chromic
this is of no consequence. acid and osmium tetroxide. The oxidizing fixatives can react
9. As with all mercuric chloride fixatives, the mercury with various side chains of proteins and other
pigment can be removed by de-zenkerization. biomolecules, allowing formation of crosslinks that stabilize
tissue structure but cause extensive denaturation despite
c. Heidenhain's Susa Solution preserving fine cell structure and are used mainly as
It is recommended mainly for tumor biopsies especially secondary fixative.
of the skin; it is an excellent cytologic fixative.
FORMULA: 1. Osmium Tetroxide (Osmic Acid; OsO4)
Mercuric chloride 45 gm This is a pale yellow powder which dissolves in
Sodium chloride 5 gm water (up to 6% at 20°C) to form a strong oxidizing solution.
Trichloroacetic acid 20 gm Fixation with osmium tetroxide or post - osmication of
Glacial acetic 40 ml glutaraldehyde-fixed tissue causes the complete
Acid Formaldehyde 40% 200 ml denaturation of protein. Potassium permanganate and
40% Distilled water 800 ml potassium dichromate are also oxidizing agents but are less
Fixation time: 3-12 hours reactive towards proteins than is osmium tetroxide.
Osmium tetroxide is traditionally used in electron
Advantages: microscopy both as a fixative and a heavy metal stain.
1. It penetrates and fixes tissues rapidly and evenly. Osmium tetroxide is a good fixative and excellent stain for
2. It produces minimum shrinkage and hardening of tissues lipids in membranous structures and vesicles. The most
due to the counter-balance of the swelling effects of prominent staining in adherent human cells (HeLa) is seen
acids and the shrinkage effect of mercury. on lipid droplets. Some intracellular structures are also
3. It permits most staining procedures to be done, including visualized. Osmium tetroxide functions as a secondary
silver impregnation, producing brilliant results with sharp fixative by reacting with lipids. It is believed that the
nuclear and cytoplasmic details. unsaturated bonds of fatty acids are oxidized by OSO4 and
4. It permits easier sectioning of large blocks of fibrous it is reduced to a black metallic osmium (MW 254.2) which
connective tissues. is electron dense and adds contrast to biological tissues
5. Susa-fixed tissues may be transferred directly to (secondary stain).
95% alcohol or absolute alcohol, thereby reducing Visualized cellular structures depend on the
processing time. fixation protocols. In glutaraldehyde fixation, the nucleoli
are visible, but overall nuclear staining is weak. In
Disadvantages: paraformaldehyde fixation, the nuclear staining becomes
1. Prolonged fixation of thick materials may produce more prominent, but some intracellular structures are lost.
considerable shrinkage, hardening and bleaching; As a first choice, fixing tissues with a combination,
hence, tissues should not be more than 1 cm. thick. glutaraldehyde and paraformaldehyde are recommended.
2. RBC preservation is poor. Osmium tetroxide penetrates slower than glutaraldehyde
3. Some cytoplasmic granules are dissolved. (0.5 mm/hr.) and the tissue takes on a progressively
4. Mercuric chloride deposits tend to form on tissues; blackened appearance depending upon their lipid content.
these may be removed by immersion of tissues in Fixation experiments with buffered OsO4 solutions have
alcoholic iodine solution. shown that the appearance of the fixed cells is conditioned

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES
by the pH of the fixative; thus, 1% OsO4 buffered at pH 7.3- 3. It is readily reduced by contact with organic matter and
7.5 with acetate-veronal buffer is recommended as an exposure to sunlight, forming a black precipitate which
appropriate fixative for electron microscopy. settles at the bottom of the container.
4. Prolonged exposure to acid vapor can irritate the eye,
Procedure: producing conjunctivitis, or cause the deposition of black
1. Wash the sample four times with PBS. osmic oxide in the cornea, producing blindness. 5. It inhibits
2. Fix with 2% Paraformaldehyde, 0.1% Glutaraldehyde in hematoxylin and makes counterstaining difficult.
PBS for 30 minutes. 5. It is extremely volatile.
3. Wash four times with PBS.
4. Wash four times with double distilled water. Precautions:
5. Prepare 0.1% OsO4 solution by diluting the 4% stock 1. Eyes and skin may be protected by working in a fume hood
solution in double distilled water. or wearing protective plastic masks or gloves while using
6. Incubate the sample with 0.1% OsO4 for 30 minutes. osmium tetroxide.
7. Wash four times with double distilled water. 2. It should be kept in a dark-colored, chemically clean bottle
to prevent evaporation and reduction by sunlight or
Advantages: organic matter.
1. It fixes conjugated fats and lipids permanently by 3. It should be kept in a cool place or refrigerated to prevent
making them insoluble during subsequent treatment deterioration.
with alcohol and xylene. Fats form hydrated osmium 4. Addition of saturated aqueous mercuric chloride solution
dioxide, are stained black and therefore are easier to (0.5 to 1 ml/100 ml of stock solution) will prevent its
identify. reduction with formation of black deposits.
2. It preserves cytoplasmic structures well, e.g. Golgi 5. Black osmic oxide crystals may be dissolved in cold water.
bodies and mitochondria. 6. To prevent contact of tissues with black precipitate formed
3. It fixes myelin and peripheral nerves well, hence, it is in the bottom of the jar, the tissues may be wrapped in
used extensively for neurological tissues. gauze and suspended in the fluid by the means of the
4. It produces brilliant nuclear staining with safranin. thread.
5. It adequately fixes materials for ultrathin sectioning in 7. Osmic acid-fixed tissues must be washed in running water
electron microscopy, since it rapidly fixes small pieces for atleast 24 hours to prevent formation of artefacts.
of tissues and aids in their staining.
