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Fixation is a procedure adopted to kill, harden and preserve materials for microscopic study, by means of a substance
known as fixative. The shape, structure, intercellular relationship and chemical constituents of tissues are preserved by
preventing degeneration, putrefaction, decomposition and distortion of tissues after death, consequent to cutting and
removal from the body.
1. They harden soft and friable tissues and make the handling and cutting of sections easier. This is usually accelerated
by the action of alcohol during the dehydration process.
2. They make the cells resistant to damage and distortion caused by hypotonic and hypertonic solution used during
tissue processing.
4. They increase the optical differentiation of cells and tissue components thereby rendering them more readily visible
during examination
5. They act as mordants or accentuators to promote and hasten staining, or they may inhibit certain dyes in favor of
another (e.g. formaldehyde intensifies while osmium tetroxide inhibits hematoxylin).
6. They reduce the risk of infection during handling and actual processing of tissues.
4. It must kill the cell thereby producing minimum distortion of cell constituents.
5. It must inhibit bacterial decomposition and autolysis.
8. It must harden tissues thereby making the cutting of sections easier.
9. It must be isotonic, causing minimal physical and chemical alteration of the cells and their constituents.
This is not, however, a strict rule since there are some hypotonic solutions (i.e. Glacial Acetic Acid) producing tissue
swelling, which are being used in conjunction with hypertonic solutions (e.g. Picric Acid) causing shrinkage of cells, to
produce a compound which would give an optimal effect on the tissue structure.
10. It must make the cellular components insoluble to hypotonic solutions and render them insensitive to subsequent
processing.
11. It must permit the subsequent application of many staining procedures to facilitate easier and more profitable
examination.
Types of Fixatives:
1. Aldehydes
Aldehyde fixatives are satisfactory for routine paraffin sections, for electron microscopy and when histochemical and
enzyme studies are indicated.
a. Formaldehyde (Formalin)
This is a gas produced by the oxidation of methyl alcohol, and is soluble in water to the extent of 37 – 40% weight in
volume. Pure stock solution of 40% formalin is unsatisfactory for routine fixation since high formaldehyde concentrates
tend to over harden the outer layer of the tissue and affect staining adversely. Therefore, it must be diluted 1:10 or 1:20
to make a 10% or 5% solution, or combined with another fixative to form a compound solution. Usual fixation time is 24
hours.
Advantages:
(1) It is cheap, readily available, easy to prepare, and relatively stable, especially if stored in buffered solution.
(2) It is compatible with many stains, and therefore can be used depending upon the need of the tissues.
(3) It does not over harden tissues, even with prolonged periods of fixation, as long as solutions are regularly changed.
(5) It preserves fat and mucin, making them resistant to subsequent treatment with fat solvents, thereby allowing tissue
enzymes to be studied.
(7) It preserves but does not precipitate proteins, thereby, allowing tissue enzymes to be studied.
(8) It does not make tissues brittle, and is therefore recommend for nervous tissue preservation.
(9) 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 photographies.
(11) It does not require washing out, unless tissues have stayed in formalin for excessively long period of time.
Disadvantages:
(1) Fumes are irritating to the nose and eyes may cause sinusitis, allergic rhinitis or excessive lacrimation.
(2) The solution is irritating to the skin and may cause allergic dermatitis on prolonged contact.
(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.
Precaution:
(1) Prolonged storage of formaldehyde especially at very low temperature, may induce precipitation of white
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 10% methanol.
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.
(2) 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.
(3) Dermatitis may be avoided by the use of rubber gloves when handling specimens fixed in formalin.
(4) Bleaching of tissues may be prevented by changing the fluid fixative every three months.
(5) Natural tissue color may be restored by immersing tissues in 70% alcohol after fixation.
(6) If fatty tissues are to be stored for a long time, cadmium or cobalt salts are added to prevent dispersion of fat out
into the fluid.
(7) Acid reaction due to formic acid formation can be buffered or neutralized by adding magnesium carbonate or calcium
carbonate to 10 – 15% formalin. This should be done on a wide mouth bottle to prevent violent explosion due to
insufficient gas space for CO2 release.
(8) To improve staining and produce firmer and harder consistency, tissues fixed in formalin for 1 – 2 hours may be
placed again in Helley’s fluid for 4 – 6 hours or in formol–sublimate for 4 – 16 hours (secondary fixation).
