MLS3B HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES
(Lect)
TOPIC 5 : CHEMICAL FIXATIVES
Instructor: Dr. Marissa A. Cauilan
solubility of protein molecules and (often) by
disrupting the hydrophobic interactions that give
many proteins their tertiary structure.
TABLE OF CONTENTS
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
X.
XI.
XII.
Chemical Fixatives
Cross-linking fixatives - aldehydes
A. Formaldehyde and formalin
B. Buffered formalin
C. Factors that influence formalin fixation
D. 10% formol-saline
E. 10% neutral buffered formalin
F. Zinc formalin
G. Formol-sublimate
H. Paraformaldehyde
I. Karnovsky’s fixative
J. Glutaraldehyde
Precipitating (Alcoholic) Fixatives
A. Methyl alcohol
B. Isopropyl alcohol
C. Ethyl alcohol
D. Carnoy’s fixative
E. Clarke’s solution
F. Alcoholic formalin
G. Formol acetic alcohol
H. Gendre’s fixative
I. Newcomer’s fluid
Metallic Fixatives
A. Mercuric chloride
B. Zenker’s solution
C. Helly’s solution
D. Lillie’s B-5 fixative
E. Heidenhain’s Susa
Oxidizing Agents
A. Osmium tetroxide
B. Fleming’s solution
Chromate fixatives
A. Chromic acid
B. Potassium dichromate
C. Muller’s fluid
D. Orth’s fluid
Picric acid fixatives
A. Bouin’s solution
B. Hollande’s solution
C. Brasil’s fixative
Glacial acetic acid
Lead fixatives
Trichloroacetic acid
Acetone
Michel’s solution
CROSS-LINKING FIXATIVES - ALDEHYDES
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FORMALDEHYDE
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Tissue preservation requires the use of a fixative that can
stabilize the proteins, nucleic acids and internal
components of the tissue by making them Insoluble.
●
Crosslinking Fixatives (e.g., Aldehydes) that
act by creating covalent chemical bonds
between proteins in tissue. This anchors soluble
proteins to the cytoskeleton, and lends
additional rigidity to the tissue.
Precipitating (or denaturing) fixatives (e.g.,
alcoholic fixatives) that act by reducing the
Pure stock solution of 40% formalin is
unsatisfactory for routine fixation since high
concentrations of formaldehyde tend to
over-harden the outer layer of the tissue and
affect staining adversely.
made with formaldehyde but the percentage
denotes a different formaldehyde concentration.
It is recommended that laboratories avoid the
use of concentrated formalin solutions by
purchasing
commercially
prepared
10%
formalin.
A. Buffered Formalin
●
●
gas produced by the oxidation of methyl alcohol,
and is soluble in water to the extent of 37-40%
weight in volume.
The commercially available solution of
formaldehyde contains 35-40% gas by weight
FORMALIN
CHEMICAL FIXATIVES
2 Major Groups:
most commonly used fixative in histology which
fixes the tissues by forming cross-linkages in the
proteins, particularly between lysine residues.
good for immunohistochemical techniques and
formaldehyde vapor can be used as a fixative for
cell smears.
The standard solution is 10% neutral buffered
formalin
or
approximately
3.7%-4.0%
formaldehyde in phosphate buffered formalin or
approximately 3.7%-4.0% formaldehyde in
phosphate-buffered saline.
10% neutral buffered formalin - most widely
used fixative for routine histology buffered to pH
7 with phosphate buffer.
effectively prevent autolysis and provide
excellent preservation of tissue and cellular
morphology
Advantages of using Formalin
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It is cheap, readily available, easy to prepare,
and relatively stable, especially if stored in
buffered solution.
It is compatible with many stains, and therefore
can be used with various staining techniques
depending upon the need of the tissues.
It does not over-harden tissues, even with
prolonged periods of fixation, as long as
solutions are regularly changed.
It penetrates tissues well.
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HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES|TOPIC 5: CHEMICAL FIXATIVES
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It preserves fat and mucin, making them
resistant to subsequent 100 treatment with fat
solvents, and allowing them to be stained for
demonstration.
It preserves glycogen.
It preserves but does not precipitate proteins,
thereby allowing tissue enzymes to be studied. It
does not make tissues brittle, and is therefore
recommended for nervous tissue preservation.
It allows natural tissue colors to be restored after
fixation by immersing formalin-fixed tissues in
70% alcohol for one hour, and is therefore
recommended for colored tissue photography.
It allows frozen tissue sections to be prepared
easily.
It does not require washing out, unless tissues
have stayed in formalin for excessively long
periods of time.
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Volume of Fixative
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Fumes are irritating to the nose and eyes and
may cause sinusitis, allergic rhinitis, or
excessive lacrimation.
The solution is irritating to the skin and may
cause allergic dermatitis on prolonged contact.
It may produce considerable shrinkage of
tissues.
It is a soft fixative and does not harden some
cytoplasmic structures adequately enough for
paraffin embedding.
If unbuffered:
a. Formalin reduces both basophilic and
eosinophilic staining of cells, thereby reducing
the quality of routine cytologic staining. Acidity of
formic acid may, however, be used to an
advantage
when
applying
the
silver
impregnation technique of staining.
b. It forms abundant brown pigment granules
on blood-containing tissues, e.g., spleen, due to
blackening of hemoglobin.
Prolonged fixation may produce:
○ Bleaching of the specimen and loss of
natural tissue colors.
○ Dispersal of fat from the tissue into the
fluid.
○ Dissolution or loss of glycogen, and
urate crystals
FACTORS THAT INFLUENCE FORMALIN FIXATION
Post-Mortem or Post-Surgical Interval
●
If there is a necessary delay in fixation, the
tissue
should
be
immersed
in
cold
phosphate-buffered saline (PBS). Tissues
should not be allowed to dry 101 before (or
after) fixation. For certain studies, vascular
perfusion with fixative may be recommended.
Tissues should be immersed in a 10-20-fold
volume of fixative.
Fixation Time
Disadvantages of using Formalin
●
Formaldehyde fixation is usually optimal near
physiological
pH
and
ionic
strength.
Unfortunately, formalin is not stable and will
gradually acidify to form more complex polymers
which can have adverse effects on the tissue.
This can be minimized by appropriate buffering
and addition of small amounts of methanol.
Fixation with 10% NBF at room temperature for
a minimum of 24 hours at room temperature is
recommended in most instances.
Some bloody, fatty or fetal tissues may require
significantly longer fixation, e.g., up to 48- 72
hours.
Temperature
●
An increase in temperature can increase the
rate of fixation but can also increase the rate of
autolysis.