6. It precipitates and gels proteins. 1.1 Flemming's Solution
7. It shows uniformly granular nuclei with clear It is the most common chrome-osmium acetic acid
cytoplasmic background. fixative used, recommended for nuclear preparation of
8. Some tissues (e.g. adrenal glands) are better fixed in such sections.
vapor form of osmium tetroxide. This eliminates FORMULA:
"washing out" of the fixed tissues. Aqueous chromic acid 15 ml.
9. Osmium tetroxide completely permeabilizes cell 1% Aqueous osmium tetroxide 4 ml.
membranes. The osmolarity of the fixative vehicle or 2% Glacial acetic acid 1 ml.
solute is relatively unimportant. Fixation time: 24 - 48 bouts
10. It penetrates tissue blocks in a gradient and in large
samples the center of the block may not be as well Advantages:
fixed as the peripheral areas. 1. It is an excellent fixative for nuclear structures, e.g.
11. Over-fixation with osmium tetroxide may result in chromosomes.
extraction of cell components during dehydration and 2. It permanently fixes fat.
increases the hardness and brittleness of the tissue (for 3. Relatively less amount of solution is required for
most tissues, 1mm blocks, should not be exposed to fixation (less than 10 times the volume of the tissues to
osmium for less than 0.5 or more than 1.5 hours). be fixed).

Disadvantages: Disadvantages:
1. It is very expensive. 1. It is a poor penetrating agent; hence, is applicable only
2. It is a poor penetrating agent, suitable only for small to small pieces of tissues.
pieces of tissues (2-3 mm. thick).

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES
2. The solution deteriorates rapidly and must be prepared 2. It hardens tissues better and more rapidly than Orth's
immediately before use. fluid.
3. Chromic-osmic acid combinations depress the staining 3. It is recommended for the demonstration for the
power of hematoxylin (especially Ehrlich's demonstration of chromatin, mitochondria, mitotic
hematoxylin). figures, Golgi bodies, RBC and colloid-containing
4. It has a tendency to form artifact pigments; these may tissues.
be removed by washing the fixed tissue in running tap
water for 24 hours before dehydration. Disadvantages:
5. It is very expensive. 1. It deteriorates and darkens on standing due to acidity;
hence, the solution must always be freshly prepared.
1.2 Flemming's solution without acetic acid 2. Penetration is slow, hence, tissues should not be
It is made up only of chromic and osmic acid, thicker than 2-3 mm.
recommended for cytoplasmic structures particularly the 3. Chromate-fixed tissues tend to produce precipitates of
mitochondria. The removal of acetic acid from the formula sub-oxide, hence should be thoroughly washed in
serves to improve the cytoplasmic detail of the cell. running water prior to dehydration.
4. Prolonged fixation blackens tissue pigments, such as
Fixation time: 24 - 48 hours melanin; this may be removed by washing the tissues
in running tap water prior to dehydration.
Advantages and Disadvantages: Same as Flemming's 5. Glycogen penetration is poor; it is therefore, generally
solution. contraindicated for carbohydrates.
CHROMATE FIXATIVES 6. Nuclear staining is poor.
Chromic Acid is used in 1-2% aqueous solution, usually 7. It does not preserve fats.
as a constituent of a compound fixative. It precipitates all 8. It preserves hemosiderin less than buffered formalin.
proteins and adequately preserves carbohydrates. It is a 9. Intensity of PAS reaction is reduced.
strong oxidizing agent; hence, a strong reducing agent (e.g.
formaldehyde) must be added to chrome-containing 1.2 Orth's Fluid
fixatives before use in order to prevent counteracting FORMULA:
effects and consequent decomposition of solution upon Potassium dichromate 2.5% 100ml
prolonged standing. Sodium sulfate (optional) 1gm
Strong formaldehyde 40% 10 ml (To be added just before
Potassium Dichromate is used in a 3% aqueous use).
solution. Fixation time: 36-72 hours
o It fixes but does not precipitate cytoplasmic structures.
o It preserves lipids. Advantages:
o It preserves mitochondria (If used in pH 4.5-5.2, 1. It is recommended for study of early degenerative
mitochondria is fixed. processes and tissue necrosis.
NOTE: If the solution becomes acidified, cytoplasm, 2. It demonstrates rickettsiae and other bacteria.
chromatin bodies and chromosomes are fixed but 3. It preserves myelin better than buffered formalin.
mitochondria are destroyed).
Disadvantages: Same as in Regaud's fluid.
1.1 Regaud’s (Muller's) Fluid
FORMULA: PICRIC ACID FIXATIVES
Potassium dichromate 3% 80 ml Picrates include fixatives with picric acid. Foremost
Strong formaldehyde 40% 20 ml (To be added just before among these is Bouin's solution that does almost as well as
use). mercurials with nuclear detail but does not cause as much
Fixation time: 12-48 hours hardness. Picrates penetrate tissue well to react with
histones and basic proteins, form crystalline picrates with
Advantages: amino acids and precipitate all proteins. It is a good fixative
1. It penetrates tissues well. for connective tissue, preserves glycogen well, and extracts
lipids to give superior results in immunostaining of biogenic
and polypeptide hormones. However, it causes a loss of

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES
basophilia unless the specimen is thoroughly washed Advantages:
following fixation. 1. It is an excellent fixative for glycogen demonstration.
Picric acid is an explosive hazard in dry form. As a 2. It penetrates tissues well and fixes small tissues rapidly.
solution, it stains everything it touches yellow, including 3. The yellow stain taken in by tissues prevents small
skin. Like mercuric chloride, picric acid enhances fragments from being overlooked.
subsequent staining, especially with anionic (“acid”) dyes. 4. It allows brilliant staining with the trichrome method.
Picric Acid is normally used in strong saturated aqueous 5. It is suitable for Aniline stains (Mallory's, Heidenhain's
solution (approximately I %). or Masson's methods).