(9) If post–fixed in osmic acid, the tissue must be washed in demineralized water to prevent hypotonicity and bleaching.
b. Glutaraldehyde – is made up of two formaldehyde residues, linked by three carbon chains, utilized for routine light
microscopic work and also satisfactory for electron microscopy.
A 2.5% solution is used for small tissue fragments and needle biopsies fixed in 2 – 4 hours at room temperature. A 4%
solution is recommended for larger tissues less than 4 mm thick, fixed in 6 – 8 hours up to 24 hours.
(1) It has a more stable effect on tissues, giving firmer texture with better tissue sections, especially of central nervous
tissues.
(4) It preserves cellular structures better, hence, is recommended for enzyme histochemistry and electron microscopy.
Disadvantages:
(4) 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.
2. Metallic fixatives
a. Mercuric chloride – is the most common fixative, used in saturated aqueous solutions of 5 – 7%; it is included in many
compound fixatives.
Advantages:
(4) It has a greater affinity to acid dyes and is preferred in lieu of formaldehyde for cytoplasmic staining.
(5) Trichome staining is excellent.
(6) It is the routine fixative of choice for preservation of cell detail in tissue photography.
(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.
(10) Compound solutions containing Mercuric chloride deteriorate rapidly upon addition of Glacial Acetic Acid to
Formalin
(11) It is extremely corrosive to metals.
Precaution
(1) Black deposits may be removed by adding saturated iodine solution in 96% alcohol, the iodine being decolorized with
absolute alcohol in the subsequent stages of dehydration.
(3) The use of metals caps to cover the bottles containing fixative should be avoided.
Just before use, Glacial Acetic Acid may be added to form Zenker’s solution or strong Formaldehyde may be added to
form Zenker – Formol solution (Helly’s).
b. Chromate fixative
(c) It preserves mitochondria (if used in pH 4.5 – 5.2, mitochondria are fixed; if solution becomes acidified, cytoplasm,
chromatin bodies and chromosomes are fixed but mitochondria are destroyed.)
(2) 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–staining fixative before use in order to prevent counteracting effects and
consequent decomposition of solution upon prolonged standing.
c. Lead fixatives
Lead fixatives are used in 4% aqueous solution of basic lead acetate
Advantage:
Disadvantage:
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.
3. Picric acid
Picric acid is normally used in strong saturated aqueous solution (approximately 1%)
Advantages:
c. The yellow stain taken in by tissue prevents small fragments from being overlooked
Disadvantages:
a. It causes RBC hemolysis and reduces the amount of demonstrable ferric iron in tissues.
b. It is not suitable for frozen sections because it causes frozen sections to crumble when cut.
c. 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).
d. Picrates are formed upon protein; precipitates are soluble in water, hence, tissues must be first rendered insoluble by
direct immersion in 70% ethyl alcohol.
Picric acid fixed tissues must never be washed in water before dehydration.
e. 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.
f. 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.
h. It interferes with azure eosin method of staining; hence, tissues should be thoroughly washed with alcohol.
4. Acetic acid
Acetic acid is normally used in conjunction with other fixatives to form a compound solution. It solidifies at 17oC, hence,
the name Glacial Acetic Acid.
Advantages:
c. 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:
a. When combined with potassium dichromate, the lipid – fixing property of the latter is destroyed (e.g. Zenker’s fluid)
b. It is contraindicated for cytoplasmic fixation since it destroys mitochondria and Golgi elements of cells.
5. Acetone
Advantages:
a. It is recommended for the study of water diffusible enzymes especially phosphatases and lipases.
c. It is used as a solvent for certain metallic salt to be used in freeze substitution technique for tissue blocks.
Disadvantages:
6. Alcohol
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.
Advantages:
Disadvantages:
b. It dissolves fats and lipids; as a general rule, alcohol containing fixatives are contraindicated when lipids are to be
studied.
7. Osmium tetroxide
This is a pale yellow powder which dissolves in water (up to 6% at 20oC) to form a strong oxidizing solution.
Advantages:
a. It fixes conjugated fats and lipids permanently by making them insoluble during subsequent treatment with alcohol
and xylene.
Fats from hydrated osmium dioxide, are stained black and therefore are easier to identify.
b. It preserves cytoplasmic structures well, e.g. Golgi bodies and mitochondria.
c. It fixes myelin and peripheral nerves well, hence, is used extensively for neurological tissues.
e. It adequately fixes materials for ultrathin sectioning in electron microscopy, since it rapidly fixes small pieces of
tissues and aids in their staining.
h. Some tissues (e.g. adrenal glands) are better fixed in vapor form of osmium tetroxide. This eliminates “washing out”
of the fixed tissue.
