HISTOPATHOLOGIC TECHNIQUES
METALLIC FIXATIVES tissues and makes cutting of frozen
Mercurial fix tissues through an unknown mechanism
that increases staining brightness and gives excellent
nuclear detail However, mercurial penetrate poorly
and produce tissue shrinkage. Their best application is
for fixation of hematopoietic and reticuloendothelial
tissues.
MERCURIC CHLORIDE
• Mercuric chloride is the most common
metallic fixative, frequently used in saturated
aqueous solutions of 5-7%.
• Tissues fived with mixtures containing
mercuric chloride (except Susa) contain black
precipitates of mercury
• Mercury deposits are removed from
deparaffinized sections before staining, by
treating the section with 0.5% iodine solution
in 70% ethanol for 5-10 minutes.
•ADVANTAGE
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.
5. Trichrome staining is excellent.
6. It is the routine fixative of choice for
preservation of cell detail in tissue
photography
• DISADVANTAGE
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 are 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
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tissues difficult.
6. It causes considerable
lysis of red blood cells and removes
much iron from hemosiderin.
Precautions:
1) Sections must be cleared of mercury deposits
before immunostaining. Black deposit may be
removed by adding saturated iodine solution
in 95% alcohol, the iodine being decolorized
with absolute alcohol in the subsequent
stages of dehydration.
2) Compound 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
4) Contact of mercuric fixatives with personal
jewelries should be avoided.
5) Mercury-containing solutions (Zenker’s or B-5)
should be always be discarded into the proper
containers. Mercury, if poured down a drain,
will form amalgams with the metal that build
up and cannot be removed.
ZENKER'S SOLUTION
• Is made up of mercuric chloride stock solution to
which glacial acetic acid has been added just before
its use to prevent turbidity and formation of a dark
precipitate.
FORMULA
Mercuric chloride-------5gm
Potassium dichromate---------2.5gm
Distilled water---------100ml
Acetic acid, glacial----------5ml (to be added just
before use) heat, cool, filter in brown bottle. Wash
sample for 24hours 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.
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 HISTOPATHOLOGIC TECHNIQUES
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.
Precautions:
1. Do not let tissues stay in solution for more than 24
hours.
2. Solutions must always be freshly prepared.
3. Tissues should be cut thin (2-3 mm.) and hollow
organs should be opened to promote complete
penetration and fixation.
4. Tissues must be washed out thoroughly in running
water to permit good staining.
5. It produces mercury pigment which should be
removed from sections prior to staining and can
produce chrome pigment if tissue is not washed in
water prior to processing. After water washing, tissue
should be stored in 70% ethanol.
6. Mercuric deposits may be removed by immersing
tissues in alcoholic iodine solution prior to staining,
through a process known as dezenkerization.
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:
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.
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Zenker-Formol (Helly’s) Solution
Zenker's solution is an excellent fixative for bone
marrow, extramedullary hematopoiesis and
intercalated discs of cardiac muscle. However, it
produces mercury pigment which should be removed
from sections prior to staining and it can produce
chrome pigment if tissue is not washed in water prior
to processing
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
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 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
•. This mixture enhances nuclear detail, which is
important for identifying
normal and abnormal cell types in bone marrow
(hematopoietic tissue)
specimens. The coagulation of nuclear chromatin is an
effect of the acetic
acid.
• The mercuric chloride ensures rapid structural
stabilization and facilitates
bright staining by many of the dyes used in micro
technique.
FORMULA:
B-5 Stock solution
Mercuric chloride: --------12 g
Sodium acetate anhydrous: --------2.5 g
Distilled water: -----------200 ml
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 HISTOPATHOLOGIC TECHNIQUES
Working solution: (prepare immediately before use)
B-5 stock solution: ---------- 20 ml
40% formaldehyde: ---------- 2 ml
Fixation time: 4 – 8 hours
Practical Applications:
1. Despite its mercuric chloride content and
consequent problems with disposal, this solution is
popular for fixation of hematopoietic, bone marrow
biopsies and lymphoid tissue.
2. Rapid fixation can be achieved in 1 1/2 - 2 hours.
3. It produces excellent nuclear detail, provides good
results with many special stains, and is recommended
for immunohisto-chemical staining.
4. Sections will require the removal of mercury
pigment prior to staining.
5. Tissue should not be stored in this fixative but
placed in 70% ethanol instead.
6. Over-fixation hardens the tissue and makes cutting
difficult.
7. The two working solutions are kept separate, since
the mixture is unstable. Mix just prior to use.
8. Some B-5 solutions will form precipitate on
standing, but this is of no consequence.
9. As with all mercuric chloride fixatives, the mercury
pigment can be removed by de-zenkerization.
Heidenhain's Susa Solution
Is recommended mainly for tumor biopsies especially
of the skin; it is an excellent cytologic fixative.
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
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.
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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.
Precautions:
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.
OXIZING FIXATIVES (OSMIC ACID;
Os04)
• Traditionally used in electron microscopy both
as a fixative and a heavy metal stain
• excellent stain for lipids in membranous
structures and vesicles
• should be kept in DARK COLORED, chemically
clean bottle to prevent evaporation
andreduction by sunlight and organic matter
• inhibits hematoxylin and makes
counterstaining difficult produces
> Prevention addition of saturated Mercuric Chloride
> Remedy: Black osmic oxide crystals may be
dissolved in cold water
Procedure:
1. Wash the sample four times with PBS.
2. Fix with 2% Paraformaldehyde, 0.1%
Glutaraldehyde in PBS for 30 minutes.
3. Wash four times with PBS.
4. Wash four times with double distilled water.
5. Prepare 0.1% OsO4 solution by diluting the 4%
stock solution in double distilled water.
6. Incubate the sample with 0.1% OsO4 for 30
minutes.
7. Wash four times with double distilled water.
Flemming's Solution
is the most common chrome-osmium acetic acid
fixative used, recommended for nuclear preparation
of such sections.
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 HISTOPATHOLOGIC TECHNIQUES
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
Is 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
Chromic Acid
• is 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.
Potassium Dichromate
❖ is used in a 3% aqueous solution.
1) It fixes but does not precipitate cytoplasmic
structures.
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2) 2. It preserves lipids.
3) 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
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 HISTOPATHOLOGIC TECHNIQUES
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: Same as in Regaud's fluid.
PICRIC ACID FIXATIVES
➢ Picrates include fixatives with picric acid.
Foremost among these is Bouin's solution
that does almost as well as mercurials
with nuclear detail but does not cause as
much hardness.
➢ Picric acid is an explosive hazard in dry
form. As a solution, it stains everything it
touches yellow, including skin.
➢ Like mercuric chloride, picric acid
enhances subsequent staining, especially
with anionic (“acid”) dyes.
➢ Picric Acid is normally used in strong
saturated aqueous solution
(approximately I %).
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.
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
Practical Applications:
➢ Bouin's Solution is recommended for fixation
of embryos and pituitary biopsies. It gives very
good results with tissue that is subsequently
stained with trichrome.
➢ It is sometimes recommended for gastro-
intestinal tract biopsies, animal embryos and
endocrine gland tissue.
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.
5
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.
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.
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.
Fixation time: 4 – 18 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.
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 HISTOPATHOLOGIC TECHNIQUES
Gendre’s solution
FORMULA:
95% Ethanol saturated with picric acid----- 800 ml
40% formaldehyde------------------------------ 150 ml
Acetic acid glacial-------------------------------- 50 ml
Fixation time: 4 - 18 hours
Practical Application:
This is an alcoholic Bouin’s solution 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:
1. It produces minimal distortion of micro-anatomical
structures and can be used for general and special
stains. (The shrinking effect of picric acid is balanced
by the swelling effect of glacial acetic acid.)
2. It is an excellent fixative for preserving soft and
delicate structures (e.g. endometrial curettings).
3. It penetrates rapidly and evenly, and causes little
shrinkage.
4. Yellow stain is useful when handling fragmentary
biopsies.
5. It permits brilliant staining of tissues.
6. 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.
7. It preserves glycogen.
8. It does not need "washing out".
Disadvantages:
1. It penetrates large tissues poorly; hence, its use is
limited to small fragments of tissue.
2. Picrates are soluble in water; hence, tissues should
not be washed in running water but rather transferred
directly from fixative to 70% alcohol.
3. It is not suitable for fixing kidney structures, lipid
and mucus.
4. It destroys cytoplasmic structures, e.g.
mitochondria.
5. It produces RBC hemolysis and removes
demonstrable ferric iron from blood pigments.
6. It reduces or abolishes Feulgen reaction due to
hydrolysis of nucleoproteins.
6
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:
1. It is better and less "messy" than Bouin's
solution.
2. 2. It is an excellent fixative for glycogen.
GLACIAL ACETIC ACID
➢ Acetic acid is 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.
➢ Acetic acid does not have much effect on
proteins, other than to enable swelling by the
absorption of water. Its major effect is to
precipitate DNA, which is split off from
nucleoprotein.
Advantages:
1. It fixes and precipitates nucleoproteins.
2. 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.
3. 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).
Disadvantages:
1. When combined with Potassium Dichromate, the
lipid-fixing property of the latter is destroyed (e.g.
Zenker's fluid).
2. It is contraindicated for cytoplasmic fixation since it
destroys mitochondria and Golgi elements of cells.
3. 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.
4. Latex gloves offer no protection, so especially
resistant gloves, such as those made of nitrile rubber,
are worn when handling the compound.
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 HISTOPATHOLOGIC TECHNIQUES

