This gives an overview of numerous fixatives used in tissue processing.

1. FIXATIVES
P.S KAAVIYA THARSINI
Ist YEAR MDS

2. Human cells remain alive
because they are connected
to the blood stream, which
provides them with the
oxygen and nutrients.
When they are removed
from the body, the cells will
be cut off from the blood
circulation
Once the cells are deprived
of oxygen for long enough,
they will die
In histology, we would like
to see them as they were in
living body and that is done
by FIXATION.

3. FIXATION
• Fixation is a complex series
of events which preserves the
tissues as close as life like state
as possible by preventing
autolysis and putrefaction

4. BASIC AIMS of Fixation
1.Preservation of tissue in a life like manner and preventing from post mortem
changes
2. Preservation of chemical compounds
3. Solidification and hardening for easy sectioning
4. Prevent shrinkage and swelling
5. Optical differentiation

5. A PERFECT FIXATIVE NEEDS
 Penetrate tissues quickly and evenly
 Prevent autolysis and putrefaction.
 Not add any extraneous material to the
tissue.
 Not swell or shrink the tissue.

6. v Prepare the tissue for later treatments such
as staining and not prevent any later
investigation that might be needed.
 Prevent desiccation and drying of tissue, which
would cause shrinkage and distortion.
 Be safe to use (non-flammable).
 Be reasonably priced.
 Be convenient to use (shelf life, storage, etc.).
A PERFECT FIXATIVE NEEDS​

7. TYPES OF
FIXATIon
PHYSICAL CHEMICAL
HEAT
MICROWAVE
FREEZE DRYING
CROSS LINKING
COAGULANTS
COMPOUND

8. CHEMICAL FIXATION
Chemical fixation utilizes organic or
non-organic solutions to
maintain adequate morphological preservation.​
METHODS:
 Immersion
 Perfusion
 Vapor
 Spraying

9. Based on no. of chemical constituents
FIXATIVES​​ EXAMPLES​​
1​​ SIMPLE​​ Eg. Formaldehyde, ethanol, osmium tetroxide​​
2​​ COMPOUND​​ Eg. Bouins fluid, alcoholic formalin, Zenkers fluid​​

10. Classification on
the Basis
of Chemical
Composition:
FIXATIVES​ EXAMPLES​
1 Aldehydes​ Formaldehyde, glutaralde
hyde​
2 Dehydrants Methyl alcohol, ethyl
alcohol, acetone
3 Oxidizing agents​ Osmium tetroxide​
4 Miscellaneous​ Picric acid, mercuric
chloride​

11. Classification of fixatives based on type of
structures fixed
1. Histochemical fixatives Formaldehyde, glutaraldehyde,
vapor fixatives
2. Microanatomical fixatives Bouin’s fluid, 10% formalin, Zenker’s
fluid, formol calcium
3. Cytologic fixatives NUCLEAR: Carnoy’s fluid, Clarke’s fluid
CYTOPLASMIC: Champy’s fluid, glacial acetic
acid, alcohol, formol saline

12. Bakers classification of
fixatives (1958)
• Coagulants
-Dehydrating
-Others
• Cross linking
• Compound

13. DEHYDRANT
COAGULANT FIXATIVES
ALCOHOLS
• The most commonly used - ethanol and methanol
• For ethanol , fixation initiates at 50 to 60%
concentration and for methanol it is at 80%
concentration, respectively.
• They are known to be coagulants that cause protein
denaturation.

14. MECHANISM
OF
FIXATION
the structure of the protein may become partially reversed,
with hydrophobic groups moving to the outside surface of the
protein

15. Methanol is
commonly used as a
fixative for blood
films
95% ethanol is used
as a fixative for
cytology smears
both alcohols are
usually combined with
other reagents when
used as fixatives for
tissue specimens

16. ADVANTAGES
tissue shrinkage
and hardening
DISADVANTAGES

17. Compound fixatives - alcohols

18. Picric Acid
• coagulant fixative.
• It forms picrates with basic protein groups,
which causes coagulation.
• Although picric acid is not able to fix most
carbohydrates and lipids, it preserves
glycogen.
• Brighter staining is seen by picric acid fixatives
OTHERS – Acid coagulants

