Types of Fixatives

Name:
MT45-GH

August 23, 2016

I. ALDEHYDE FIXATIVES
A.Formaldehyde (Formalin)
Advantages

1. It is cheap, readily available, easy to

prepare, and relatively stable, especially if stored
in buffered solutions.
2. It is compatible with many stains, and
therefore can be used with various staining
techniques depending upon the need of the tissues.
3. It does not overharden tissues, even with
prolonged periods of fixation, as long as solutions
are regularly changed.
4. It penetrates tissues well.
5. It preserves fat and mucin, making them
resistant to subsequent treatment with fat solvents,
thereby allowing them to be stained for
demonstration.
6. It preserves glycogen.
7. 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.
8. 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 sections to be
prepared easily.
9. It allows frozen tissue sections to be
prepared easily.
10. It does not require washing out, unless
tissues have stayed in formalin for excessively
long periods of time.
11. It is a tolerant fixative, used for mailing
specimen because specimen can be left in formalin
indefinitely.

Disadvantages

1. Fumes are irritating to the nose and eyes

and may cause sinusitis, allergic rhinitis, or
excessive lacrimation.
2. The solution is irritating to the skin and may
cause Allergic Dermatitis on prolonged contact.
3. It may produce considerable shrinkage of
tissues.
4. It is a soft fixative and does not harden
some cytoplasmic structures adequately enough for
paraffin embedding.
5. 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.
6. Prolonged fixation may produce:
a.
Bleaching of the specimen and loss
of natural tissue colors.
b.
Dispersal of fat from the tissues into
the fluid.
c.
Dissolution or loss of glycogen,
biurate of sodium crystals and uric acid.

B. 10% Formol-Saline
Advantages
1. It penetrates and fixes tissue evenly.
2. It preserves microanatomic and cytologic
details with minimum shrinkage and distortion.
3. Large specimens may be fixed for a long
time provided that the solution is changed every
three months.
4. It preserves enzymes and nucleoproteins.
5. It demonstrates fats and mucin.
6. It does not overharden tissues, thereby
facilitating dissection of the specimen.

Disadvantages
1. It is a slow fixative. The period of fixation
is required to be 24 hours or longer.
2. Formol-saline fixed tissues tend to shrink
during alcohol dehydration; this may be reduced by
secondary fixation.
3. Metachromatic reaction of amyloid is
reduced.
4. Acid dye stains less brightly than when
fixed with mercuric chloride.

7. It is ideal for most staining techniques,

including silver impregnation.
8. It allows natural tissue color to be restored
upon immersion in 70% alcohol.
C. 10% Neutral Buffered Formalin (NBF) or Phosphate-Buffered Formalin (pH 7)
1. It prevents precipitation of acid formalin
pigments on post-mortem tissue.
2. It is the best fixative for tissues containing
iron pigments and for elastic fibers which do not
stain well after Susa, Zenker or Chromate fixation.
3. It requires no post-treatment after fixation
goes directly into 80% alcohol for processing.

1. It is longer to prepare; hence, is timeconsuming

2. Positivity of mucin to PAS is reduced.
3. It may produce gradual loss in basophilic
staining of cells.
4. Reactivity of myelin to Weigerts iron
hematoxylin stain is reduced.
5. It is inert towards lipids, especially neutral
fats and phospholipids.

D. Formal-Corrosive (Formal-Sublimate)
1. It penetrates small pieces of tissues rapidly,
2. It produces minimum shrinkage and hardening.
3. It is excellent for many staining procedures
including silver reticulum methods.
4. It brightens cytoplasmic and metachromatic
stains better than with formalin alone.
5. Cytological structures and blood cells are well
preserved.
6. There is no need for washing-out. Tissues
can be transferred directly from fixative to alcohol.
7. It fixes lipids, especially neutral fats and
phospholipids.

1. Penetration is slow; hence, tissue sections

should not be more than 1 cm thick.
2. It forms mercuric chloride deposits.
3. It does not allow frozen tissue sections to be
made.
4. It inhibits the determination of the extent of
tissue decalcification.

E. Alcoholic Formalin (Gendres Fixative)

Advantages
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 microincineration 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-lining.

F. Glutaraldehyde
1. It has a more stable effect on tissues,
giving a firmer texture with better tissue sections,
especially of central nervous tissues.
2. It preserves plasma proteins better.
3. It produces less tissue shrinkage.
4. It preserves cellular structures better;
hence, is recommended for enzyme histochemistry
and electron microscopy.

1. It is more expensive.
2. It is less stable.
3. It penetrates tissues more slowly.
4. It tends to make tissue (i.e. renal biopsy)
more brittle.
5. It reduces PAS positivity of reactive mucin.
This may be prevented by immersing
glutaraldehyde-fixed tissues in a mixture of

5. It is more pleasant and less irritating to the

nose.
6. It does not cause dermatitis.

concentrated glacial acetic acid and aniline oil.

II. METALLIC FIXATIVES

A. MERCURIC CHLORIDE
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.
5. Trichrome staining is excellent.
6. It is the routine fixative of choice for
preservation of cell detail in tissue photography.
7. It permits brilliant metachromatic staining
of cells.
8. It is recommended for renal tissues, fibrin,
connective tissues and muscles.

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.

A1. Zenkers Fluid

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 compatible with most stains.
6. It may act as a mordant to make certain
special staining reactions possible.

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.

A.2 Zenker-Formol (Hellys solution)

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 is better than
Zenkers.

The disadvantages of Hellys solution are similar to

Zenkers 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.

4. It preserves cytoplasmic granules well.

A.3 Heidenhains Susa Solution
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 the 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.