Tissue Thickness
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tissues should be no thicker than 3-5 mm
Careful slicing of tissues and solid organs before
transferring them to fixative can greatly influence
the efficiency of fixation by increasing exposed
surface area and decreasing total thickness.
Post-Fixation Storage
●
If there is to be a delay in processing after
complete fixation (usually 24 hours or more), the
fixed tissue can be stored for up to 3 days in the
cold in 70% ethanol. However, it is essential that
the tissue is completely fixed prior to transfer to
the alcohol.
MIXTURE OF FIXATIVES
Two aldehyde fixative mixtures have been particularly
useful for electron cytochemistry. The best known mixture
of
fixative
is
Karnovsky's
paraformaldehyde-glutaraldehyde solution. Acrolein is
another aldehyde which has been introduced as a
mixture with glutaraldehyde or formaldehyde.
Precautions:
1. Prolonged storage of formaldehyde, especially at very
low temperature, may induce precipitation of white
para-formaldehyde deposits and produce turbidity
although this, in itself, does not impair the fixing property
of the solution. Precipitates may be removed by filtration
or by addition of I0% methanol.
Composition of Fixative
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HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES|TOPIC 5: CHEMICAL FIXATIVES
2. Methanol added as a preservative to formaldehyde will
prevent its decomposition to formic acid or precipitation to
paraformaldehyde, but it serves to denature protein,
thereby rendering formalin unsuitable as a fixative for
electron microscopy.
3. Concentrated solutions of formaldehyde must NEVER
be neutralized since this might cause violent explosions.
4. Room should be properly ventilated with adequate
windows and preferably with an exhaust fan to prevent
inhalation of fumes and consequent injury to the eyes and
nose.
5. Dermatitis may be avoided by the use of rubber gloves
when handling specimens fixed in formalin.
6. The bleaching of tissues may be prevented by
changing the fluid fixative every three months.
7. Natural tissue colors may be restored by immersing
tissues in 70% alcohol after fixation.
8. Brown or black crystalline precipitate formed by the
action of formic acid with blood can be removed from the
sections prior to staining by treatment with saturated
alcoholic picric acid or a 1% solution of potassium
hydroxide in 80% alcohol. The use of neutral (phosphate)
buffered formalin will prevent the pigmentation.
9. If fatty tissues are to be stored for a long time,
cadmium or cobalt salts can be added to prevent
dispersion of fat out into the fluid.
10. After use, formalin should be collected, sealed in
appropriately labelled containers and disposed of by a
commercial waste service.
11. All empty containers should be washed thoroughly
with water.
12. Formalin should not be released into soil, drains and
waterways.
13. Acid reaction due to formic acid formation can be
buffered or neutralized by adding magnesium carbonate
or calcium in a wide-mouth bottle to prevent violent
explosion due to insufficient gas space for CO2 release.
14. Calcium acetate may be used to buffer formalin but it
leaves a calcium deposit. The greatest amount of calcium
deposit appears wherever the tissue is most exposed to
the fixative (i.e., periphery of the block, within and around
the walls of blood vessels, or in the proximity of hollow
structures).
15. To improve staining and produce firmer and harder
consistency, tissues fixed in formalin for 1 -2 hours may
be placed again in Helly's fluid for 4-6 hours or in
formol-sublimate for 4-16 hours (secondary fixation).
16. Formic acid develops over time producing an acidic
solution, requiring a buffer to remain neutral pH (7.0).
17. Acidic formalin causes hematin pigment deposition in
tissues particularly in hematogenous tissue after storage
for extended periods of time. Methods are available to
remove hematin but it is better to prevent its deposition
by maintaining a neutral pH.
20. If post-fixed in osmic acid, the tissue must not be
washed in demineralized water to prevent hypotonicity
and bleaching.
22. Fixation of tissue blocks not exceeding 5 mm. in
thickness is usually complete in 6-12 hours at room
temperature.
10% FORMAL-SALINE
FORMULA:
● 40% formaldehyde: 100 ml
● Distilled water: 900 ml
● Sodium dihydrogen phosphate monohydrate: 4
gm
● Disodium hydrogen phosphate anhydrous 6.5
gm
The solution should have a pH of 6.8
Fixation time: 12 – 24 hours
Recommended Application:
● Widely used for routine histopathology prior to
the introduction of phosphate buffered formalin.
● It is recommended for fixation of central
nervous tissues and general post-mortem
tissues for histochemical examination. It is
also recommended for the preservation of lipids,
especially phospholipids.
● Tends to prevent the formation of formalin
pigment.
Advantages
1.
2.
3.
4.
5.
6.
7.
8.
It penetrates and fixes tissues evenly.
It preserves microanatomic and cytologic details
with minimum shrinkage and distortion.
Large specimens may be fixed for a long time
provided that the solution is changed every three
months.
It preserves enzymes and nucleoproteins.
It demonstrates fats and mucin.
It does not over-harden tissues, thereby
facilitating dissection of the specimen.
It is ideal for most staining techniques, including
silver impregnation.
It allows natural tissue color to be restored upon
immersion in 70%alcohol.
Disadvantages
Similar to formaldehyde with ff. additions
1.
2.
3.
It is a slow fixative. The period of fixation is
required to be 24 hours or longer.
Formal-saline fixed tissues tend to shrink during
alcohol dehydration; this may be reduced by
secondary fixation.
Metachromatic reaction of amyloid is reduced.
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HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES|TOPIC 5: CHEMICAL FIXATIVES
4.
Acid dye stains less brightly than when fixed
with mercuric chloride.
4.
5.
10% NEUTRAL-BUFFERED FORMALIN
FORMULA:
Mix together:
● Sodium Dihydrogen Phosphate (Na2HPO4),
anhydrous, 6.5 gm
● Sodium Dihydrogen Phosphate (NaH2PO4•H20)
4 gm
● Distilled water 900 ml
○ Adjust pH to 7.4, then add:
● 40% formaldehyde 100 ml
Advantages
Similar to Formal-Saline with ff. additions:
1.
2.
3.
It prevents precipitation of acid formalin
pigments on postmortem tissue.
It is the best fixative for tissues containing iron
pigments and for elastic fibers which do not stain
well after Susa, Zenker’s or chromate fixation.
It requires no post-treatment after fixation and
goes directly into 80% alcohol for processing.
6.
Disadvantages
1.
2.
3.
4.
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Disadvantages
1.
2.
3.
4.
5.
It is longer to prepare; hence, is
time-consuming.
Positivity of mucin to PAS is reduced.
It may produce gradual loss in basophilic
staining of cells.
Reactivity of myelin to Weigert's iron
hematoxylin stain is reduced.
It is inert towards lipids, especially neutral fats
and phospholipids.