19. Compound fixatives – Picric acid

20. Acetic acid
• Acetic acid coagulates nucleic acids but
does not fix or precipitate proteins; it is
therefore added to other fixatives to
prevent the loss of nucleic acids.
• Basic mechanism:
These acids insert a lipophilic
anion into a hydrophilic region and disrupt
the tertiary structures of proteins

21. • Formaldehyde was
discovered in
1859 by Butlerov.
In 1889 Ttrillat was the first
who manufactured formalde
hyde commercially as
industrial reagent.
• In 1892, Ferdinand
Blum recognized that
formalin could give
benefit when used as a
fixative.
•Formaldehyde is found to
be the most commonly
used fixative.
CROSSLINKING FIXATIVES

22. • It is a pungent toxic gas that is soluble in water
• The most widely used fixative is 10% formalin solution for
routine light microscopic sections.
• Formalin = 37-40% of formaldehyde + 60-63% of water
• 10% formalin has 4% of formaldehyde

23. REACTION WITH PROTEINS
Reactive hydroxy methyl group
1
2

24. • The side chains of peptides or proteins which
are most reactive with methylene
hydrate are lysine, cysteine, histidine,
arginine, tyrosine and the reactive hydroxyl
groups of serine and threonine
REVERSIBILITY OF FORMALIN FIXATION
• Washing for 24 hours removes approximately
half of the reactive groups and after 4 weeks
up to 90% are removed

25. FORMALDEHYDE FIXATION
Formaldehyde primarily
preserves protein and the
general structure of
cellular organelles.
It can interact with nucleic
acids but has little effect
on carbohydrates and
preserves lipids if the
solutions contain calcium

28. Neutral buffered 10%
formalin
1. Tap water 900 ml
2. Formalin (37% formaldehyde solution) 100 ml
3. Sodium phosphate, monobasic, monohydrate 4 g
4. Sodium phosphate, dibasic, anhydrous 6.5 g
The pH should be 7.2–7.4

29. Various
formulations
of Formalin

30. Compound fixatives - Dehydrant cross linking

31. GLUTARALDEHYDE
• Glutaraldehyde is a bifunctional aldehyde which probably combines with the
similar groups as formaldehyde.
Glutaraldehyde was found in 1963 by Sabatini
et al as particular fixative for ultrastructural
researches.

32. Although penetration rate is
slower compared to
formaldehyde,
glutaraldehyde is more
effective cross-linker for
proteins than formaldehyde
Crosslinking is irreversible
and withstands acids, urea ,
semicarbazide and heat
Does not react with
carbohydrates or lipids
unless they contain free
amino group
GLUTARALDEHYDE

33. MECHANISM
OF FIXATION

34. exposure may result in respiratory tract, skin, and
digestive tract irritation
On exposure to oxygen, glutaraldehyde becomes
unstable and breaks down with decrease in pH.
Glutaraldehyde preserves the ultrastructure of the
tissue, thereby it is used in electron microscopy studies, but owing
to poor penetration and overhardening properties, it is not used as
tissue fixatives for light microscopy.
For stability, it requires pH of 5 and storage at 4°C. At
room temperature, glutaraldehydes are not able to cross-link
the nucleic acids.

35. • For the purpose of electron
microscopy:
• Glutaraldehyde is used 2.5% glutaraldehyde
in 100 mM phosphate buffer at pH 7.0.
• Glutaraldehyde comes commercially as 25%
or 50% solutions in 10 ml.​

36. Advantages:
1. Better fixation of ultrastructure.
2. Less cell shrinkage.
3. Preservation of protein is better.
4. Good cross-linking with collagen.
Disadvantages:
1. Poor penetration and tissue
should be less than 0.5 mm thick.
2. Less stable compound.
3. No lipid fixation.
4. Costly.

37. OSMIUM TETROXIDE
• Osmium tetroxide is type of fixative that is soluble in water
and nonpolar solvents.
• Osmium tetroxide seems to react with proteins side chains that
cause cross-linking.
• It is traditionally sold as crystalline solid that is sealed in glass
ampule. It is seen that osmium tetroxide crystals convert from
solid state to vapor state.