Picric acid also dyes the tissues, but the yellow 6. It precipitates all proteins.
color may be removed by treatment with another acid dye 7. It is stable.
or lithium carbonate. Tissues fixed with picric acid retain
little affinity for basic dyes. It preserves glycogen well but Disadvantages:
causes considerable shrinkage of tissue. Washing with 1. It causes RBC hemolysis and reduces the amount of
changes of 50% and 70% ethanol will remove most of the demonstrable ferric iron in tissue.
yellow color, but the excess picrate may be removed more 2. It is not suitable for frozen sections because it causes
easily from the sections when the paraffin wax has been frozen sections to crumble when cut.
removed. Picric acid is only sold in the aqueous state. When 3. Prolonged fixation makes tissues hard, brittle and
it dries out, it becomes explosive. difficult to section. Tissues should not be allowed to
Paraffin sections of formaldehyde fixed tissues are remain in the fluid for more than 12-24 hours
usually immersed for a few hours in a picric acid solution (depending on size).
(Bouin’s fluid is commonly used) before Trichrome staining. 4. Picrates form protein precipitates that are soluble in
Trichrome stains use combinations of anionic dyes with water; hence, tissues must be first rendered insoluble
phosphotungstic or phosphomolybdic acid to impart by direct immersion in 70% ethyl alcohol.
contrasting colors to cytoplasm, collagen fibers and other 5. Picric acid fixed tissues must never be washed in water
components of tissues. before dehydration.
6. Picric acid will produce excessive staining of tissues; to
1. Bouin’s Solution remove the yellow color, tissues may be placed in 70%
The complementary effects of the three ethyl alcohol followed by 5% sodium thiosulfate and
ingredients of Bouin’s solution work well together to then washed in running water.
maintain morphology. Specimens are usually fixed in 7. Picric acid is highly explosive when dry, and therefore
Bouin’s solution for 24 hours. Prolonged storage in this must be kept moist with distilled water or saturated
acidic mixture causes hydrolysis and loss of stainable DNA alcohol at 0.5 to 1% concentration during storage.
and RNA. Thorough washing after fixation is necessary. 8. It alters and dissolves lipids.
9. It interferes with Azure eosin method of staining;
FORMULA: hence, tissues should be thoroughly washed with
Picric acid saturated aqueous soln. (2.1%) 75 ml alcohol.
40% formaldehyde 25 ml
Acetic acid glacial 5 ml 2. Hollande’s Solution
Fixation time: 4 – 18 hours Store at room temperature FORMULA:
Copper acetate: 25 gm Picric acid: 40 gm
Practical Applications: 40% formaldehyde: 100 ml Acetic acid: 15 ml
Bouin's Solution is recommended for fixation of embryos Distilled water: 1000 ml
and pituitary biopsies. It gives very good results with tissue Dissolve chemicals in distilled water without heat.
that is subsequently stained with trichrome. It preserves Fixation time: 4– 18 hours
glycogen well but usually lyses erythrocytes. It is sometimes
recommended for gastro-intestinal tract biopsies, animal Practical Applications:
embryos and endocrine gland tissue. It stains tissue bright It is recommended for gastro-intestinal tract specimens and
yellow due to picric acid. Excess picric should be washed fixation of endocrine tissues. It produces less lysis than
from tissues prior to staining with 70% ethanol. Because of Bouin’s Solution. It has some decalcifying properties. The
its acidic nature, it will slowly remove small calcium
deposits and iron deposits.

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES
fixative must be washed from tissues if they are to be put 6. It reduces or abolishes Feulgen reaction due to
into phosphate buffered formalin on the processing hydrolysis of nucleoproteins.
machine because an insoluble phosphate precipitate will
form. 4. Brasil's Alcoholic Picroformol Fixative
FORMULA:
3. Gendre’s solution Formaldehyde 37% 2040 ml.
FORMULA: Picric acid 80 gm.
95% Ethanol saturated with picric acid 800 ml Ethanol or isopropyl alcohol 6000 ml.
40% formaldehyde 150 ml Trichloroacetic acid 65 gm.
Acetic acid glacial 50 ml
Fixation time: 4 - 18 hours Overnight tissue fixation by automatic processing
technique may utilize 3-4 changes of Brasil's fixative at 1/2
Practical Application: to 2 hours each, succeeded directly by absolute alcohol.
This is an alcoholic Bouin’s solution that appears to improve
upon ageing. It is highly recommended for the preservation Advantages:
of glycogen and other carbohydrates. After fixation the 1. It is better and less "messy" than Bouin's solution.
tissue is placed into 70% ethanol. Residual yellow color 2. It is an excellent fixative for glycogen.
should be washed out before staining.
GLACIAL ACETIC ACID
Advantages: Acetic acid is a colorless liquid that when undiluted is
also called “Glacial” Acetic Acid because it is a water-free
1. It produces minimal distortion of micro-anatomical
(anhydrous) acetic acid that freezes and solidifies at about
structures and can be used for general and special stains. 16°C. Acetic acid does not have much effect on proteins,
(The shrinking effect of picric acid is balanced by the other than to enable swelling by the absorption of water.
swelling effect of glacial acetic acid.) Its major effect is to precipitate DNA, which is split off from
2. It is an excellent fixative for preserving soft and delicate nucleoprotein. For this reason, acetic acid is valuable for
structures (e.g. endometrial curettings). the preservation of nuclei, and is often added to fixatives
3. It penetrates rapidly and evenly, and causes little specifically to do that. Acetic acid is not used alone for
fixation but is incorporated into other fixatives to form a
shrinkage.
compound solution, most commonly at a concentration of
4. Yellow stain is useful when handling fragmentary approximately 5%.
biopsies.
5. It permits brilliant staining of tissues. Advantages:
6. It is the preferred fixative for tissues to be stained by 1. It fixes and precipitates nucleoproteins.
Masson's trichrome for collagen, elastic or connective 2. It precipitates chromosomes and chromatin materials;
tissue. (If tissue is fixed in formalin, a pre-treatment in hence, is very useful in the study of nuclear
components of the cell. In fact, it is an essential
Bouin’s solution (as mordant prior to trichrome stain) is
constituent of most compound nuclear fixatives.
recommended. 3. It causes tissues (especially those containing collagen)
7. It preserves glycogen. to swell. This property is used in certain compound
8. It does not need "washing out". fixatives to counteract the shrinkage produced by
other components (e.g., mercury).
