Fixation is the process of preserving tissues using chemicals called fixatives. This is done to prevent post-mortem changes in tissues and maintain the tissues' morphology. The goal of fixation is to preserve tissues as closely as possible to their in-vivo state. Formalin is commonly used as a fixative due to its ability to penetrate tissues quickly and uniformly while minimizing shrinkage, and allowing most staining protocols. However, formalin fixation can result in artifacts like brown pigments if the fixative is not buffered properly. Other classes of fixatives include alcohols, aldehydes, oxidizing agents, picrates and mercurials. Factors affecting fixation include temperature, tissue size, fixative-to

1. 1

2. FIXATION
• The purpose of fixation is to preserve the tissue
in such a way that it appears the same in a
microscopic preparation as it appeared while
living and functioning.
• We must acknowledge that this is an impossible
aim, as the very act of fixation stops all functions
and distorts in some fashion the appearance.
• Nevertheless, the aim should be to approach this
ideal as closely as possible.
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3. FIXATION
A fixative is a substance which preserves the
shape, structures, relationship and chemical
constituents of tissues and cells after death.
Therefore fixation is the process of maintaining
tissue or cell morphology in a life like state as
much as possible.
N.B: Fixation helps prevent post mortem
changes like autolysis and putrefaction.
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4. A fresh, unfixed specimen after surgical removal. To prevent
degeneration or drying-out the specimen should be fixed as soon as
possible
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5. TERMINOLOGIES USED
Secondary fixation.
After fixation in formal saline, the tissue is treated with
an appropriate fixative e.g. zenkers formal and
Heidenhains susa. The second fixation or treatment
improves the preservation and staining of specific
tissue elements.
Post fixation.
Some proteins are masked by fatty substances which
do not allow fixative to penetrate tissue.
To be able to demonstrate such proteins, the tissue is
fixed, dehydrated, cleared and then later fixed in
ethanol to preserve the proteins.
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6. Washing out.
• Washing of tissue in running tap water to remove
the fixative used.
• This is because some fixatives if not washed out
can react with the dehydrating agent during
tissue processing e.g. potassium dichromate and
osmium tetroxide can react with ethanol.
Post chromatisation
This refers to treatment of tissue with 3%
potassium dichromate after normal fixation. This is
important since it links specific tissue elements to
the stains being applied e.g mitochondria.
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7. ,
It can be done by treating tissue sections with 3%
potassium dichromate for 12-24 hours.
N.B:
Post chromatization, also known as post chroming
and usually applies to tissue fixed in formal saline.
The purpose of post chroming is to mordant the
tissue.
However, post chroming is different from post
mordanting as the latter is carried out after staining
e.g. application of iodine in gram staining.
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8. POST MORTEM CHANGES
• Changes that take place on a cell or tissue after
death of an organism i.e. changes that take place on
cell or tissue after tissue has been removed from a
living person e.g. skin and breast.
• These changes include; autolysis and putrefaction.
Autolysis refers to destruction of cell or tissue caused
by enzymes produced by lysosomes which digest a
way the different cellular components following death.
Putrefaction refers to destruction of cell or tissue by
normal flora found in normal and pathological
conditions e.g. T.B found in pathological condition
leading to production of gas leading to a filthy smell.
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9. .
N.B; post mortem changes are most prominent in
highly specialized tissue e.g. liver, intestinal tract,
kidney and brain.
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10. PURPOSES OF FIXATION
1. To kill tissues by denaturing proteins(enzymes)
so that postmortem activities such as
putrefaction (bacterial attack) and
autolysis(enzyme attack) can be prevented.
Bacterial attack can be prevented by observing
very strict antiseptic techniques while autolysis
by altering the shape of the enzyme by fixatives
which leads to destruction of tissue biological
activity.
Note: severely autolyzed tissues fail to stain.