38. • During fixation by osmium tetroxide, either due to
slow rate of reaction or due to restricted
penetration of osmium tetroxide into tissue, large
amounts of carbohydrates as well as proteins are
eradicated.
• Osmium tetroxide is helpful for staining of lipids
in frozen sections.
• In addition to its use as a secondary fixative for
electron microscope examinations, OsO4 can be
used to stain lipids in frozen sections.

39. • It is observed that fixation by osmium
tetroxide causes swelling in tissue,
which can be decreased by adding
sodium chloride or calcium chloride to
fixatives.
• Continued exposure to osmium
tetroxide vapors can cause deposition
into cornea, which eventually leads to
blindness.

40. Advantages:
1. This is a very good fixative for lipid.
2. It preserves cytoplasmic organelles such as Golgi
bodies and mitochondria,
3. Does not make the tissue hard

42. OSMIUM
TETROXIDE
• It is commercially available in
sealed vial 0.1–1 g.
• Aqueous solution of 4% OsO4 is
made.
• This should be stored in clean glass
vial away from sunlight.
• In laboratory 2–4% OsO4 in buffer
solution of pH 7.2 is used.

43. MERCURIC CHLORIDE
• It is now rarely used due to the health and
safety issues of mercury-containing fixatives
• special stains e.g. B5, used in some laboratories
for fixing hematopoietic tissue

44. MERCURIC CHLORIDE
• These fixatives have slow
penetration capacity, so
the sections of specimens should
be thin.
The chemistry of fixation is not well
understood but it is known that it reacts
with ammonium salts, amines, amides,
amino acids and sulfydryl groups to
harden tissues.

45. Compound fixatives – Mercuric chloride

46. POTASSIUM
DICHROMATE
• It is an ionic compound with
two potassium ions(k+) and the
negatively charged dichromate
ion ( Cr2O7–)
• The two chromium atoms are in
hexavalent state(with oxidation
state +6) each attached to three
oxygen atoms as well as a
bridging oxygen atom.

47. • The fixation and hardening reactions are not
fully understood, but probably involve in the oxidation
of proteins
• Chromate is reported to make unsaturated lipids insoluble upon
prolonged (>48 hours) fixation and hence mitochondria are
well preserved by these fixatives
Fixatives containing chromate at a pH of 3.5–5.0
make proteins insoluble without coagulation.

48. • During this procedure
the chromium attaches
to the lipid and may
be used
as a mordant for some
dyes, including
hematoxylin.
• Potassium dichromate is a
very valuable fixing agent
where lipids are concerned.
This is best observed with
the postchroming procedure,
which makes many
unsaturated lipids insoluble
which take weeks to be fully
effective.
For faster demonstration of
lipids in paraffin
sections, post osmication
should be used.

49. primarily been used to
prepare neuroendocrine
tissues especially normal adrenal medulla
and its related tumors, e.g.
pheochromocytomas.
Reliance on the chromaffin reaction are now
being replaced by immunohistochemistry with
a range of neuroendocrine markers
including chromagranin A and synaptophysin

50. Various formulations of
Potassium dichromate

51. HEAT FIXATION
The simplest form of fixation is heat.
It precipitates the proteins making
it insoluble in water after heat fixation
Even though adequate morphology could be
obtained by boiling tissue in normal saline, in
histopathology heat is primarily used to
accelerate other forms of fixation as well as
the steps of tissue processing.
PHYSICAL
METHODS

52. MICROWAVE FIXATION
• Microwaves are electromagnetic wave with frequencies between 300 MHz and
300 GHz
• Microwave creates electromagnetic field, and the molecules rapidly oscillate
generating heat by motion.
• Microwave heating speeds fixation and can reduce times for fixation of some
gross specimens and histological sections from more than 12 hours to less
than 20 minutes.

53. MICROWAVE FIXATION
• Microwaving tissue in formalin results in the production of large amounts of
dangerous vapors, so in the absence of a hood for fixation, or a microwave
processing system designed to handle these vapors, this may cause safety
problems.
• Recently, commercial glyoxal-based fixatives which do not form vapors when
heated at 55°C have been introduced as an efficient method of microwave
fixation.

