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I. ALDEHYDE FIXATIVES
A.Formaldehyde (Formalin)
Advantages
Disadvantages
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.
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.
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
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.
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.
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
C. LEAD FIXATIVES
1. It is recommended for acid
mucopolysaccharides.
2. It fixes connective tissue mucin.
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.
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.
B. Brasils Alcoholic Picroformol Fixative - it is better and less messy than Bouins solution and it is an
excellent fixative for glycogen.
Disadvantages
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.
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.
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.
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.
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.
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.)
B. Flemmings solution without acetic acid- the advantages and disadvantages are same as Flemmings solution.
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.
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.