Disadvantages:
b. It is a poor penetrating agent, suitable only for small pieces of tissues.
c. 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.
d. Prolonged exposure to acid vapor can irritate the eye, producing conjunctivitis, or cause the deposition of black osmic
oxide in the cornea, producing blindness.
Precaution:
a. It should be kept in a dark–colored, chemically clean bottle to prevent evaporation and reduction by sunlight or
organic matter.
b. Eyes and skin may be protected by using a fume hood or wearing protective plastic masks or gloves while using
osmium tetroxide.
c. Addition of saturated aqueous mercuric chloride solution (0.5 to 1ml / 100 ml of stock solution) will prevent its
reduction with formation of black deposits.
e. Osmic acid – fixed tissues must be washed in running water for at least 24 hours to prevent formation of artifacts.
f. To prevent contact of tissues with black precipitate formed in the bottom of the jar, the tissue may be wrapped in
cotton gauze and suspended in the fluid by means of a thread.
8. Heat
This procedure involves thermal coagulation of tissue proteins for rapid diagnosis, usually employed for frozen tissue
sections and preparation of bacteriologic smears.
Advantages:
a. Fixation is better
Disadvantages:
a. It produces considerable tissue shrinkage and distortion
B. Compound fixatives – are made up of two or more fixatives which have been added together to obtain the optimal
combined effect of their individual actions upon the cells and tissue constituents.
A. Microanatomical fixatives
Are those which permit the general microscopic study of tissue structures without altering the structural pattern and
normal intercellular relationship of the tissues in question.
1. 10% formol saline – is a simple microanatomical fixative made up of saturated formaldehyde (40% grams by weight
volume) diluted to 10% with sodium chloride; it is recommended for fixation of central nervous tissues and general
post–mortem tissues for histochemical examination.
48 hours at 20 – 25oC
Advantages:
b. It preserves microanatomic and cytologic details with minimum shrinkage and distortion.
c. Large specimen may be fixed for a long time provided that the solution is changed every three months.
f. It does not over harden tissue, thereby facilitating dissection of the specimen.
h. It allows natural tissue color to be restored upon immersion in 70% alcohol.
Disadvantages:
b. Formol–saline fixed tissues tend to shrink during alcohol dehydration; this may be reduced by secondary fixation.
d. Acid dye stains less brightly than when fixed with mercuric chloride
2. 10% Neutral Buffered Formalin – is recommended for preservation and storage of surgical, post–mortem and
research specimens.
Advantages:
Disadvantages:
3. Heidenhain’s Susa – recommended mainly for tumor biopsies especially of the skin; it is an excellent cytologic
fixative.
Advantages:
b. 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.
c. It permits most staining procedures to be done, including Silver Impregnation, producing brilliant result with sharp
nuclear and cytoplasmic details.
e. Susa–fixed tissues may be transferred directly to 95% alcohol or absolute alcohol, thereby reducing processing time.
Disadvantages:
a. Prolonged fixation of thick materials may produce considerable shrinkage, hardening and bleaching; hence, tissues
should not be more than 1 cm thick.
d. Mercuric chloride deposits tend to form on tissues; these may be removed by immersion of tissues in alcohol iodine
solution.
Precaution:
a. 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.
4. Formol Sublimate (Formol Corrosive) – formol–mercuric chloride solution is recommended for routine post–mortem
tissues.
Advantages:
c. It is excellent for many staining procedures including Silver Reticulum methods.
d. It brightens cytoplasmic and metachromatic stains better than with formalin alone.
f. There is no need for “washing–out”; tissues can be transferred directly from fixative to alcohol.
Disadvantages:
a. Penetration is slow; hence, tissue sections should not be more than 1cm thick.
5. Zenker’s solution – is made up of Mercuric Chloride stock solution to which Glacial Acetic acid has been added just
before its use. It is recommended for fixing small pieces of liver, spleen, connective tissue fibers and nuclei.
Advantages:
f. It may act as a mordant to make certain special staining reactions possible.
Disadvantage:
a. Penetration is poor.
c. Prolonged fixation (for more than 24 hours) will make tissues brittle and hard.
d. It causes lysis of red blood cells and removes iron from hemosiderin.
f. It has the tendency to form mercuric pigment deposits or precipitates.
Precaution:
a. Do not let tissues stay in solution for more than 24 hours.
c. Tissues must be washed out thoroughly in running water to permit good staining.
