Tissue samples undergo autolysis or microbial degradation after removal from the body. Fixation uses chemicals to preserve tissues by terminating biochemical reactions and maintaining the tissue's shape and size. Effective fixatives prevent autolysis and bacterial growth, allow long-term storage, maintain cell volume, and enable clear staining. Tissues can be fixed through various methods including immersion, coating, vapor, perfusion, freeze-drying, and microwave fixation. Factors like fixation speed and uniformity, impact on antigenicity and staining, and safety risks vary between methods.

2. Once the tissue is removed from the body it will
go through a process of self-destruction.
This process is known as autolysis.
If tissue is left without any preservation, then a
Microbial attack will occur, the process is known
as putrefaction.
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3. In the fields of histology, pathology, and cell
biology, fixation is a chemical process by which
biological tissues are preserved from decay, either
through autolysis or putrefaction.
Fixation terminates any ongoing biochemical
reactions, and may also increases the mechanical
strength or stability of the treated tissues.

4. The basic aims of fixation are the following:
To preserve the tissue nearest to its living state
To prevent any change in shape and size of the tissue at
the time of processing
To prevent any autolysis
To make the tissue firm to hard
To prevent any bacterial growth in the tissue
To make it possible to have clear stain
To have better optical quality of the cells

5. 1.Prevention of autolysis of the cells or tissue
and prevention of decomposition of the tissue
by bacteria.
Allow long storage of tissues

6. 2. Maintaining the volume and shape
of the cell as far as possible
It must kill the cell quickly without
shrinkage or swelling

7. 3. Consistently high-quality staining
particularly routine stain.
Must allow staining

8. 4. Rapid action.
It must penetrate the tissue rapidly
5.Cheap
6.Non-toxic

9. 1. Volume changes:
Fixatives may change the volume of the cells.
Osmium tetroxide cause cell swelling.
Volume change may be due to:
a) Altered membrane permeability,
b)Inhibition of the enzymes responsible for respiration
c)change of transport Na+ ions.

10. 2. Hardening of tissue:
The fixation changes the
of the tissue.

11. 3. Interference of
staining:
Fixation may cause hindrance of
of enzymes.
–Formaldehyde inactivates 80% of ribonuclease
enzyme (RNAse).
–osmium tetroxide inhibits haematoxylin and
staining.

12. 4. Changes of optical density by
fixation:
The fixation may cause changes in the
nuclei.
The nuclei may look like condensed and
hyperchromatic.
Change of optical density of the nuclei

13. The fixative can be classified on the basis of the
following criteria :
A.Nature of fixation
•Immersion
•Coating
•Vapor
•Freeze – Drying
•Microwave

14. B. Chemical properties
Aldehyde
Oxidizing agent
Protein denaturing
Cross – Linkage

15. C. Component present
Only one chemical present or
More than one

16. D. Action on tissue protein
Coagulalative
Non – coagulative

17. 1.Immersion fixation
2.Coating fixation
3.Vapour fixation
4.Perfusion fixation
5.Freeze – dry fixation
6.Microwave fixation
7.Heat fixation

18. The whole specimen is immersed in the liquid
fixative
Examples:
Tissue samples are immersed in 10% neutral
buffered formalin or
Cytology smear immersed in 95% ethyl
alcohol.
The most used fixation in the laboratories.
(1) Immersion fixation

19. Spray fixation
The spray fixative usually consists of alcohol and wax.
This is commonly used in the cytology samples.
 The spray fixative is used for easy transportation of
the slide.
(2) Coating fixation

20. Advantages:
a)Fixation of the cells
b)To impart a protective covering over the
smear
c)No need to carry liquid fixative in bottle or
jar

21. Notes:
The spraying over the smear should be smooth
and steady, and the optimum distance of 10–12
inches should be maintained between the nozzle
of the spray and the smear.
The spray fixative usually consists of alcohol and
wax.
Therefore, this wax should be removed before the
staining procedure.

22. Both Histology & Cytology samples.
The vapour of chemical is used to fix either a
smear or tissue section.
The vapor converts the soluble material to
insoluble material, and these materials are
retained when the smear comes in contact with
liquid solution.
(3) Vapour fixation

23. Vapour Fixative examples:
Formaldehyde.
Osmium tetroxide.
Glutaraldehyde.
Ethyl alcohol.

24. The fixative solution is infused in the
arterial system of the animal, and the
whole animal is fixed.
The organ such as the brain or spinal cord
can also be fixed by perfusion fixation.
Mainly used in research purpose.
(4) Perfusion fixation

25. Used for research purpose
This technique is useful to study:
soluble material
very small molecules.
(5) Freeze – drying fixation

26. The tissue is cut into thin sections.
Immersed in liquid coolant “Quenching”.
liquid nitrogen, Propane & Isopentane.
Then rapidly frozen into a very low
temperature.
keeping it in close contact with chilled metal.
How to perform Freeze – Drying fixation?

27. Then ice within the tissue is removed
with the help of vacuum chamber in higher
temperature (−30 °C).

28. Advantages:
Excellent for enzyme study
No changing proteins
No shrinkage tissue
Preservation of glycogen

29. Microwave is a type of electromagnetic
wave.
Its frequencies between 300 MHz and 300
GHz.
Its wavelength varies from centimetre to
nanometre.
(6) Microwave fixation

30. It operates with a frequency of 2.45 GHz and 0.915 GHz.
The electromagnetic field is created by the microwave, and
Dipolar molecules (water) rapidly oscillate in this
electromagnetic field.
This rapid kinetic motion of these molecules generates uniform
heat.
Homogeneous increase of temperature within the tissue
─every part of the tissue is heated.
The generated heat accelerates the fixation.

31. Factors controlling the temperature
rise:
The rise of temperature in the microwave
heated media depends mainly on:
The dielectric property of the media
Thermal properties of the material
Radiation level
Orientation and shape of the object

32. Advantages:
 Rapid processing.
No change in volume of tissue.
Preservation of the tissue antigen and
good for immunohistochemistry.
Other fixation methods may prevent
antibody bindings.
It facilitates the staining reaction
any bad effect.

33. Disadvantages:
Safety problems
1.The tissue immersed in formalin during
during microwave fixation may generate
generate large amount of toxic gas.
Solution:
Overhead hood  for the removal of this
substance from the microwave.
2.Heat injury.
Overhead hood

34. Applications:
Surgical pathology laboratory
Electron microscopy after
tetroxide fixation
Urgent processing of biopsy (e.g.
kidney biopsy)

35. The simplest form of fixation.
boiling tissue in normal saline.
Boiling an egg
• precipitates the proteins
Cutting an egg  the yolk and egg white can be
identified separately.
Each component is less soluble in water after heat fixation than
the same component of a fresh egg.
(7) Heat fixation

36. The tissue should be free from
excessive blood before putting it
into fixative.

37. Tissue must be very thin.
Tissue should be thinly cut in 3–5
mm thickness.

38. The amount of fixative must be
more than the tissue.
The amount of fixative fluid should be
20 times more than the volume of the
tissue. So that it can penetrate all parts.

39. The tissue with fixative should be
in a tightly screw-capped bottle.

40.  "Basic and Advanced Laboratory
Techniques in Histopathology and
Cytology" by Pranab Dey
Bancroft’s Theory and Practice of
Histological Techniques