ZINC FORMALIN (UNBUFFERED)
FORMULA:
● Zinc sulphate 1 gm
● Deionized water 900 ml
○ Stir until dissolved, then add –
● 40% formaldehyde 100 m
Recommended Applications:
● Devised as alternatives to mercuric chloride
formulations. They are said to give improved
results with immunohistochemistry.
FORMOL-CORROSIVE (FORMOL-SUBLIMATE)
FORMULA:
● Sat. Aq. Mercuric chloride 90 ml.
● Formaldehyde 40% 10 ml.
Fixation time: 3-24 hours
Formol-mercuric chloride solution is
recommended for routine post-mortem tissues.
It penetrates small pieces of tissues rapidly.
It produces minimum shrinkage and hardening.
It is excellent for many staining procedures
including silver reticulum methods.
Penetration is slow; hence, tissue sections
should not be more than 1 cm thick.
It forms mercuric chloride deposits.
It does not allow frozen tissue sections to be
made.
It inhibits the determination of the extent of
tissue decalcification.
PARAFORMALDEHYDE
Polymerized form of formaldehyde, usually
obtained as a fine white powder, which
depolymerizes back to formalin when heated.
Suitable for paraffin embedding and sectioning,
and also for immunocytochemical analysis.
Allows for subsequent immuno-detection of
certain antigens and should therefore be used
when the objective is to study morphology and
protein expression simultaneously.
Other benefits include: Long term storage and
good tissue penetration.
KARNOVSKY’S FIXATIVE(4% Paraformaldehyde1% Glutaraldehyde in 0.1M Phosphate Buffer)
● Mixture of paraformaldehyde and
glutaral-dehyde.
● Suitable for use when preparing samples for
light microscopy in resin embedding and
sectioning, and for electron microscopy.
To prepare Karnovsky’s Fixative (for 100 ml add the
following together)
FORMULA:
● 8 % paraformaldehyde 25 ml
● 25 % glutaraldehyde 10 ml
● 0.2 M phosphate buffer 50 ml.
● Make up to 100 ml with distilled water.
This is to be used fresh.
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Advantages
1.
2.
3.
It brightens cytoplasmic and metachromatic
stains better than with formalin alone.
Cytological structures and blood cells are well
preserved. There is no need for "washing-out".
Tissues can be transferred directly from fixative
to alcohol.
It fixes lipids, especially neutral fats and
phospholipids.
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GLUTARALDEHYDE
Made up of two formaldehyde residues, linked
by a three carbon chain.
Glutaraldehyde fixation causes rapid and
irreversible changes, fixes quickly, is well suited
for electron microscopy, it fixes well at 4C, and it
gives best overall cytoplasmic and nuclear
detail.
The tissue must be as fresh as possible and
preferably sectioned and fixed in glutaraldehyde
at a thickness of no more than 1 mm to enhance
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HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES|TOPIC 5: CHEMICAL FIXATIVES
fixation. It penetrates very poorly, but gives best
overall cytoplasmic and nuclear detail.
2.
Advantages of Glutaraldehyde over Formalin:
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1.
2.
3.
4.
5.
6.
It has a more stable effect on tissues, giving a
firmer texture with better tissue sections,
especially of central nervous tissues.
It preserves plasma proteins better.
It produces less tissue shrinkage.
It preserves cellular structures better; hence, is
recommended for electron microscopy.
It is more pleasant and less irritating to the nose.
It does not cause dermatitis.
Disadvantages of Glutaraldehyde over Formalin:
1.
2.
3.
4.
5.
It is more expensive.
It is less stable.
It penetrates tissues more slowly.
It tends to make tissue (i.e. renal biopsy) more
brittle.
It reduces PAS positivity of reactive mucin. This
may be prevented by immersing
glutaraldehyde-fixed tissues in a mixture of
concentrated glacial acetic acid and aniline oil.
Precautions:
1. The specimen vial must be kept refrigerated during the
fixation process.
2. Solution may be changed several times during fixation
by swirling the vials to make sure that the specimen is in
contact with fresh solution all the time.
PRECIPITATING (ALCOHOLIC) FIXATIVES
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●
Alcohols are protein denaturants and are not
used routinely for tissues because they cause
too much brittleness and hardness. The protein
denaturants - methanol, ethanol and acetone are rarely used alone for fixing blocks unless
studying nucleic acid. They are also very good
for cytologic smears because they act quickly
and give good nuclear detail.
Alcohol rapidly denatures and precipitates
proteins by destroying hydrogen and other
bonds. It must be used in concentrations ranging
from 70 to 100% because less concentrated
solutions will produce lysis of cells. Ethanol
(95%) is fast and cheap.
Methyl Alcohol 100%
Advantages:
1. It is excellent for fixing dry and wet smears,
blood smears and bone marrow tissues.
2. It fixes and dehydrates at the same time.
Disadvantages:
1. Penetration is slow.
If left in fixative formorethan48hours,issues may
be over-hardened and difficult to cut.
Isopropyl Alcohol
is used for fixing touch preparations, although
some touch preparation are air dried and not
fixed, for certain special staining procedures
such as Wright-Giemsa.
Ethyl Alcohol
Is used at concentrations of 70-100%. If the
lower concentrations are used, the RBC's
become
hemolyzed
and
WBC's
are
inadequately preserved. It may be used as a
simple fixative. It is, however, more frequently
incorporated into compound fixatives for better
results.
Fixation Time: 18-24 hours
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Advantages
1.
2.
3.
4.
5.
6.
It preserves but does not fix glycogen.
It fixes blood, tissue films and smears.
It preserves nucleoproteins and nucleic acids,
hence, are used for histochemistry, especially
for enzyme studies.
It fixes tissue pigments fairly well.
It is ideal for small tissue fragments.
It may be used both as a fixative and
dehydrating agent.
Disadvantages
1.
2.
3.
4.
5.
6.
7.
8.
Hemosiderin preservation is less than in
buffered formaldehyde.
It is a strong reducing agent; hence, should not
be mixed with chromic acid, potassium
dichromate and osmium tetroxide which are
strong oxidising agents.
Lower concentrations (70-80%) will cause RBC
hemolysis
and inadequately preserve
leukocytes.
It dissolves fats and lipids, as a general rule.
Alcohol-containing fixatives are contraindicated
when lipids are to be studied.
It causes glycogen granules to move towards
the poles or endsof the cells (polarization).
Tissue left in alcohol too long will shrink, making
it difficult or impossible to cut.
It causes polarization of glycogen granules.
It produces considerable hardening and
shrinkage of tissue
Carnoy’s Fixative
FORMULA:
●
Absolute alcohol 60 ml.