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11. CONT’…
2. Fixation helps stabilize tissue elements
(maintains relationship between cells and
extracellular substances such as connective
tissue fibers and amorphous ground substances)
so that the effect of any subsequent procedures
will be minimal.
A well fixed tissue is almost impervious/resistant
to abuse during the processing and staining
procedures.
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12. …
3. It brings out differences in refractive index and
to increase the visibility of or the contrast between
different tissue elements.
Therefore, enhancing differences in the refractive
indexes of various tissue structures will increase
the contrast between those structures.
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13. …
4. Most staining is enhanced by fixation and
frequently tissue that has not been fixed well stain
poorly.
5. Fixatives also aid in rendering tissue constituents
such as lipids and carbohydrates insoluble so that
they are studied.
6. Fixation makes tissue firm so that gross
dissection and collection of thin sections required
for processing becomes much easier.
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14. CRITERIA OF A GOOD FIXATIVE
1. Be able to maintain cell or tissue morphology.
2. Should not affect staining.
3. Be able to evenly and quickly penetrate tissue.
4. Aid attachment of the smear onto the slide to
prevent washing off during staining.
5. Ability to inactivate enzymes especially
lysosomal enzymes to avoid autolysis.
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15. .
6. Must prepare the tissue/cell for the reagents
that would be applied during staining or after
staining e.g xylene, alcohol and DPX.
7. Able to kill bacteria and molds to avoid
putrefaction.
8. Be simple to prepare and economical in use.
9. Ability to make tissue firm so that grossing and
collection of thin sections required for possessing
become easier.
10. Ability to act as a mordant which serves to link
the dye to the tissue/cell.
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16. HALLMARKS OF GOOD FIXATION
A well fixed cell will show the following features;
1. Nuclei with various crisp chromatin pattern and
a crisp blue nuclear chromatin membrane. i.e.
the nuclei should not show any smudginess,
bubbling, or fading.
2. Nuclei should not show fading.
3. There should not be cell shrinkage
4. Cell cytoplasm should be preserved and should
stain well with eosin. Smudge bubbling
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17. Crispy Chromatin
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18. FACTORS THAT AFFECT THE RATE OF FIXATION
1.Temperature; the temperature at which fixation is
carried may affect tissue morphology. In general,
increasing the temperature of the fixative up to
approximately 45-550C has little effect on tissue
morphology.
Note;
1. Traditionally, refrigerator temperatures of 0°c-4°c
was considered the ideal temperature for the
fixation of specimens for electron microscopy.
2. Formaldehyde fixation performed at room
temperature instead of refrigerator temperature
produces proper tissue preservation and less tissue
effect.
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19. Factors cont’
2.Size
• This should be considered when the gross tissue
specimen is placed in a fixative.
• Specimens such as segments of colon or small
intestines should be surgically opened to expose
all tissue layers before placed in the fixative.
• Solid organs e.g. breast, spleen and kidney
should be covered with adequate fixative and
bread-loafed to allow faster penetration of the
fixative.
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20. Factors Cont’
Volume ratio; the fixative volume should be at least 15-
20x greater than the tissue volume.
• Fixative molecules are bound chemically to the tissue
and thus the solution is gradually depleted of these
molecules.
• Tissues also contain soluble salts that are dissolved by
fixative solution.
• The 2 way exchange does not greatly alter the
characteristics of the fixative if a large volume ratio is
used.
• However if the volume of tissue is greater than that
of the solution, the fixative composition can be
altered leading to poor fixation.
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21. …
Time; ideally, tissue should be placed in a fixative
immediately after surgical removal and autopsies
should be performed immediately after death.
The more time that elapses between interruption of
blood supply and fixation, the more postmortem
changes occur.
Note;
1. Tissue that is not well fixed does not process well
and subsequently will not stain well. Therefore,
adequate fixation time is of primary importance
in quality assurance.
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22. …
Choice of fixative; this depends on the
investigation to be carried out e.g. if
immunohistochemistry is to be done on tissues,
the ideal fixation is achieved by rapid freezing.