Disadvantages:
1. It penetrates large tissues poorly; hence, its use is Disadvantages:
1. When combined with Potassium Dichromate, the
limited to small fragments of tissue.
lipid-fixing property of the latter is destroyed (e.g.,
2. Picrates are soluble in water; hence, tissues should not Zenker's fluid).
be washed in running water but rather transferred 2. It is contraindicated for cytoplasmic fixation since it
directly from fixative to 70% alcohol. destroys mitochondria and Golgi elements of cells.
3. It is not suitable for fixing kidney structures, lipid and 3. Concentrated acetic acid is corrosive to skin and must,
mucus. therefore, be handled with appropriate care, since it
can cause skin burns, permanent eye damage, and
4. It destroys cytoplasmic structures, e.g. mitochondria.
irritation to the mucous membranes. These burns or
5. It produces RBC hemolysis and removes demonstrable
blisters may not appear until hours after exposure.
ferric iron from blood pigments.

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES
4. Latex gloves offer no protection, so especially smears, cryostat sections) to several hours (1-24 hours for
resistant gloves, such as those made of nitrile rubber, small tissue blocks).
are worn when handling the compound.
Advantages:
LEAD FIXATIVES 1. It is recommended for the study of water diffusible
Lead fixatives are used in 4% aqueous solution of basic enzymes especially phosphatases and lipases.
lead acetate. Led oxaloacetate, a primary reaction product 2. It is used in fixing brain tissues for diagnosis of rabies.
precipitate for the visualization of the activity of glutamic 3. It is used as a solvent for certain metallic salts to be
oxaloacetic transaminase in tissue section, is stable at a used in freeze substitution techniques for tissue blocks.
slightly alkaline pH. At concentrations which are used for
tissue fixation, a slightly inhibitory effect on glutamic Disadvantages:
oxaloacetic transaminase activity is produced by acetone 1. It produces inevitable shrinkage and distortion.
while a glutaraldehyde-formaldehyde mixture results in 2. It dissolves fat.
marked reduction of activity. 3. It preserves glycogen poorly.
4. It evaporates rapidly.
Advantages:
1. It is recommended for acid mucopolysaccharides. MICHEL’S SOLUTION
2. It fixes connective tissue mucin. Michel’s Solution provides a stable medium for
transport of fresh unfixed tissues, such as renal, skin and
Disadvantage oral mucosa biopsies, which will undergo subsequent
It takes up C02 to form insoluble lead carbonate frozen section and immunofluorescence studies. Michel’s
especially on prolonged standing. This may be removed by Transport Medium is not suitable for transporting cells for
filtration or by adding acetic acid drop by drop to lower the flow cytometry or for tissues used for fluorescent insitu
pH and dissolve the residue. hybridization (FISH). It is not a fixative, and is not suitable
for any other use (particularly, for transporting living cells
TRICHLOROACETIC ACID for flow cytometry). It should be kept refrigerated (not
Trichloroacetic Acid (TCA) is a reagent that is used for frozen) until use. Specimens may be kept in it at room
the precipitation of proteins and nucleic acids. It is also temperature for 5 days while in transport until they can be
used as a decalcifier and fixative in microscopy. Addition of delivered to the reference laboratory. This simple salt
TCA to a final concentration of 10% (w/v) will precipitate solution maintains pH, but does not kill most pathogens.
most proteins from solution. The excess TCA can be Specimens received in transport medium should be washed
removed from protein pellets by washes with buffer. For in three changes of washing solution (10 minutes for each
the precipitation of nucleic acids, a 5% solution of ice-cold wash). It is not suitable for FISH studies.
TCA has been used. It is sometimes incorporated into
compound fixative. Procedure:
Advantages: 1. Bring aliquot of Michel Transport Medium or a pre-filled
1. It precipitates proteins. Michel Transport Medium Vial to room temperature
2. Its marked swelling effect on tissues serves to prior to use. Michel Transport Medium is routinely
counteract shrinkage produced by other fixatives. stored at 4°C. During refrigerated storage some
3. It may be used as a weak decalcifying agent. precipitation may develop on the container bottom.
4. Its softening effect on dense fibrous tissues facilitates Precipitation should re-dissolve by allowing the solution
preparation of such sections. to reach room temperature prior to use.
2. Place fresh tissue in an adequate amount of Michel
Disadvantage: Transport Medium, ensuring that the specimen will be
It is a poor penetrating agent, hence, is suitable only totally submerged in fluid during transport. Tissue that
for small pieces of tissues or bones. has been previously frozen will not provide optimal
testing results and should not be used with Michel
Transport Medium.
ACETONE 3. Store and/or transport tissue in Michel Transport
Acetone is not recommended as morphological fixative Medium up to a maximum of five days. Care should be
for tissue blocks, mainly because of its shrinkage and poor taken to maintain cool to ambient temperatures of 4°C
preservation effects. Its use is reserved for the fixation of to 22°C during transport. Tissues transported in Michel
cryostat sections or for tissues in which enzymes have to be Transport Medium at room temperature for 5 days prior
preserved. Acetone is almost always used alone and to fixation and routine processing should provide
without dilution; it fixes by dehydration and precipitation. It satisfactory histological results although morphology
is used to fix specimens at cold temperatures (0 to 4°C). detail will not be equal to that of expediently fixed and
Fixation time may vary from several minutes (for cell processed tissue.

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES
4. Upon receipt, wash the tissue held in Michel Transport to prevent drying of surface layers and ultimate tissue
Medium in three changes of Michel Wash Solution for distortion.