●
Chloroform 30 ml.
● Glacial acetic acid 10 ml.
Fixation Time: 1-3 hours
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HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES|TOPIC 5: CHEMICAL FIXATIVES
sometimes used during processing to complete fixation
following incomplete primary formalin fixation. It can be
used for fixation or post-fixation of large fatty specimens
(particularly breast), because it will allow lymph nodes to
be more easily detected as it clears and extracts lipids. If
used for primary fixation specimens, it can be placed
directly into 95% ethanol for processing.
Advantages
1. It is considered to be the most rapid fixative and may
be used for urgent biopsy specimens for paraffin
processing within 5 hours.
2. It fixes and dehydrates at the same time.
3. It permits good nuclear staining and differentiation.
4. It preserves Nissl granules and cytoplasmic granules
well.
5. It preserves nucleoproteins and nucleic acids.
6. It is an excellent fixative for glycogen since aqueous
solutions are avoided.
7. It is very suitable for small tissue fragments such as
curettings and biopsy materials.
8. Following fixation for one hour, tissues may be
transferred directly to absolute alcohol-chloroform
mixture, thereby shortening processing time.
9. It is also used to fix brain tissue for the diagnosis of
rabies.
Formol-acetic alcohol
FORMULA:
● Ethanol absolute: 85 ml
● 40% formaldehyde: 10 ml
● Acetic acid glacial: 5 ml
Fixation time: 1 - 6 hours
Recommended Applications
Formol-acetic acid alcohol is a faster-acting agent than
alcoholic formalin due to the presence of acetic acid that
can also produce formalin pigment. It is sometimes used
to fix diagnostic cryostat sections. If used for primary
fixation, the specimens can be placed directly into 95%
ethanol for processing.
Disadvantages
1. It produces RBC hemolysis, dissolves lipids and can
produce excessive hardening and shrinkage.
2. It causes considerable tissue shrinkage.
3. It is suitable only for small pieces of tissues due to slow
penetration.
4. It tends to harden tissues excessively and distorts
tissue morphology.
5. It dissolves fat, lipids, and myelin.
6. It leads to polarization unless very cold temperatures
(-70°C) are used.
7. It dissolves acid-soluble cell granules and pigments.
Gendre's Fixative
FORMULA:
● 95% Ethyl alcohol saturated with picric acid 80
ml.
● Strong formaldehyde solution 15 ml.
● Glacial acetic acid 5 ml.
Post-fixation with phenol-formalin for 6 hours or
more can enhance immunoperoxidase studies on the
tissues, and in some cases, electron microscopy, if it is
necessary at a later time to establish a diagnosis.
Clarke’s solution
FORMULA:
● Ethanol (absolute) 75 ml
● Acetic acid glacial 25 ml 112
Fixation time: 3 - 4 hours
Advantages
Recommended Applications
Clarke’s solution has been used on frozen sections and
smears. It can produce fair results after conventional
processing if fixation time is kept very short. It preserves
nucleic acids but extracts lipids. Tissues can be
transferred directly into 95% ethanol.
Alcoholic formalin
FORMULA:
● 40% Formaldehyde: 100 ml
● 95% Ethanol: 900 ml
● 0.5 g calcium acetate can be added to ensure
neutrality
Fixation time: 12 – 24 hours
Recommended Applications:
Alcoholic-formalin combines a denaturing fixative with the
additive and cross-linking effects of formalin. It is
1. Fixation is faster (fixation time is reduced to one-half).
2. It can be used for rapid diagnosis because it fixes and
dehydrates at the same time, e.g., in the frozen section
room.
3. It is good for preservation of glycogen and for
micro-incineration technique (the burning of a minute
tissue specimen for identification of mineral elements
from the ashes).
4. It is used to fix sputum, since it coagulates mucus.
Disadvantages
1. It produces gross hardening of tissues.
2. It causes partial lysis of RBC.
3. Preservation of iron-containing pigments is poor.
4. Formaldehyde does not give as good a morphological
picture as glutaraldehyde.
5. Formaldehyde causes little cross-linking under usual
fixation conditions where low concentrations of proteins
are used, while glutaraldehyde is most effective at
cross-linking.
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HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES|TOPIC 5: CHEMICAL FIXATIVES
Newcomer's Fluid
FORMULA:
● Isopropyl alcohol 60 ml.
● Propionic acid 30 ml.
● Petroleum 30 ml. Ether 10 ml.
● Acetone 10 ml. Dioxane 10 ml.
Fixation time: 12-18 hours at 3°C
Advantages
1. It is recommended for fixing mucopolysaccharides and
nuclear proteins.
2. It produces better reactions in Feulgen stains than
Carnoy's fluid.
3. It acts both as a nuclear and histochemical fixative.
METALLIC FIXATIVES
Mercurials fix tissues through an unknown
mechanism that increases staining brightness and gives
excellent nuclear detail. However, mercurials 114
penetrate poorly and produce tissue shrinkage. Their best
application is for fixation of hematopoietic and
reticuloendothelial tissue.
●
●
1. Mercuric Chloride
Mercuric chloride is the most common metallic
fixative, frequently used in saturated aqueous
solutions of 5-7%. Mercuric chloride is widely
used as a secondary fixative reacting with a
number
of amino acid residues and
accompanied by spectroscopic changes,
probably due to reaction with histidine residues.
Mercuric chloride-based fixatives are used as an
alternative to formaldehyde-based fixatives to
overcome poor cytological preservation and
include such well-known fixatives as B-5 and
Zenker's solution. They penetrate relatively
poorly and cause some tissue hardness, but
give excellent nuclear detail.
Mercuric chloride penetrates poorly and
produces shrinkage of tissues, so it is usually
combined with other fixative agents.
Advantages
1. It penetrates and hardens tissues rapidly and well.
2. Nuclear components are shown in fine detail.
3. It precipitates all proteins.
4. It has a greater affinity to acid dyes and is preferred in
lieu of formaldehyde for cytoplasmic staining.
116-121
Disadvantages
1. It causes marked shrinkage of cells (this may be
counteracted by addition of acid).
2. It rapidly hardens the outer layer of the tissue with
incomplete fixation of the center; therefore, thin sections
should be made.
3. Penetration beyond the first 2-3 millimeters is slow;
hence, not more than 5 mm. thickness of tissues should
be used.
4. If left in fixative for more than 1-2 days, the tissue
becomes unduly hard and brittle.
5. It prevents adequate freezing of fatty tissues and
makes cutting of frozen tissues difficult.
6. It causes considerable lysis of red blood cells and
removes much iron from hemosiderin.
7. It is inert to fats and lipids.
8. It leads to the formation of black granular deposits in
the tissues.