Penetration; fixatives solutions penetrate at
vastly/massively different rates.
Research shows that formaldehyde penetrates
faster than any other fixative.
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23. …
Tissue storage: The method of wet tissue storage is
very important because some tissues may be
needed for additional studies otherwise if a tissue
is not fixed and stored properly, additional studies
may be impossible.
Neutral buffered formalin has been seen as the
best solution for tissue storage for long which is
not true with many other fixatives.
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24. …
PH; the PH of the fixative is very important in
preventing formation of pigments. Maintaining
fixative PH. between 7.2-7.4 prevents formation of
pigments in tissues which can lead to false positive
results.
Osmolality of fixative and ionic composition; The
osmolality of the buffer and fixative is important;
hypertonic and hypotonic solutions.
The best morphological results are obtained with
solutions that are slightly hypertonic
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25. …
Suspension; fixation properties are displayed in line
with the way the tissue is suspended in the fixative.
Agitation; increased agitation increases the rate at
which tissue is fixed.
Concentration of fixative; Effectiveness and
solubility primarily determine the appropriate
concentration of fixatives.
• Concentrations of formalin above 10% tend to
cause increased hardening and shrinkage.
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26. Cont’
Additives use
• The addition of electrolytes such calcium
chloride, potassium thiocyanate, ammonium
sulfate, and potassium dihydrogen phosphate
and non-electrolytes like sucrose and dextran in
fixatives improves the morphology of the fixed
tissue.
• These additives may react either directly with
proteins/enzymes causing denaturation, or
independently with the fixatives and cellular
constituents.
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27. CLASSIFICATION OF FIXATIVES
Fixatives are classified according to their mode
of action
ALCOHOLS: These fix tissue by coagulating
proteins forming a semi-solid structures that
hold intracellular structures and prevent them
from leaking out of the tissue.
Examples include; 100% methanol and 95%
ethanol.
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28. CLASSIFICATION cont’
• ALDEHYDES: These fix tissue by formation of
cross linkages and metylene bridges. These
appear like wire mesh and hold intracellular
structures with in the cell.
• They are formed as a result of the interaction
between the covalent bonding of the fixative and
hydrogen bonding with in the proteneous
structures of the tissue.
• Aldehydes also fix tissue by formation of
methylene bridges (similar to cross linkages).
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29. …
• They are responsible for masking epitopes in
biological tissue and therefore tissue fixed
with aldehydes need to be un masked before
immunohistochemistry procedures are done.
Examples of aldehydes include;
formaldehyde/formalin, glutaraldehyde and
glyoxal.
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30. Note
1. They usually cause extensive cross linking hence
causing adverse effects in immunohistochemical
methods.
2. Glyoxal is used as a 40% aqueous solution and
buffered at PH of 4.0
3. Do not cause formation of artifacts like smudgy
nuclei and distorted staining as with formalin.
4. Causes slight reduction of staining when tissue is
stored for long in it.
5. Most special stains are satisfactory after glyoxal
fixation.
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31. Formaldehyde solutions used in the lab
1. 10% neutral buffered formalin; this is the most
widely used solution for routine formalin fixation.
Its a hypotonic solution with a PH of
approximately 6.8.
2. 10% neutralized formalin; though it has been
widely used, its not recommended because the
Solution becomes acidic after withdrawal from the
storage bottle.
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32. …
3. 10% formalin saline/formal saline; this solution
is isotonic exclusive of the formaldehyde but some
times produces formalin pigments.
4. Calcium formalin; recommended especially for
the fixation and preservation of phospholipids in
tissues.
5. Formalin ammonium bromide; recommended
for tissue specimens of the central nervous system.
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33. …
6. Modified millonig formalin
7. Alcoholic formalin
8. Phosphate buffered paraformaldehyde
9. Acetate formalin
10. 10% aqueous formalin
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34. WHY 10% FORMALIN IS ROUTINELY IN HISTOLOGY
1. Cause less shrinkages to tissue as compared to
other fixatives.