10 minutes each change. 3. If fixation is not immediately possible, refrigerate but do
5. Freeze tissue per laboratory protocol. not freeze. Slow freezing of unfixed tissues near 0°C
6. Tissue placed in Michel Transport Medium may be must be avoided since this may promote formation of
subsequently processed for light or electron ice crystal artefacts. Repeated freezing and thawing, on
microscopy. Wash tissue for 2-3 minutes in tap water the other hand, will destroy cellular organelles, release
and place in appropriate fixative prior to processing. enzymes and diffuse soluble components of the cell.
4. Fresh tissue may be infectious. Consider any fresh or
- Secondary Fixation is the process of placing an already incompletely-fixed tissue as potentially infectious to you
fixed tissue in a second fixative in order: and other workers in your lab.
 To facilitate and improve the demonstration of 5. Drying should be avoided to prevent shrinkage and
particular substances. distortion of tissue with loss of cellular detail. Drying of
 To make special staining techniques possible (with specimen surfaces will cause permanent damage and
secondary fixative acting as a mordant). may mask any pathological change. Small endoscopic
 To ensure further and complete hardening and specimens are particularly susceptible to this type of
preservation of tissues. damage. Small tissue biopsies may be placed in a petri
dish with moistened filter paper to prevent drying.
Secondary fixation may be done before dehydration 6. Distortion, rough handling or other mechanical damage
and on deparaffinized sections before staining, usually with to the tissue will cause permanent morphological
10% formalin or 10% formol saline as a primary fixative. The changes which can make interpretation difficult.
tissue may have been placed in a primary fixative for 7. If placed in normal saline solution (NSS) during the
storage, or may require further fixation for special staining. operation, autolysis may occur before fixation is carried
For example, tissue fixed in 10% buffered neutral formalin on.
may require secondary fixation with Zenker's solution (that 8. Tissues should not be more than 5 mm. thick except in
acts as a mordant) prior to staining with Masson's lung edema (in which case tissue slices may be 1-2 cm.
trichrome (for connective tissue), Mallory's aniline blue thick), with minimum squeezing and handling.
stain (for collagen), or phosphotungstic acid-hematoxylin Thinsections allow complete penetration by fixative in a
(PTAH) stain (for striated muscle). short time.
9. Purulent material, exudates or transudates should be
- Post-Chromatization is a form of secondary fixation marked and kept for possible cultures, smears and other
whereby a primarily fixed tissue is placed in aqueous bacteriologic examination.
solution of 2.5-3% potassium dichromate for 24 hours to 10. The amount of fixative must be adequate,
act as mordant for better staining effects and to aid in approximately 10-20 times the volume of the tissue
cytologic preservation of tissues. specimen except in osmium tetroxide which is very
expensive, requiring only 5-10 times that of tissue
- Washing out is the process of removing excess fixative volume for fixation.
from the tissue after fixation in order to improve 11. For prolonged fixation (e.g. museum preparation)
staining and remove artefacts from the tissues. Several volume of fixing fluid should not be less than 50 times
solutions may be used. that of the tissue.
12. Contamination of fixed tissue with precipitates (e.g. in
a. Tap water is used to remove: osmium tetroxide), should be avoided. In most
o excess chromates from tissues fixed in Kelly's, instances, fixed tissues must be washed thoroughly to
Zenker's, and Flemming's solutions remove salts and/or pigments before staining.
o excess formalin 13. The required period for fixation should not be exceeded
o excess osmic acid since this may cause excessive hardening or brittleness
b. 50-70% alcohol is used to wash out excess amount of of tissues.
picric acid (Bouin's solution). 14. Hollow organs (e.g. stomach, intestines) should be
c. Alcoholic iodine is used to remove excessive mercuric packed with cotton soaked in fixative or
fixatives. completelyopened before being immersed in adequate
fixing solution.
General Precautions in Handling and Fixation of 15. Air-filled lungs may float on fixative. To avoid this, the
Specimens organ may be covered with several layers of gauze to
1. All tissue specimens must be properly labeled and maintain it under surface.
identified. 16. Human brains may be suspended by a cord tied under
2. Surgical specimens should be fixed as soon as possible the Circle of Willis to prevent flattening. Intravascular
after removal, or refrigerated if fixation is to be delayed, perfusion (washing out of blood with Ringer's lactate)
maylead to artifact formation with loss of blood

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 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES
content. Dense tissues such as brains are poorly formulated fixatives should be prepared from stock
penetrated, hence require long fixation (usually 2 weeks solutions immediately before use because they are
prior to sectioning). unstable (e.g., Helly’s fluid).
17. Eyes should not be dissected before they are fixed since 31. If a specimen is received in fixative of dubious quality, it
this may lead to immediate tissue collapse and wrinkling must be replaced with fresh fixative.
due to the escape of vitreous humor. They are not, 32. Fixatives should be used only once. Specimens shed
however, easily penetrated due to tough sclera. Formal- cells and tissue fragments into the fixative solution
alcohol must be injected before immersing the organ in which could contaminate any subsequent specimen.
the fixative. 33. Avoid metal lids. Some fixatives are highly corrosive and
18. Frozen sections may lead to formation of ice crystal will attack metals (e.g., mercury salts).
artifacts. 34. Some fixatives require that specimens be washed in
19. To avoid rigor contraction and staining of artifacts when water prior to processing (e.g., Zenker’s or Helly’s) or
fixing muscle, the fresh biopsy material may be some other requirement may exist (phosphate may
stretched by sutures on each end, or laid flat in a moist precipitate from buffer in concentrations of alcohol of
filter paper before suspending in fixative. greater than 70%).
20. Water should not be used for glycogen-containing 35. All fixatives are toxic and irritant. Anyone using fixatives
tissues because glycogen is soluble in water. should be aware of their potential hazards.