LEAD FIXATIVES Advantages:

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.
Advantages:
1. It is recommended for acid mucopolysaccharides.
2. It fixes connective tissue mucin.
Disadvantage:
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.
1. It is recommended for the study of water diffusible
enzymes especially phosphatases and lipases.
2. It is used in fixing brain tissues for diagnosis of
rabies.
3. It is used as a solvent for certain metallic salts to be
used in freeze substitution techniques for tissue
blocks.
Disadvantages:
1. It produces inevitable shrinkage and distortion.
2. It dissolves fat.
3. It preserves glycogen poorly.
4. It evaporates rapidly.

TRICHLOROACETIC ACID
➢ Trichloroacetic Acid (TCA) is 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.

Advantages:
1. It precipitates proteins.
2. Its marked swelling effect on tissues serves to
counteract shrinkage produced by other fixatives.
3. It may be used as a weak decalcifying agent.
4. Its softening effect on dense fibrous tissues
facilitates preparation of such sections.

Disadvantage: It is a poor penetrating agent,

hence, is suitable only for small pieces of tissues or
bones.

ACETONE
➢ 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.
➢ It is used to fix specimens at cold
temperatures (0 to 4°C). Fixation time may
vary from several minutes (for cell smears,
cryostat sections) to several hours (1-24 hours
for small tissue blocks).

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