9. It reduces the amount of demonstrable glycogen.
10. Compound solutions containing mercuric chloride
deteriorate rapidly upon addition of glacial acetic acid to
formalin.
11. It is extremely corrosive to metals.
Zenker’s Solution
FORMULA:
● Mercuric chloride 5 gm
● Potassium dichromate 2.5 gm
● Distilled water 100 ml
● Acetic acid, glacial 5 ml (to be added just before
use)
○ Heat, cool, filter in brown bottle. Wash
sample for 24 hours with distilled water
after fixation.
Fixation time: 12-24 hours
Advantages
1. It produces a fairly rapid and even fixation of tissues.
2. Stock solutions keep well without disintegration.
3. It is recommended for trichrome staining.
4. It permits brilliant staining of nuclear and connective
tissue fibers.
5. It is recommended for congested specimens (such as
lung, heart and blood vessels) and gives good results
with PTAH and trichrome staining.
6. It is compatible with most stains.
7. It may act as a mordant to make certain special
staining reactions possible.
8. It is a stable fixative that can be stored for many years.
Disadvantages
1. Penetration is poor.
2. It is not stable after addition of acetic acid.
3. Prolonged fixation (for more than 24 hours) will make
tissues brittle and hard.
4. It causes lysis of red blood cells and removes iron from
hemosiderin.
5. It does not permit cutting of frozen sections.
6. It has the tendency to form mercuric pigment deposits
or precipitates.
7. Tissue must be washed in running water for several
hours (or overnight) before processing. Insufficient
washing may inhibit or interfere with good cellular
staining.
● Mercuric deposits may be removed by
immersing tissues in alcoholic iodine solution
prior to staining, through a process known as
dezenkerization.
Chemically, de-zenkerization is done by oxidation with
iodine to form mercuric iodide, which can be
subsequently removed by treatment with sodium
thiosulfate, using the following procedure:
Novesteras, Rivera, Nicolas, Dumayas, Suyam, Waminal | 7
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES|TOPIC 5: CHEMICAL FIXATIVES
1. Bring slides to water.
2. Immerse in Lugol's iodine (5 minutes).
3. Wash in running water (5 minutes).
4. Immerse in sodium thiosulfate 5% (5 minutes).
5. Wash in running water (5 minutes).
6. Proceed with required water soluble stain.
● 40% formaldehyde: 2 ml
Fixation time: 4 – 8 hours
Zenker-Formol (Helly’s) Solution
FORMULA:
● Mercuric chloride, 5 gm
● Potassium dichromate, 2.5 gm
● Distilled water, 100 ml
● 40% formaldehyde 5 ml (to be added
immediately before use)
● Heat, cool, filter in brown bottle.
● Wash sample for 24 hours with distilled water
after fixation.
Fixation time: 4 – 24 hours
FORMULA:
● Mercuric chloride 45 gm
● Sodium chloride 5 gm
● Trichloroacetic acid 20 gm
● Glacial acetic 40 ml
● Acid Formaldehyde 40% 200 ml
● 40% Distilled water 800 ml
Fixation time: 3-12 hours
Zenker’s solution is an excellent fixative for bone marrow,
extramedullary hematopoiesis and intercalated discs of
cardiac muscle. However, it produces mercury pigment
Never use metal forceps to handle tissue.
Advantages
1. It is an excellent microanatomic fixative for pituitary
gland, bone marrow and blood containing organs such as
spleen and liver.
2. It penetrates and fixes tissues well.
3. Nuclear fixation and staining with Helly’s solution is
better than with 118 Zenker's.
4. It preserves cytoplasmic granules well.
Disadvantages
The disadvantages of Helly's solution are similar to
Zenker's except that brown pigments are produced if
tissues (especially blood containing organs) are allowed
to stay in the fixative for more than 24 hours due to RBC
lysis. This may be removed by immersing the tissue in
saturated alcoholic picric acid or sodium hydroxide.
Lillie’s B-5 Fixative
● 4% aqueous formaldehyde with 0.22M mercuric
chloride and 0.22M acetic acid.
● This mixture enhances nuclear detail, which is
important for identifying normal and abnormal
cell types in bone marrow (hematopoietic tissue)
specimens.
● A dirty looking brown crystalline precipitate,
probably mercurous chloride (Hg2Cl2) forms in
all parts of tissues fixed in mixtures containing
HgCl2 (Mercury pigment)
○ Removed by treatments with iodine and
sodium thiosulfate solutions. Because it
contains mercury, B-5 is subject to toxic
waste disposal regulations
FORMULA:
● B-5 Stock solution
● Mercuric chloride: 12 g
● Sodium acetate anhydrous: 2.5 g
● Distilled water: 200 ml
● Working solution: (prepare immediately before
use)
● B-5 stock solution: 20 ml
Heidenhain’s Susa Solution
Recommended mainly for tumor biopsies especially of
the skin; it is an excellent cytologic fixative.
Advantages
1. It penetrates and fixes tissues rapidly and evenly.
2. It produces minimum shrinkage and hardening of
tissues due to the counter-balance of the swelling effects
of acids and the shrinkage effect of mercury.
3. It permits most staining procedures to be done,
including silver impregnation, producing brilliant results
with sharp nuclear and cytoplasmic details.
4. It permits easier sectioning of large blocks of fibrous
connective tissues.
5. Susa-fixed tissues may be transferred directly to 95%
alcohol or absolute alcohol, thereby reducing processing
time.
Disadvantages
1. Prolonged fixation of thick materials may produce
considerable shrinkage, hardening and bleaching; hence,
tissues should not be more than 1 cm. thick.
2. RBC preservation is poor.
3. Some cytoplasmic granules are dissolved.
4. Mercuric chloride deposits tend to form on tissues;
these may be removed by immersion of tissues in
alcoholic iodine solution.
5. Weigert's method of staining elastic fibers is not
possible in Susa fixed tissues.
After using Heidenhain's Susa fixative, the tissue should
be transferred directly to a high-grade alcohol, e.g. 96%
or absolute alcohol, to avoid undue swelling of tissues
caused by treatment with low-grade alcohol or water.
OXIDIZING AGENTS
●
●
Can react with various side chains of proteins
and other biomolecules, allowing formation of
crosslinks that stabilize tissue structure but
cause extensive denaturation despite preserving
fine cell structure
Used mainly as secondary fixative.
Osmium Tetroxide (Osmic Acid; OsO4)
●
●
a pale yellow powder which dissolves in water
(up to 6% at 20°C) to form a strong oxidizing
solution
traditionally used in electron microscopy both as
a fixative and a heavy metal stain.