54. FREEZE DRYING AND FREEZE SUBSTITUITION
 Freeze-drying is a useful technique for studying soluble materials and small molecules
 tissues are cut into thin sections, immersed in liquid nitrogen, and the water is removed in a
vacuum chamber at −40°C.
 The tissue can be post-fixed with formaldehyde vapor.
 These methods of fixation are used primarily in the research environment and are rarely used in
the clinical laboratory setting

55. FACTORS AFFECTING FIXATION
TEMPERATURE BUFFERS AND PH
OF THE FIXATIVE
DURATION SIZE
OSMOLALITY CONCENTRATION ADDITIVES

56. Temperature
Room temperature
suitable for routine
work.
High temperature
facilitates fixation.
Low temperature
(0–4 °C) suitable for
enzyme
histochemistry.

57. pH of the fixative
Buffers
High acidity or alkalinity
interferes fixation.
pH 6–8 is the best range.
Neutral pH is
preferable.
The pH for the
ultrastructure preservation
should be buffered between
7.2 to 7.4.
•phosphates,​
• cacodylate,​
• bicarbonate,
• Tris, and
• acetate.​

58. DURATION AND SIZE
the depth (d) reached by a fixative is directly proportional to the
square root of duration of fixation (t) and expressed this relation as:
D = k/t
the time of fixation is
approximately equal to
the square of the
distance which the
fixative must penetrate
unfixed gross specimens
which are to be cut and
stored in fixative prior to
processing should not
be thicker than
0.5cm

59. Osmolality of the fixative solution
Hypertonic
cell shrinkage
Hypotonic
cell swelling
Best
mild hypertonic
(400–450 mOsm)

60. CONCENTRATION
Mild lower concentration with
neutral pH is preferable.
Very low concentration
prolongs the time of fixation
Higher concentration causes
rapid fixation
with undesirable effect.

61. CONCENTRATION OF FIXATIVES
Concentrations of
formalin above 10%
tend to cause
increased hardening
and shrinkage.
• Ethanol
concentrations
below 70% do not
remove free water
from tissues
efficiently
In addition, higher
concentrations result in
formalin being present
in its polymeric form,
which can be deposited
as white precipitate.

62. Additives
improves the morphology of
the fixed tissue.
The electrolytes may react either directly with proteins
causing denaturation, or with the fixatives and cellular
constituents
calcium chloride
potassium thiocyanate
ammonium sulfate
potassium
dihydrogen phosphate.

63. • The choice of electrolytes to be
added to fixatives used on
a tissue processor may vary.
• electrolytes such as phosphates
may cause problems with some
processors due to precipitation of
the salts.
• non-electrolyte substances such as
sucrose, dextran, and detergent has
also been reported to
improve fixation
Additives

64. Choice of fixative based on technique
TECHNIQUE FIXATIVE OF CHOICE
ROUTINE HISTOPATHOLOGY 10% neutral buffered formalin
ELECTRON MICROSCOPY Glutaraldehyde solution
Osmium tetroxide
IMMUNOHISTOCHEMISTRY 10% neutral buffered formalin, alcoholic
formalin,glyoxal
FNAC 95% ethanol
ENZYME HISTOCHEMISTRY Fresh frozen sections

65. TARGET SUBSTANCE FIXATIVE OF CHOICE
Protein 10% buffered formalin
Lipid Frozen section , osmium tetroxide, potassium
dichromate, formal calcium
Glycogen Alcohol based fixative , Picric acid
Mucopolysaccaharide Frozen section
Enzyme Frozen section
DNA and RNA HOPE, UMFix, PAXGene tissue fix,RCL2,Alcohol
based
Choice of Fixative based on target substance

66. Choice of
fixative
based on
tissue

67. H&E pics of tissues fixed
by different fixatives
Formaline fixed small intestine
mucosal tissue
Ethanol fixed small
intestine mucosal tissue
Bouins fixed testicular
tissue
Glyoxal fixed uterus

68. ARTEFACTS
Pigment artefact by mercuric
chloride
Pigment artefact due to formalin

69. THANK
YOU