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. Weigerts method of staining elastic fibers is
not possible in Susa-fixed tissues.

A.4 B-5 Fixative

Advantages
1. Rapid fixation can be achieved in 1 1/2 -2
hours.
2. It is a good fixative for cytology of bone
marrow biopsies.

Disadvantages
1. Overfixation hardens the tissue and makes
cutting difficult.
2. The two working solutions are kept separate,
since the mixture is stable. Mix just prior to use.
3. Some B-5 solutions will form precipitate on
standing, but this is of no consequence.
4. As with all mercuric chloride fixatives, the
mercury pigments can be removed by dezenkerization.

B. CHROMATE FIXATIVES
B1. 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.
B2. Potassium Dichromate- is used in a 3% aqueous solution . It fixes but does not precipitate cytoplasmic
structures. It preserves lipids. 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).
B3. Regards (Mullers) Fluid
Advantages
1. It penetrates tissues well.
2. It hardens tissue better and more rapidly
than Orths fluid.
3. It is recommended for 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,
e.g. 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.
B.4 Orths Fluid
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.

Same as in Regauds Fuid

C. LEAD FIXATIVES
1. It is recommended for acid
mucopolysaccharides.
2. It fixes connective tissue mucin.

It takes up CO2 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.

III. PICRIC ACID FIXATIVES

PICRIC ACID
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 (Mallorys,
Heidenhains or Massons 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 are formed upon protein; precipitates
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.
A. Bouins Solution
Advantages

Disadvantages

1. It produces minimal distortion of microanatomical 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.
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 Massons trichrome for collagen,
elastic or connective tissue. ( if tissues is fixed in
formalin, a pre=treatment in Bouin (as mordant
prior to trichrome stain) is recommended.
7. It preserves glycogen.
8. It does not need washing out.

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.

B. Brasils Alcoholic Picroformol Fixative - it is better and less messy than Bouins solution and it is an
excellent fixative for glycogen.

IV. GLACIAL ACETIC ACID

Advantages

Disadvantages

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).

1. When combined with Potassium

Dichromate, the lipid-fixing property of the
latter is destroyed (e.g. Zenkers fluid).
2. It is contraindicated for cytoplasmic
fixation since it destroys mitochondria and
Golgi elements of cells.

V. ALCOHOL FIXATIVES
Advantages
1. It is ideal for small tissue fragments.
2. It may be used both as a fixative and
dehydrating agent.
3. It is excellent for glycogen preservation.
4. It preserves nuclear stains.

Disadvantages
1. Lower concentrations (70-80%) will cause
RBC hemolysis and inadequately preserve leukocytes.
2. It dissolves fats and lipids, as a general rule.
Alcohol-containing fixatives are contraindicated when
lipids are to be studied.
3. It causes glycogen granules to move towards
the poles or ends of the cells (polarization).
4. Tissue left in alcohol too long will shrink,
making it difficult or impossible to cut.

A. 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.
2. If left in fixative for more than 48 hours,
tissues may be overhardened and difficult to cut.

B. Isopropyl Alcohol 95% - is used for fixing touch preparations, although some touch preparations are air dried and
not fixed, for certain special staining procedures such as Wright-Giemsa.
C. Ethyl Alcohol
1. It preserves but does not fix glycogen.
2. It fixes blood, tissue films and smears.
3. It preserves nucleoproteins and nucleic
acids, hence, is used for histochemistry,
especially for enzyme studies.
4. It fixes tissue pigments fairly well.

1. It causes polarization of glycogen granules.

2. It produces considerable hardening and
shrinkage of tissues.
3. Hemosiderin preservation is less than in
buffered formaldehyde.
4. It is a strong reducing agent; hence, should not
be mixed with chromic acid, potassium dichromate
and osmium tetroxide which are strong oxidizing
agents.

D. Carnoys Fluid
1. It is considered to be the most rapid
fixative.
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.
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, tissues may be
transferred directly to absolute alcohol, thereby
shortening processing time.

1. It produces RBC hemolysis.

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 degree Celsius) are used.
7. It dissolves acid-soluble cell granules and
pigments.

E. Newcomers Fluid- is recommended for fixing mucopolysaccharides and nuclear proteins, produces better
reaction in Feulgen stain than Carnoys fluid and it acts both as a nuclear and histochemical fixative.

VI. OSMIUM TETROXIDE (OSMIC ACID)

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, is used extensively for neurological

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

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.

deposition of black osmic oxide in the cornea,

producing blindness.
5. It inhibits hematoxylin and makes
counterstaining difficult.
6. It is extremely volatile.

A. Flemmings Solution
1. It is an excellent fixative for nuclear
structures, e.g. chromosomes.
2. It permanently fixes fats.
3. Relatively less amount of solution is
required for fixation (less than 10 times the
volume of the tissues to be fixed.)

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
Ehrlichs 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.

B. Flemmings solution without acetic acid- the advantages and disadvantages are same as Flemmings solution.

VII. TRICHLOROACETIC ACID

Advantages

Disadvantages

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.

1. It is a poor penetrating agent;

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

VIII. ACETONE
1. It is recommended for the study of water
diffusible enzymes especially phosphatases and
lipases.
2. Itis 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.

1. It produces inevitable shrinkage and

distortion.
2. It dissolves fat.
3. It preserves glycogen poorly.
4. It evaporates rapidly.

IX. HEAT FIXATION

1. Fixation is better.
2. It preserves nuclear and cytoplasmic detail.
3. It is suitable for frozen tissue preparation.

1. It produces considerable tissue

shrinkage and distortion.
2. It destroys RBC.

3. It dissolves starch and glycogen.