21. To assure rapid access of the fixative to all parts of the
tissue, the tissue may be minced, that is, small pieces of Difficulties Caused by Improper Fixation:
specimen may be divided into small fragments and 1. Failure to arrest early autolysis of cells
transferred to the vial of a fixative by means of a tooth CAUSE: Failure to fix immediately (the tissue was
pick. probably allowed to dry before fixing); Insufficient
22. Hard tissues (e.g. cervix, uterine, fibroids, fixative
hyperkeratotic skin, fingernails, etc.) may be washed 2. Removal of substances soluble in fixing agent
out in running water overnight and immersed in 4% CAUSE: Wrong choice of fixative
aqueous phenol solution for 1-3 days (Lendrum's 3. Presence of artefact pigments on tissue sections
method). This will soften the tissue and allow easier CAUSE: Incomplete washing of fixative
sectioning without producing any marked distortion of 4. Tissues are soft and feather-like in consistency
the inner structures. CAUSE: Incomplete fixation
23. Presence of mucus prevents complete penetration of 5. Loss or inactivation of enzymes needed for study
fixative; hence, tissues that contain mucus are fixed CAUSE: Wrong choice of fixative
slowly and poorly. Excess mucus may be washed away 6. Shrinkage and swelling of cells and tissue structure
with normal saline solution. CAUSE: Overfixation
24. Fatty tissues should be cut in thin sections and fixed 7. Tissue blocks are brittle and hard
longer. CAUSE: Prolonged fixation
25. Tissues containing large amount of blood (e.g. blood
vessels and spleen) should be flushed out with saline Fixation Artifacts
before fixing. Formalin pigment is a well-known artifact that may be
26. The position or shape that is desired for sectioning produced under acid conditions. The pigment may be
should be maintained before fixation. For example, a eliminated or reduced by fixation in phenol - formalin. The
piece of intestine may be stretched out on a piece of use of neutral buffered formalin and phenol-formalin
paper and placed in the proper sized container, before almost completely stops the formation of formalin pigment
pouring the fixative over it. which occurs with nonbuffered formaldehyde solutions,
27. The fixative should penetrate from all sides. Always and also fixes tissues more rapidly.
place specimens into containers that already "Crush artifact" may be found in surgical specimens
containfixative. This will prevent adhesion of the particularly in liver biopsies, associated with an intense
specimen to the container. eosinophilic staining at the center of the tissue in H&E-
28. Where possible, hollow organs or specimens with stained sections. This may be due to partial coagulation of
natural cavities should be opened to allow immediate partially fixed protein by ethanol or by incomplete wax
access to the fixative. impregnation during subsequent histological processing.
29. Gentle agitation (swirling) of the specimen during its
first few minutes in fixative will facilitate penetration. Lipid Fixation
An adequate volume is vital (at least 20:1). An excess of In conventional histopathological techniques, lipids
fixative is required as its effective components may be are largely removed during preparation of tissues. Cryostat
depleted as part of the reactions of fixation. or frozen sections should be used for demonstrating lipid in
30. Fixatives should be carefully made from reagents of tissues, followed by a general lipid stain. Fixatives
suitable quality, and fresh if so specified. Poor quality containing mercuric chloride and potassium dichromate
reagents can produce poor quality fixation. Some can be effective for preservation of lipids in cryostat

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES
sections. In general, phospholipids, which contain amino instances, formalin-fixed and paraffin embedded sections
groups, are fixed by aldehydes. However, formaldehyde has may be used. In more sensitive cases, the tissue must be
been shown to react with unsaturated fatty acids during prepared as a cryostat section and fixation limited to a few
histological fixation, so that less lipid can be demonstrated seconds in absolute methanol or acetone. Insufficient
histochemically in tissue that has been stored in fixation will result in unfixed tissues being damaged by
formaldehyde for a long time. Baker's formal-calcium may dehydrating fixative effect of ethanol during processing,
be used to preserve phospholipids. Improved causing loss of immunohistochemical antigenicity and
ultrastructural demonstration of lipids has been achieved poorer microtomy. Antigen-retrieval techniques are
by post-fixing in imidazole osmium tetroxide. Cholesterol required for successful immunohistochemistry.
may be fixed with digitonin for ultrastructural
demonstration. Fixation for Immunohistochemistry
Fixation is often an important consideration when
Carbohydrate Fixation preserving tissue for immunohistochemistry. Some
Carbohydrates are hydrophilic; they hold much water antibodies cannot recognize antigens if they are cross-
in the extracellular space by hydrogen bonding. There is linked in a certain manner. Antigen retrieval may be used to
also, of course, much water inside cells, since water uncover antigens after fixation. To ensure free access of the
accounts for about 60% of the weight of the human body. antibody to its antigen, the cells must be fixed and
Alcoholic fixatives are generally recommended for glycogen permeabilized. In general, fixation strengths and times are
fixation. Glycogen can be demonstrated satisfactorily considerably shorter for cells than on the thicker,
enough for diagnosis, although losses of glycogen can be structurally complex tissue sections. For
high (60-80%) in aqueous solution. Alcoholic formaldehyde immunocytochemistry, sample preparation essentially
is a better fixative in human skin compared with neutral entails fixing the target cells to the slide. Perfect fixation
buffered formaldehyde. The most useful fixatives for would immobilize the antigens, while retaining authentic
preserving glycogen are alcohol-based, such as Rossman's cellular and subcellular architecture and permitting
fluid or cold absolute alcohol. This is essential when unhindered access of antibodies to all cells and subcellular
processing tissues from patients with glycogen storage compartments. Wide ranges of fixatives are commonly
disease. There is better retention of glycogen if the section used, and the correct choice of method will depend on the
is coated with celloidin. nature of the antigen being examined and on the
properties of the antibody used.