Novesteras, Rivera, Nicolas, Dumayas, Suyam, Waminal | 8
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES|TOPIC 5: CHEMICAL FIXATIVES
●
●
a good fixative and excellent stain for lipids in
membranous structures and vesicles.
The most prominent staining in adherent human
cells (HeLa) is seen on lipid droplets
wrapped in cotton gauze and suspended in the fluid by
means of a thread.
7. Osmic acid-fixed tissues must be washed in running
water for at least 24 hours to prevent formation of
artefacts.
Advantages
1. It fixes conjugated fats and lipids permanently by
making them insoluble during subsequent treatment with
alcohol and xylene. Fats form hydrated osmium dioxide,
are stained black and therefore are easier to identify.
2. It preserves cytoplasmic structures well, e.g. Golgi
bodies and mitochondria.
3. It fixes myelin and peripheral nerves well, hence, it is
used extensively for neurological tissues.
4. It produces brilliant nuclear staining with safranin.
5. It adequately fixes materials for ultrathin sectioning in
electron microscopy, since it rapidly fixes small pieces of
tissues and aids in their staining.
6. It precipitates and gels proteins.
7. It shows uniformly granular nuclei with clear
cytoplasmic background.
8. Some tissues (e.g. adrenal glands) are better fixed in
vapor form of osmium tetroxide. This eliminates "washing
out" of the fixed tissues.
9. Osmium tetroxide completely permeabilizes cell
membranes. The osmolarity of the fixative vehicle or
solute is relatively unimportant.
10. It penetrates tissue blocks in a gradient and in large
samples the center of the block may not be as well fixed
as the peripheral areas.
11. Over-fixation with osmium tetroxide may result in
extraction of cell components during dehydration and
increases the hardness and brittleness of the tissue (for
most tissues, 1mm blocks, should not be exposed to
osmium for less than 0.5 or more than 1.5 hours).
Disadvantages
1. It is very expensive.
2. It is a poor penetrating agent, suitable only for small
pieces of tissues (2-3 mm. thick).
3. It is readily reduced by contact with organic matter and
exposure to sunlight, forming a black precipitate which
settles at the bottom of the container.
4. Prolonged exposure to acid vapor can irritate the eye,
producing conjunctivitis, or cause the deposition of black
osmic oxide in the cornea, producing blindness.
5. It inhibits hematoxylin and makes counterstaining
difficult.
6. It is extremely volatile.
Precautions:
1. Eyes and skin may be protected by working in a fume
hood or wearing protective plastic masks or gloves while
using osmium tetroxide.
2. It should be kept in a dark-colored, chemically clean
bottle to prevent evaporation and reduction by sunlight or
organic matter. 3. It should be kept in a cool place or
refrigerated to prevent deterioration.
4. Addition of saturated aqueous mercuric chloride
solution (0.5 to 1 ml/100 ml of stock solution) will prevent
its reduction with formation of black deposits.
5. Black osmic oxide crystals may be dissolved in cold
water.
6. To prevent contact of tissues with black precipitate
formed in the bottom of the jar, the tissues may be
●
Flemming's solution
Most common chrome-osmium acetic acid
fixative used, recommended for nuclear
preparation of such sections.
FORMULA:
● Aqueous chromic acid 15 ml.
● 1%Aqueous osmium tetroxide 4 ml.
● 2%Glacial acetic acid 1 ml.
Fixation time: 24- 48 bouts
Advantages
1. It is an excellent fixative for nuclear structures, e.g.
chromosomes.
2. It permanently fixes fat.
3. Relatively less amount of solution is required for
fixation (less than 10 times the volume of the tissues to
be fixed).
Disadvantages
1. It is a poor penetrating agent; hence, is applicable only
to small pieces of tissues.
2. The solution deteriorates rapidly and must be prepared
immediately before use.
3. Chromic-osmic acid combinations depress the staining
power of hematoxylin (especially Ehrlich's hematoxylin).
4. It has a tendency to form artifact pigments; these may
be removed by washing the fixed tissue in running tap
water for 24 hours before dehydration.
5. It is very expensive.
Flemming's solution without acetic acid
Made up only of chromic and osmic acid,
recommended for cytoplasmic structures
particularly the mitochondria.
● The removal of acetic acid from the formula
serves to improve the cytoplasmic detail of the
cell.
Fixation time: 24- 48 hours
●
Advantages and Disadvantages: same as Flemming's
solution.
●
●
CHROMATE FIXATIVES
Used in 1-2% aqueous solution, usually as a
constituent of a compound fixative.
It precipitates all proteins and adequately
preserves carbohydrates. It is a strong oxidizing
agent; hence, a strong reducing agent (e.g.
formaldehyde)
must
be
added
to
chrome-containing fixatives before use in order
to prevent counteracting effects and consequent
decomposition of solution upon prolonged
standing.
Novesteras, Rivera, Nicolas, Dumayas, Suyam, Waminal | 9
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES|TOPIC 5: CHEMICAL FIXATIVES
●
Potassium Dichromate
Used in a 3% aqueous solution
1. It fixes but does not precipitate cytoplasmic structures.
2. It preserves lipids.
3. It preserves mitochondria (If used in pH 4.5-5.2,
mitochondria is fixed. If the solution becomes acidified,
cytoplasm, chromatin bodies and chromosomes are fixed
but mitochondria are destroyed).
Regaud's (Muller's Fluid)
FORMULA:
● Potassium dichromate 3% 80 ml
● Strong formaldehyde 40% 20 ml (To be added
just before use).
Fixation time: 12-48 hours
Advantages
1. It penetrates tissues well.
2. It hardens tissues better and more rapidly than Orth's
fluid.
3. It is recommended for the demonstration of chromatin,
mitochondria, mitotic figures, Golgi bodies, RBC and
colloid-containing tissues.
Disadvantages
1. It deteriorates and darkens on standing due to acidity;
hence, the solution must always be freshly prepared.
2. Penetration is slow, hence, tissues should not be
thicker than 2-3 mm.
3. Chromate-fixed tissues tend to produce precipitates of
sub-oxide, hence should be thoroughly washed in running
water prior to dehydration.
4. Prolonged fixation blackens tissue pigments, such as
melanin; this may be removed by washing the tissues in
running tap water prior to dehydration.
5. Glycogen penetration is poor; it is therefore, generally
contraindicated for carbohydrates.
6. Nuclear staining is poor.
7. It does not preserve fats.
8. It preserves hemosiderin less than buffered formalin.
9. Intensity of PAS reaction is reduced.
Orth's Fluid
FORMULA:
● Potassium dichromate 2.5% 100ml
● Sodium sulfate (optional) 1gm
● Strong formaldehyde 40% 10 ml (To be added
just before use).