Advantages:
a. It is an excellent microanatomic fixative for pituitary gland, bone marrow and blood containing organs such as spleen
and liver.
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.
Advantages:
Disadvantages:
a. It penetrates poorly; hence, its use is limited to small fragments of tissue.
b. Picrates are soluble in water, hence, tissues should not be washed with running water but rather, transferred directly
from fixative to 70% alcohol.
e. It produces RBC hemolysis and remove demonstrable ferric iron from blood pigments.
8. Brasil’s solution
Advantages:
Overnight tissue fixation by automatic processing techniques may utilize 3 – 4 changes of Brasil’s fixative at 1 ½ to 2
hours each, succeeded directly by absolute alcohol.
B. Cytological fixatives
1. Nuclear fixatives
a. Flemming’s fluid is the most common Chrome–Osmium Acetic Acid fixative used, recommended for nuclear
preparation of such sections.
Advantages:
(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.
b. Carnoy’s fluid – recommended for fixing chromosomes, lymph glands and urgent biopsies.
(6) It is an excellent fixative for glycogen since aqueous solutions are avoided.
(7) It is very suitable for small tissue fragments such as curettage and biopsy materials.
(8) Following fixation, tissues may be transferred directly to absolute alcohol, thereby shortening processing time.
Disadvantages:
(6) It leads to polarization unless very cold temperature (–70oC) are used.
c. Bouin’s fluid (see discussions above)
d. Newcomer’s fluid
Advantages:
2. Cytoplasmic fixatives
a. Fleming’s fluid without Acetic acid – made up only of chromic and osmic acid, recommend for cytoplasmic structures
particularly the mitochondria. The removal of acetic acid from the formula serves to improve the cytoplasmic detail of
the cell.
b. Helly’s fluid
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.
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.
Advantages:
(2) It hardens tissues better and more rapidly than Orth’s 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.
(4) Prolonged fixation blackens tissue pigments, e.g., melanin; this may be removed by washing the tissue in running tap
water prior to dehydration.
e. Orth’s fluid
Advantages:
(1) It is recommended for study of early degenerative process and tissue necrosis.
Disadvantages:
This is a simple microanatomical fixative made up of Standard Formaldehyde diluted to 10% with sodium chloride; it is
recommended for fixation of Central Nervous Tissues and general post–mortem tissues for histochemical examinations.
48 hours at 20 – 25oC
Advantages:
a. It is recommended for the study of water diffusible enzymes especially phosphatases and lipases.
c. It is used as a solvent for certain metallic salts to be used in freeze substitution techniques for tissue blocks.
Disadvantages:
Advantages:
1. Secondary fixation – is the process of placing an already fixed tissue in a second fixative in order to:
b. To make special staining techniques possible (with secondary fixative reacting as mordant).
c. To ensure further and complete hardening and preservation of tissues.
This maybe done before dehydration and on deparaffinized sections before staining, usually with 10% formalin or 10%
formol saline as a primary fixative.
2. Post–chromatization – secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5 – 3%
potassium dichromate for 24 hours, to act as a mordant for better staining effects and to aid in cytologic preservation of
tissues.
3. Washing out – is the process of removing excess fixative from the tissue after fixation in order to improve staining
and remove artifacts from the tissues. Several solutions may be used.
a. Tap water – is used to remove:
(1) Excess chromates from tissues fixed in Helly’s, Zenker’s and Fleming’s solution.
b. 50 – 70% alcohol – is used to washout excess amount of picric acid (Bouin’s solution)
A. Retarded by
1. Size and thickness of the tissue specimen – larger tissues require more fixatives and longer fixation time.
2. Presence of mucus – prevents complete penetration of fixative; hence, tissues that contain mucus and fixed slowly
and poorly. Excess mucus may be washed away with normal saline solution.
3. Presence of fat – fatty tissues should be cut in thin sections and fixed longer.
4. Presence of blood – tissues containing large amount of blood (e.g. blood vessels and spleen) should be flushed out
with saline by arterial cannulization before fixing.
B. Enhanced by
1. Size and thickness of tissues – smaller and thinner tissues require less fixative and shorter fixation time.
2. Agitation – fixation is accelerated when automatic or mechanical tissue processing is used.
3. Moderate heat (37 – 56oC) accelerates fixation but hastens autolytic changes and enzyme destruction.
1. Autopsy materials should be fixed as soon after death as possible to prevent decomposition and autolysis due to
deprived oxygen and metabolism. If this is not possible, the body should be placed in a mortuary refrigerator (kept at
temperature of 4oC) or undergo arterial embalming for better tissue preservation.