Protein fixation Fixation methods fall generally into two classes:
Neutral buffered formal saline or formaldehyde vapor organic solvents and crosslinking reagents. Organic solvents
are the most commonly used fixatives for amino acid such as alcohols and acetone remove lipids and dehydrate
histochemistry. the cells, while precipitating the proteins on the cellular
architecture. Cross-linking reagents (such as
Fixation for Electron Microscopy paraformaldehyde) form intermolecular bridges, normally
The most useful primary fixatives for electron through free amino groups, thus creating a network of
microscopy are osmium tetroxide, glutaraldehyde and linked antigens. Cross-linkers preserve cell structure better
paraformaldehyde, with the whole procedure performed at than organic solvents, but may reduce the antigenicity of
4°C. For routine studies, glutaraldehyde or osmium some cell components, and require the addition of a
tetroxide is adequate. For electron histochemistry and permeabilization step, to allow access of the antibody to
electron immunocytochemistry, Karnovsky's the specimen. Fixation with both methods may denature
paraformaldehyde-glutaral-dehyde is useful. Post-fixation protein antigens, and for this reason, antibodies prepared
may be achieved with osmium tetroxide. against denatured proteins may be more useful for cell
staining. The mechanism of fixation is dependent on the
Fixation for Enzyme Histochemistry reagent used. Alcohol based fixation dehydrates
The general aim of fixation for enzyme histochemistry cells/tissues, causing proteins to denature and precipitate
is to preserve the maximum enzyme activity at its original in situ. Paraformaldehyde causes covalent cross-links
localization, while also preserving structural integrity. The between molecules, effectively gluing them together into
tissue for study may be fixed in 4% formaldehyde or formal- an insoluble meshwork.
saline overnight, or fresh frozen cryostat sections may be Many researchers believe that tissue may be stored
fixed in acetone or formaldehyde and washed in distilled indefinitely in 10% neutral buffered formalin without harm.
water prior to enzyme staining. This is partly true; sections cut from such material and
stained with a routine histological stain (e.g., Hematoxylin
Fixation for Immunofluorescence and Eosin) will look fine. Problems, however, will likely arise
Immunofluorescence techniques are commonly used when such material is used for immunostaining since the
in pathology for the demonstration of various antibodies. cross-linking action continues and may irreversibly change
The preservation of antigenicity has become a very the conformation (i.e., reduce the antigenicity) of antibody
important consideration when choosing a fixative. In many targets. Do not over-fix the tissue if it is intended to be

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES
used for immunostaining. Fixation method, time, and Permeabilize with 0.5% Triton X-100 for 10 minutes.
temperature are variables that need to be considered when 8. Paraformaldehyde-Triton Fixation
developing immunohistochemistry protocols. Significantly Fix in 3-4% paraformaldehyde for 10-20 minutes.
altering fixation times between batches of specimens can Rinse briefly with PBS.
have major consequences on the quality of Permeabilize with 0.5% Triton X-100 for 10 minutes.
immunostaining. 9. Paraformaldehyde-Methanol Fixation
Tissue fixation will permeabilize the cells and allow Fix in 4% paraformaldehyde for 10-20 minutes.
antibodies to access intracellular structures. There are Rinse briefly with PBS.
several good reasons to use cryo-preservation rather than Permeabilize with cooled methanol for 5-10 minutes at
chemical fixation, including minimal alteration of the –20 °C.
structure of proteins to be identified by immunostaining.
Cryopreservation will avoid or reduce the ‘auto Practical Consideration:
fluorescence’ (i.e., high background) that is inherent to  Do not reuse or save diluted antibodies. The
formalin fixed paraffin embedded tissue when performing antibodies adhere to the walls of the container and
immunofluorescent microscopy. It promotes the this effectively reduces the titer to zero.
preservation of delicate enzymes that are easily destroyed  Freeze / thaw cycles will destroy antibodies.
by fixation. Lipid stains (e.g., Oil Red O) require frozen  'Dirty' (high background) polyclonal antibodies can
sections since the ethanols used in tissue processing will sometimes be 'cleaned up' by storing the antibody
extract the lipids. The morphology of cryosections, over pieces of fixed tissue. Irrelevant antibodies in the
however, is often inferior to paraffin sections. serum may be adsorbed onto the tissue, effectively
Formalin-fixed-paraffin-embedded tissue sections raising the titer of the specific antibodies. Another
typically 'auto fluoresce' when viewed under a fluorescent way to approach this is to incubate the antibody on a
microscope (some tissues are worse than others); for this tissue section and then reuse the antibody on a
reason, immunofluorescent (IF) staining is best done on second section.
frozen sections. Background auto fluorescence, to a greater
or lesser degree, can be suppressed by a post-staining Antigen Retrieval for Immunostaining
Sudan Black treatment. Antigen-retrieval methods in immunohistochemistry
have shown that some of the reactions of fixation are
The appropriate fixation method should be chosen reversible, particularly those of formaldehyde, but there is
according to the relevant application. considerable variation in the quality of antigen
preservation with various agents. However, these
1. Acetone Fixation techniques do not work for all antigens. There are a
Fix cells in -20°C acetone for 5-10 minutes. number of methods commonly used to restore
No permeabilization step needed following acetone antigenicity. These methods are not generally used when
fixation. working with cryo-preserved sections.
2. Methanol Fixation Microwave antigen retrieval is one of several so-called
Fix cells in -20°C methanol for 5-10 minutes. ‘HIER’ (heat induced epitope retrieval) methods. Others
Permeabilization step is needed following methanol include steaming, pressure cookers and autoclaves.
fixation. Typically, this type of retrieval involves boiling the slides in
3. Ethanol Fixation a pH 6 citric acid buffer for ten minutes, followed by an
Fix cells in cooled 95% ethanol, 5% glacial acetic acid for additional fifteen minutes in the hot buffer. There are
5-10 minutes. instances where changing the pH to 3 (in citric acid buffer)
4. Methanol-Acetone Fixation or to 8-9 (in a Tris EDTA buffer) are successful.
Fix in cooled methanol, 10 minutes at –20 °C. Partial digestion of tissue sections by enzymatic
Remove excess methanol. Permeabilize with cooled method with one (or several) enzymes can sometimes be
acetone for 1 minute at –20 °C. effective. Protocols are provided for pepsin, protease and
5. Methanol-Acetone Mix Fixation trypsin. The enzyme concentrations and incubation times
1:1 methanol and acetone mixture. given may have to be adjusted.