2. It's chemical composition is stable and can be used
for a long time.
3. Relatively cheap and readily available as compared
to other fixatives.
4. Hardens tissue more than any other fixative except
acetone and ethanol.
5. Allows most staining protocols to be performed on
tissue fixed in it.
6. Quickly and evenly penetrates tissue compared to
other fixatives,
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35. DISADVANTAGES OF USING 10% FORMAL
SALINE
• When used in addition with Nacl to achieve the
correct osmolarity, the solution becomes acidic
by reacting with atmospheric oxygen leading to
formation of formic acid which leads to black
acid hematin ( formalin pigment)
• This is common in tissue containing a lot of
blood. The formalin pigments are formed when
the PH of formalin drops below 6.0.
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36. OXIDIZING AGENTS
• These also fix tissue by formation of cross links
with the proteins in the tissue .
• However, they are not routinely used because
they cause extensive denaturation of proteins.
• Examples include; Potassium dicromate and
Osmium tetroxide.
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37. Note
1. Osmium tetroxide is primarily used for fixation of
specimens for electron microscopy.
2. Used in fixation and preservation of fats and
lipids in tissues
3. It penetrates only a few cell layers so the
sections must be extremely thin.
4. It cause tissue swelling which can be minimized
by addition of calcium or sodium chloride in
osmium containing fixatives.
5. Potassium dichromate also preserves lipids but
not at the same degree as osmium tetroxide.
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38. PICRATES
These are fixatives that contain picric acid.
examples include; bouin’s and hollande’s solutions.
N.B;
1. Most picrates work on coagulation principle.
2. Picrates like bouin’s solution stain everything
they come into contact with yellow.
3. However it can be removed with 50- 70%
ethanol, lithium carbonate. separately or during
the staining sequence.
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39. CONT’
3. Bouin’s solution is an excellent general fixative
for connective tissues(glycogen) and hollande’s
solution is excellent for gastrointestestinal biopsies
and endocrine tissues.
4. Picrates leaves tissue very receptive to acid dyes
like eosin and gives tissue a very good soft
consistency.
5. The only disadvantage with picrates is either
causing extreme shrinkage or allows extreme
shrinkage to occur in the subsequent processing
steps.
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40. MERCURIALS
These are fixatives that contain mercuric
chloride e.g. Zenkers ,Helly’s,
schaudinn’s,ohlmacher’s, Barnoy-lebrun and B5
solutions.
Note;
1. Fixatives containing mercuric chloride are
mostly used on haemopoetic tissues like bone
marrow, liver, spleen , lymph node and kidney.
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41. MERCURIALS
2. Major advantage of using mercury as a
fixative is that it leaves tissue highly receptive to
staining.
3. Mercury is highly poisonous. Therefore,
fixatives containing it are not routinely used.
4. Mode of action of mercurials is un known.
5.Presence of mercury in tissue inhibits its
freezing . Therefore, frozen sections are difficult
to prepare.
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42. FIXATION ARTIFACTS
Artifacts are structures or features in tissue that
interfere with normal histological examination
under the microscopic.
Fixation artifacts are pigments that end up being
deposited in tissues after using histological
fixatives.
Examples include;
1. Formalin pigments,
2. Pink disease artifacts,
3. Chrome deposited
4. Mercury chloride pigments.
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43. FORMALIN PIGMENTS
• Also called acid formalin hematin. They appear as
brown granules in tissue and the concentration of
the pigments is highest in tissue containing blood
vessels due to the presence of hemoglobin which
reacts with formic acid.
• The formic acid is as a result of a reaction between
formalin and atmospheric oxygen.
• Formalin pigments can be avoided by using buffered
formalin at a PH of 7.0. the buffer breaks down
formic acid to water and hydrogen ions. However, if
they are already present in tissue, they can be
removed using barrets method.