2. Surgical specimens should be fixed as soon as possible after removal or refrigerated if fixation is to be delayed, to
prevent drying of surface layers and ultimate tissue distortion.
If placed in NSS during the operation, autolysis may occur before fixation is carried on.
4. If tissues are refrigerated, slow freezing of unfixed tissues near 0oC must be avoided since this may promote
formation of ice crystal artifacts. Repeated freezing and thawing, on the other hand, will destroy cellular organelles,
release enzymes and diffuse soluble components of the cell.
5. Tissue slices should be taken at right angles to the surface of the organ and should be sufficiently deep to show the
normal anatomic components.
6. Tissues should not be more than 5 mm thick except in lung edema (in which case tissue slices may be 1 – 2 cm thick),
with minimum squeezing and handling.
7. Purulent materials, exudates or transudates should be marked and kept for possible cultures, smears and other
bacteriologic examinations.
8. The amount of fixative must be adequate, approximately 20 – 50 times the volume of the tissue specimen except in
osmium tetroxide which is very expensive requiring only 5 – 10 times that of tissue volume for fixation.
9. Contamination of fixed tissue with precipitate (e.g. Osmium tetroxide), should be avoided.
10. In most instances, fixed tissues must be washed thoroughly to remove salts and/or pigments before staining.
11. Low temperature retards fixation but prevents autolysis, therefore tissues should be fixed at a temperature near the
freezing point of the fixative.
12. The required period for fixation should not be exceeded since this may cause excessive hardening or brittleness of
tissues.
14. Drying should be avoided to prevent shrinkage and distortion of tissue with loss of cellular detail.
Small tissue biopsies may be placed in a petri dish with moistened filter paper to prevent drying.
15. Solid organs should be injected with, as well as immersed in, enough fixatives to ensure complete penetration and
fixation.
16. Hollow organs (e.g. stomach, intestines) should be packed with cotton soaked in fixative or completely opened
before being immersed in adequate fixing solution.
17. Air–filled lungs may float on fixative. To avoid this, the organ may be covered with several layers of gauze to
maintain it under surface.
18. Human brains should undergo intravascular perfusion (washing out of blood with Ringer’s lactate). This may,
however, lead to artifact formation with loss of blood content. They may be suspended by a cord tied under the Circle of
Willis to prevent flattening.
20. Eyes should not be dissected before they are fixed since this may lead to immediate tissue collapse and wrinkling
due to escape of vitreous humor. They are not, however, easily penetrated due to tough sclera. Formol – alcohol must
be injected before immersing the organ in the fixative.
21. Frozen sections may lead to formation of ice crystals artifacts.
21. When fixing muscles, to avoid rigor contraction and staining of artifacts, the fresh biopsy material may be stretched
for 30 minutes by sutures on each end, and left in a moist filter paper placed in a petri dish or suspended in fixative.
22. Water should not be used for glycogen–containing tissues because glycogen is soluble in water.
23. To assure rapid access of the fixative to all parts of the tissue, the tissue may be minced, that is, small pieces of
specimen may be divided into small fragments (½ to 1 x 1 mm) and transferred to the vial of a fixative by means of a
toothpick.
24. Hard tissues (e.g. cervix, uterine, fibroids, hyperkeratotic skin, fingernails, etc.) may be washed out in running water
overnight and immersed in 4% aqueous phenol solution for 1 – 3 days (Lendrum method). This will soften the tissue and
allow easier sectioning without producing any marked distortion of the cell structure.
Proper tissue processing should start with proper fixation and preservation since a badly preserved tissue will later on
yield a badly processed specimen and may prove to be unsuitable for study. The choice of fixative and mode of
processing must therefore vary depending upon the following factors:
Good cutting and staining of section is greatly dependent upon proper fixation. Errors in the choice and use of fixative
have to a very large extent adversely affected and caused ultimate failure in the processing of tissues under
investigation.
Cause:
Failure to fix immediately (the tissues was probably allowed to dry before fixing); Insufficient fixative
Cause:
Cause:
Incomplete fixation
Cause:
Incomplete fixation
Cause:
Overfixation
Cause:
Prolonged fixation
An incompletely fixed tissue may lead to improper and incomplete clearing and impregnation and may later prove to be
a hindrance to normal sectioning and staining of specimen.