Fixation time: 36-72 hours
3. Chromate-fixed tissues tend to produce precipitates of
sub-oxide, hence should be thoroughly washed in running
water prior to dehydration.
4. Prolonged fixation blackens tissue pigments, such as
melanin; this may be removed by washing the tissues in
running tap water prior to dehydration.
5. Glycogen penetration is poor; it is therefore, generally
contraindicated for carbohydrates.
6. Nuclear staining is poor.
7. It does not preserve fats.
8. It preserves hemosiderin less than buffered formalin.
9. Intensity of PAS reaction is reduced.
●
●
●
●
●
●
●
●
●
●
●
●
●
●
Advantages
1. It is recommended for study of early degenerative
processes and tissue necrosis.
2. It demonstrates rickettsiae and other bacteria.
3. It preserves myelin better than buffered formalin.
Disadvantages
1. It deteriorates and darkens on standing due to acidity;
hence, the solution must always be freshly prepared.
2. Penetration is slow, hence, tissues should not be
thicker than 2-3 mm.
●
PICRIC ACID FIXATIVES
Picrates include fixatives with picric acid.
Penetrates tissue well to react with histones and
basic protein, form crystalline picrates with
amino acids and precipitate all proteins.
Good fixative for connective tissue and it
preserves glycogen well.
Extracts lipids to give superior result in
immunostaining of biogenic and polypeptide
hormones.
However, it causes a loss of basophilia unless
the specimen is thoroughly washed.
Only sold in aqueous state. When it dries out, it
become an explosive hazard in dry form.
Stains everything it touches yellow, including
skin. Hence, it can be removed by treatment
with another acid dye or lithium carbonate
Same with mercuric chloride, it enhances
subsequent staining, especially with anionic
dyes.
Normally used in strong saturated aqueous
solution, approximately I%.
Tissue fixed with picric acid retain little affinity for
basic
dyes. It preserves glycogen well but causes
considerable
shrinkage of tissue.
Washing with changes of 50% to 70% of ethanol
will removed most of the yellow color, but the
excess picrate may be removed more easily
form the sections when the paraffin wax has
been removed.
Paraffin sections of formaldehyde fixed tissues
are usually immersed for few hours in picric acid
solution which is
Bouin’s fluid is commonly used.
Bouin's Solution
The complementary effects of the three
ingredients of Bouin’s solution work well
together to maintain morphology.
Specimens are usually fixed in Bouin’s solution
for 24 hours. Prolonged storage in this acidic
mixture causes hydrolysis and loss of stainable
DNA and RNA. Thorough washing after fixation
is necessary.
FORMULA:
● Picric acid saturated aqueous soln. (2.1%) 75 ml
● 40% formaldehyde 25 ml Acetic acid glacial
● 5 ml
Fixation time: 4– 18 hours
Store at room temperature
Novesteras, Rivera, Nicolas, Dumayas, Suyam, Waminal | 10
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES|TOPIC 5: CHEMICAL FIXATIVES
Practical Applications:
●
●
●
●
●
Recommended for fixation of embryos and
pituitary biopsies.
Gives very good results with tissue that is
subsequently stained with trichrome.
It preserves glycogen well but usually lyses
erythrocytes.
Recommended
for
gastro-intestinal
tract
biopsies, animal embryos and endocrine gland
tissue.
It stains tissue bright yellow due to picric acid.
Excess picric should be washed from tissues
prior to staining with 70% ethanol. Because of its
acidic nature, it will slowly remove small calcium
deposits and iron deposits
Advantages
1. It is an excellent fixative for glycogen demonstration.
2. It penetrates tissues well and fixes small tissues
rapidly.
3. The yellow stain taken in by tissues prevents small
fragments from being overlooked.
4. It allows brilliant staining with the trichrome method.
5. It is suitable for Aniline stains (Mallory's, Heidenhain's
or Masson's methods).
6. It precipitates all proteins.
7. It is stable.
Fixation time: 4– 8 hours
Practical
Applications:
It
is
recommended
for gastro-intestinal
tract
specimens
and fixation of
endocrine tissues. It produces less lysis
than Bouin’s
Solution. It has some decalcifying properties.The fixative
must be washed from tissues if they are to be put into
phosphate buffered formalin on the processing machine
because an insoluble
phosphate
precipitate
will form.
Gendre’s
FORMULA:
● 95%
picric
● 40%
● Acetic
Fixation time:
solution
Ethanol saturated
acid
800
ml
formaldehyde
150
acid
glacial 50
4
18
with
ml
ml
hours
Practical
Application:
This is an alcoholic Bouin’ssolution that appears to
improve upon
ageing. It is highly recommended for
the preservation of glycogen and other carbohydrates.
After fixation the tissue is placed
into
70%
ethanol. Residual yellow color should
be
washed out before staining.
Advantages
Disadvantages
1. It causes RBC hemolysis and reduces the amount of
demonstrable ferric iron in tissue.
2. It is not suitable for frozen sections because it causes
frozen sections to crumble when cut.
3. Prolonged fixation makes tissues hard, brittle and
difficult to section. Tissues should not be allowed to
remain in the fluid for more than 12-24 hours (depending
on size).
4. Picrates form protein precipitates that are soluble in
water; hence, tissues must be first rendered insoluble by
direct immersion in 70% ethyl alcohol.
5. Picric acid fixed tissues must never be washed in water
before dehydration.
6. Picric acid will produce excessive staining of tissues; to
remove the yellow color, tissues may be placed in 70%
ethyl alcohol followed by 5% sodium thiosulfate and then
washed in running water.
7. Picric acid is highly explosive when dry, and therefore
must be kept moist with distilled water or saturated
alcohol at 0.5 to 1% concentration during storage.
8. It alters and dissolves lipids.
9. It interferes with Azure eosin method of staining;
hence, tissues should be thoroughly washed with alcohol.
●
●
●
●
●
●
●
●
Disadvantages
●
Hollande’s Solution
●
FORMULA:
● Copper acetate: 25 gm
● Picric
acid:
40 gm
● 40%
formaldehyde:
100 ml
● Acetic acid:
15 ml
● Distilled water: 1000 ml
● Dissolve chemicals in distilled water without
heat.
It produces minimal distortion
of
micro-anatomical structures and can 128 be
used
for general and special stains. (The
shrinking
effect
of picric acid
is
balanced
by
the
swelling
effect
of
glacial acetic acid.)
It
is
an excellent
fixative for
preserving soft and
delicate structures
(e.g endometrial curettings).
It penetrates rapidly and evenly, and causes little
shrinkage.
Yellow stain is useful when handling fragmentary
biopsies.
It permits brilliant staining of tissues.