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44. BARRET'S METHOD
1. Take sections to xylene to remove cover slip and dpx
2. Put in 100% alcohol to remove the xylene
3. Hydrate to water.
4. Rinse in absolute ethanol
5. Flood the sections with saturated alcohol picric acid
for 5-10 minutes.
6. Wash with 95% ethanol to remove the yellow color
of picric acid and control this microscopically.
7. Wash with tape water.
8. Stain as desired.
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45. MERCURY CHLORIDE PIGMENTS
• This is a dark to brown pigment found in sections
of tissue fixed using fixatives containing mercuric
chloride.
• Found uniformly distributed in sections.
• It can be removed from tissues during tissue
processing by adding 0.25% sodium iodine in the
80% ethanol used for dehydration.
• Pigments are removed by converting mercuric
chloride into mercuric iodine which is water
soluble.
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46. CHROME DEPOSITS
These are found in tissue fixed using potassium
dichromate.
They are fine granules that are removed from
tissue by washing sections in running tap water
or
washing in 1% acid alcohol then water.
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47. PINK DISEASE ARTIFACTS
These are artifacts found in tissue rapidly fixed in
buffered formal saline.
This is common in lymphode and epithelial cells
and characterized by the nucleous failing to stain
with hematoxylin and instead taking up eosin.
These artifacts are avoided by adding 2% acetic
acid to the fixative
or by adding 1% HCL to the absolute ethanol used
to dehydrate sections.
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48. TROUBLE SHOOTING IN FIXATION
Autolysis caused by delayed fixation which is
solved by;
 Placing specimens in fixatives solution as soon as
possible and ensuring that the volume is 15-20x
that of the tissue.
 Surgically opening large specimens such as
segments of colon or small intestines to expose
all layers such that the inner epithelial surfaces
are covered by the fixatives.
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49. .
Slicing /bread loafing solid/large organs such as
the kidney, spleen and breasts to ensure
adequate fixation.
Note;
Autolysis may be shown by a loss or total
disappearance of nuclear chromatin.
Some cells may disappear such as epithelial cells in
intestinal specimens or there may be cell shrinkage
with artifactual space around the cells.
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50. Incomplete fixation
• Because of rapid turnaround time deemed
necessary, specimens are not usually fixed
adequately.
• If tissue is not well fixed when processing has
begun, fixation continues in the alcohol and the
center of tissue will often be more eosinophillic
than the peripheral.
• If signs of incomplete fixation is noted on H and E
stained section, the following corrective actions
should be taken;
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51. Corrective actions
Increase the time allowed in fixative solution
Change to another fixative such as zinc formalin
which still requires several hours for complete
fixation or Glyoxal which is an extremely rapid
fixative.
Place formalin alcohol in the first 3 changes of
the processing cycle to decrease fixation time
and also begin dehydration.
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52. Corrective actions
Ensure that the gross sections are thin enough
for good reagent penetration and the amount
of fixative is 15-20x that of the tissue.
Ensure that the formalin solution is not
depleted because of overuse; change the
solutions frequently.
Use agitation of cassettes in fixatives holding
solutions during or after grossing.
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53. Ischemia time
• Cold ischemia time, is defined as the time from
the removal of the tissue from the patient to the
initiation of tissue fixation.
• This time should be shortened as much as
possible, specifically, no more than 1h.
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54. REVISION QUESTIONS
1. Define; fixation, coagulative fixative, non-
coagulative fixative, additive fixative, non-
additive fixative.
2. List down the functions of fixation.
3. Discuss the factors that affect the rate of tissue
fixation.
4. Describe the Barrets method of removing
pigments from tissues.
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55. .
5. Explain the mode of action of;
a) Aldehydes fixatives
b) Picrate fixatives
c) Mercurial fixatives
d) Alcohols
6. List down 4 reasons why 10% formalin is the
routinely used fixative in a histopathology lab.
7.Discuss the classification of fixatives according
to composition.
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