Make the mixture fresh and fix cells at -20 C for 5-10
minutes. Effect of heat during fixation
6. Methanol-Ethanol Mix Fixation When the temperature of a fixative is raised or
1:1 methanol and ethanol mixture. lowered (as is sometimes recommended for particular
Make the mixture fresh and fix cells at -20 C for 5-10 histochemical procedures), the rate of diffusion into the
minutes. specimen is affected, as is the rate of the chemical fixation
7. Formalin Fixation reactions occurring with the various tissue components.
Fix cells in 10% neutral buffered formalin for 5-10 Increasing temperature accelerates the process of fixation.
minutes. Excessive heat however, particularly if it is prolonged, can
Rinse briefly with PBS.

GEN. PATH | TRANSCRIBED BY: CANARIA, ALONZO, BANDAYREL, ESPIRITU, GALINATO, LAVARO, QUILALA, SIVILA
 MODULE 4: CONVENTIONAL TISSUE PROCESSING: FIXATION
1st SEMESTER | A.Y. 2022-2023
BSMLS 3A INSTRUCTOR: MR. RODS ANTHONY REYES
damage the cells and cause substantial shrinkage and composition, shape and number of containers (including
hardening of the specimen. cassettes), the agitation or movement of the containers,
Today, most laboratories carry out primary fixation of and the number, volume and dimensions of the specimens
specimens at ambient temperature and only after being fixed. After microwaving they should immediately be
specimens are loaded onto the processor. Temperatures of sssliced to 2 mm and placed in 70% ethanol. For
between 37°C and 45°C are commonly employed. microwave assisted fixation, 2 mm thick slices should be
Another problem encountered in using hot fixative prepared from tissues initially fixed in formalin prior to
solutions to initially fix larger specimens (greater than microwave treatment. Because of the many variables
3mm thick), is that the outside of the specimen fixes involved in microwave fixation, it is vital that every aspect
rapidly while it may take quite some time for the fixative of the technique is fully standardized (including consistent
to penetrate to the center of the block which then may be specimen dimensions), that the microwave oven is
poorly fixed or not fixed at all. The blocks will then show properly calibrated, and that the staff performing the
an exaggerated “zonal” fixation effect with different fixation step fully understands the factors that will
morphological and staining characteristics on the outside influence its outcome.
as compared to the inside of the specimen.
Advantages:
MICROWAVE FIXATION 1. The chief advantage of microwave fixation is that the
The introduction ·of commercial microwave ovens has tissue is heated right through the block in a very short
allowed easy access to controlled heating, thereby time, thereby potentially allowing the study of cellular
overcoming the previous problems of erratic heating processes that proceed very rapidly.
brought about by direct flaming. Microwave fixation allows 2. It is a non-chemical technique that is useful in
light microscopic techniques used in routine preserving neurochemical substances in brain, such
histopathology to be performed adequately. These include acetylcholine.
the standard stains, special stains, histochemistry for 3. It may also be used for the rapid fixation of routine
mucous substances, some enzyme histochemistry, and surgical specimens.
immuno-cytochemical techniques. 4. It significantly reduces the time taken for immuno-
Heat is considered to be the major factor responsible histochemistry and insitu hybridization.
for the effects of microwaves during tissue fixation. Apart 5. Microwave treated tissue (at 50oC), post-fixed in
from increasing diffusion rates, heat will increase osmium tetroxide also gives satisfactory results for
molecular kinetics and speed up chemical reactions. It is electron microscopy.
necessary to achieve a temperature in excess of 60°C
within the specimen for primary microwave fixation, while Disadvantages:
lower temperatures may be acceptable for microwave- 1. Microwaves generated by commercial ovens only
assisted fixation. penetrate tissue to a thickness of 10-15 mm.
Broadly, there are two ways in which microwave 2. There is no significant cross-linking of protein
technology is used for tissue fixation. Fresh tissue, placed molecules, and subsequent chemical fixation may be
in saline or other isotonic solution, can be irradiated to needed.
produce primary fixation (referred to as “microwave 3. Viable spores and other pathogens may remain in
fixation” or “microwave stabilization”). No chemical tissues processed with alcohol-based fixatives or
fixative is used at this stage. Alternatively, specimens can microwaving alone.
be placed in buffered formalin or some other fixative and,
at a later stage, microwaved to assist the fixative action of FIXATIVE:
the fixing agent (referred to as ‘microwave-assisted M Phosphate buffered, 40% Formaldehyde (10%
fixation”). In this latter case, the microwaving may be formalin buffered to pH 7.3)
carried out while the tissue is in fixative, in which case Expose tissue to formaldehyde, followed by 1.5
there may be some hazard from toxic fumes produced, or minutes of microwave.
the tissue may be transferred back to saline or buffer for
the microwave step. Either way, the fixative solution must PROCEDURE:
be present within the tissue for microwave-assisted 1. Fix tissue (no more than 5 mm) in formalin solution for
fixation to occur. Microwave-assisted fixation is much 4 hours.
more commonly used than primary microwave fixation. 2. Soak blocks in water at room temperature for 1
Proprietary fixatives of relatively low toxicity containing minute in 100 ml of formalin.
glyoxal, have been developed for use in microwave- 3. Place in microwave, 450 watts, at 55°C for 1.5 to 4
assisted fixation. minutes.
The rate at which the microwave energy will generate 4. Remove blocks and slice tissue to 2 mm thick.
heat during tissue fixation depends on a number of factors 5. Place directly in alcohol.
including the power setting and power output of the oven,
the volume and nature of the holding solution, the

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