It is the preferred fixative for tissues to be
stained by Masson's trichrome for collagen,
elastic or connective tissue. (If tissue is fixed in
formalin, a pre-treatment in Bouin’s solution (as
mordant
prior
to
trichrome
stain)
is
recommended.
It preserves glycogen.
It does not need "washing out".
●
●
●
It penetrates large tissues poorly; hence, its use
is limited to small fragments of tissue.
Picrates are soluble in water; hence, tissues
should not be washed in running water but
rather transferred directly from fixative to 70%
alcohol.
It is not suitable for fixing kidney structures, lipid
and mucus.
It destroys cytoplasmic structures, e.g.
mitochondria.
It produces RBC hemolysis and removes
demonstrable ferric iron from blood pigments.
Novesteras, Rivera, Nicolas, Dumayas, Suyam, Waminal | 11
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES|TOPIC 5: CHEMICAL FIXATIVES
●
It reduces or abolishes Feulgen reaction due to
hydrolysis of nucleoproteins.
●
Brasil's Alcoholic Picroformol Fixative
FORMULA
● Formaldehyde 37% 2040 ml.
● Picric acid 80 gm.
● Ethanol or isopropyl alcohol 6000 ml.
● Trichloroacetic acid 65 gm.
● Overnight
tissue
fixation
by automatic
processing technique may utilize 3-4 changes of
Brasil's fixative at 1/2 to 2 hours each,
succeeded directly by absolute alcohol.
●
Advantages
●
●
●
It is better and less "messy" than Bouin's
solution.
It is an excellent fixative for glycogen.
●
a colorless liquid that when undiluted is also
called “Glacial” Acetic Acid because it is a
water-free (anhydrous) acetic acid that freezes
and solidifies at about 16°C.
major effect is to precipitate DNA, which is split
off from nucleoprotein. For this reason, acetic
acid is valuable for the preservation of nuclei,
and is often added to fixatives specifically to do
that.
●
●
●
●
●
●
●
●
Disadvantages
When combined with Potassium Dichromate, the
lipid-fixing property of the latter is destroyed
(e.g. Zenker's fluid).
It is contraindicated for cytoplasmic fixation
since it destroys mitochondria and Golgi
elements of cells.
Concentrated acetic acid is corrosive to skin and
must, therefore, be handled with appropriate
care, since it can cause skin burns, permanent
eye damage, and irritation to the mucous
membranes. These burns or blisters may not
appear until hours after exposure.
Latex gloves offer no protection, so especially
resistant gloves, such as those made of nitrile
rubber, are worn when handling the compound.
●
●
acid
It takes up C02 to form insoluble lead carbonate
especially on prolonged standing. This may be
removed by filtration or by adding acetic acid
drop by drop to lower the pH and dissolve the
residue.
a reagent that is used for the precipitation of
proteins and nucleic acids.
It is also used as a decalcifier and fixative in
microscopy. Addition of TCA to a final
concentration of 10% (w/v) will precipitate most
proteins from solution.
The excess TCA can be removed from protein
pellets by washes with buffer. For the
precipitation of nucleic acids, a 5% solution of
ice cold TCA has been used. It is sometimes
incorporated into compound fixatives.
Advantages
●
●
●
●
It precipitates proteins.
Its marked swelling effect on tissues serves to
counteract shrinkage produced by other
fixatives.
It may be used as a weak decalcifying agent.
Its softening effect on dense fibrous tissues
facilitates preparation of such sections.
Disadvantages
●
It is a poor penetrating agent, hence, is suitable
only for small pieces of tissues or bones.
ACETONE
●
●
LEAD FIXATIVES
for
TRICHLOROACETIC ACID
●
Advantages
It fixes and precipitates nucleoproteins.
It precipitates chromosomes and chromatin
materials; hence, is very useful in the study of
nuclear components of the cell. In fact, it is an
essential constituent of most compound nuclear
fixatives.
It causes tissues (especially those containing
collagen) to swell. This property is used in
certain compound fixatives to counteract the
shrinkage produced by other components (e.g.
mercury).
Advantages
It
is
recommended
mucopolysaccharides.
It fixes connective tissue mucin.
Disadvantages
GLACIAL ACETIC ACID
●
Lead fixatives are used in 4% aqueous solution
of basic lead acetate. Lead oxaloacetate, a
primary reaction product precipitate for the
visualization of the activity of glutamic
oxaloacetic transaminase in tissue sections, is
stable at a slightly alkaline pH. At concentrations
which are used for tissue fixation, a slight
inhibitory effect on glutamic oxaloacetic
transaminase activity is produced by acetone
while a glutaraldehyde-formaldehyde mixture
results in marked reduction of activity.
Acetone is not recommended as morphological
fixative for tissue blocks, mainly because of its
shrinkage and poor preservation effects. Its use
is reserved for the fixation of cryostat sections or
for tissues in which enzymes have to be
preserved.
Acetone is almost always used alone and
without dilution; it fixes by dehydration and
precipitation. It is used to fix specimens at cold
temperatures (0 to 4°C). Fixation time may vary
Novesteras, Rivera, Nicolas, Dumayas, Suyam, Waminal | 12
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES|TOPIC 5: CHEMICAL FIXATIVES
from several minutes (for cell smears, cryostat
sections) to several hours (1-24 hours for small
tissue blocks).
●
●
●
Advantages
It is recommended for the study of water
diffusible enzymes especially phosphatases and
lipases.
It is used in fixing brain tissues for diagnosis of
rabies.
It is used as a solvent for certain metallic salts to
be used in freeze substitution techniques for
tissue blocks.
Disadvantages
●
●
●
●
It produces inevitable shrinkage and distortion.
It dissolves fat.
It preserves glycogen poorly.
It evaporates rapidly.
MICHEL’S SOLUTION
●
●
●
●
provides a stable medium for transport of fresh
unfixed tissues, such as renal, skin and oral
mucosa
biopsies,
which
will
undergo
subsequent
frozen
section
and
immunofluorescence studies.
not suitable for transporting cells for flow
cytometry or for tissues used for fluorescent
in-situ hybridization (FISH).
not a fixative, and is not suitable for any other
use (particularly, for transporting living cells for
flow cytometry). It should be kept refrigerated
(not frozen) until use.
This simple salt solution maintains pH, but does
not kill most pathogens. Specimens received in
transport medium should be washed in three
changes of washing solution (10 minutes for
each wash). It is not suitable for FISH studies.
REFERENCE:
Bruce-Gregorios, J. (2017).
Techniques (U.S. edition)
Histopathologic
Novesteras, Rivera, Nicolas, Dumayas, Suyam, Waminal | 13