This document discusses fixation and fixatives used in histopathology. It defines fixation as a chemical process that preserves tissues from decay by terminating biochemical reactions and increasing stability. The objective of fixation is to preserve cells and tissues in a lifelike state to allow for further processing without change. It then discusses various types of fixatives, how to choose a fixative, fixation methods, and factors that affect fixation quality. Key fixatives mentioned include formaldehyde, glutaraldehyde, mercuric chloride, picric acid, and osmium tetroxide.

1. Fixation & Fixatives
Dr mohamed serar
Msc, Bsc,Histopathologist

2. • Fixation is a chemical process by which tissues
are preserved from decay.
• Fixation terminates any ongoing biochemical
reactions, and may also increase the
mechanical strength or stability of the treated
tissues.

3. • The objective of fixation is to preserve cells and
tissue constituents in as close a life-like state as
possible and to allow them to undergo further
preparative procedures without change.

4. • Prevent autolysis and bacterial
decomposition.
• Harden the tissue against the later chemical
and physical events.
• Preserve tissue as live as possible or as near to
it.
• Enhance the refractive index.
• Ensure a good processing and staining.
• Preserve the tissue arrangement.
Functions Of a Good Fixative

5. 1. The cost and the availability.
2. The procedure to be applied.
3. The sample or the substances to be fixed.
4. The speed of fixation.
How to Chose a Good Fixative

6. • Preserve the Microantomical relationships.
• Preserve cellular components.
• Preserve the important chemical structures.
Methods Of Fixation

7. • Perfusion fixation.
• Immersion fixation.
• Vapor fixation.
• Microwave fixation.
• Freeze Drying Fixation.
Types Of Fixation Procedures

8. • Temperature.
• Size and penetration of fixative.
• Changes in volume.
• pH and Buffers.
• Osmolality.
• Concentration of fixative.
• Duration.
• Agitation and suspension.
Factors Affecting Fixation

9. • Nucleus:
- Pyknosis.
- Karryorhexis.
- Karyolysis.
Effects of bad Fixation

10. • Cytoplasm:
- Granulation.
- Vacculation.

11. • General:
- Swelling.
- Shrinkage.

12. • Bad fixation effects the staining reaction by causing hypo -
chromatin or hyper -chromatin staining, but it doesn't affect
the appearance reaction –lose of stain reaction.
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13. Ideal fixative or how fixative should
work
• A fixative should conver structural, stability to the
tissues and in order to do so, it changes the chemical
composition of the tissues.
• The fixative must not be proteolytic, that is, it must
not break the peptide linkage of the protein causing
hydrolysis of proteins freeing it is soluble amino acid.
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14. CLASSIFICATION OF FIXATIVES

15. According to the Mechanisms
• Aldehydes: Cross linking fixatives react with
the basic amino acid Lysine.
• Oxidizing agents: React with various sides of
protein making cross links. (Potassium
dichromate, chromic acid, potassium
permanganate).

16. • Protein denaturing agents. (Methyl Alcohol,
Ethyl Alcohol, Acetic Acid)
• Unknown mechanisms (Picric Acid, Mercuric
Chloride).

17. According to Components
1. Simple Fixatives: Contain one chemical
compound.
2. Compound Fixatives: Contain more than one
simple fixative.

18. All of these are Compound Fixatives:
- Aldehyde containing fixatives.
- Alcohol containing fixatives.
- Mercuric Chloride containing fixatives.
- Picric acid containing fixatives.

19. CLASSIFICATION OF FIXATIVES

20. According to Components
• Simple Fixatives: one chemical compound.
• Compound Fixatives: more than one simple
fixative.

21. According to their use
1. Microanatomical Fixatives: preserve
relationship between tissue layers.
2. Cytological Fixatives: preserves cell
constituents.
3. Histochemical Fixatives: preserve special and
certain components in the tissue.

22. According to their action
• Additive Fixatives: contain atoms of the
fixative join protein and form new compound.
(Coagulation and precipitation) e.g. Aldehyde
fixatives.
• Non-additive Fixatives: no obvious
combination is happening but the nature of
the protein is denatured and form coagulation
(e.g. egg boiling) e.g. mercuric chloride.

23. According to the active group
1. Aldehyde containing Fixatives.
2. Alcoholic containing Fixatives.
3. Picric acid containing Fixatives.
4. Mercuric chloride containing Fixatives.
5. Chromic acid containing Fixatives.

24. Other Classifications
1. Cross-linking Fixatives.
2. Aldehydes Fixatives.
3. Oxidizing agents.
4. Precipitating agents.
5. Others.

25. Secondary Fixation

26. The primary fixation process may not be
optimal; the wet tissue may be subjected
to secondary fixation. For example,
tissues fixed in buffered formalin may be
subjected to further fixation with a
mercuric chloride-formaldehyde solution
for a period of several hours to improve
staining of sections.

27. • OR
• Is fixative used after the primary fixative to
help demonstration of specific structure.
• e.g ( Mercuric chloride, absolute alcohol )
• Use after primary fixative (10% formalin).
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28. Post Fixation

29. It is all the processes done to the fixed
tissue after the fixation is completed.
Washing in water, neutralization,
secondary fixation and the removal of
fixative pigments and artifacts all can
be said it is a post fixation procedures.

30. Fixation pigments and artifacts and them
removal

31. - Formalin pigments: it occurs in blood rich
tissues such as blood vessels. It appears as
brown to black deposition in the tissue. It can be
removed by using Saturated alcoholic picric acid.
- Mercuric chloride pigments: also brown to
black pigments can be removed by using
alcoholic Iodine or Na thiosulphate.
- Picric acid pigments: a yellowish pigments can
be removed by using Alcohol

32. Fixatives

33. Simple Fixatives
• Formaldehyde.
• Glutraldehyde.
• Mercuric chloride.
• Potassium dichromate.
• Chromic acid.
• Picric acid.
• Osmium tetroxide.
• Acetic acid.
• Ethyl alcohol.

34. Formaldehyde
• Gas soluble in water to 40%.
• Contain 10-14% methanol (stabilizer).
• Nearly acidic (formic acid).
• Neutralized by Mg, Ca carbonate or buffer.
• Production of Para-formaldehyde (cold).
• Yellow means contamination with ferric ions.

35. Advantages
• Good hardening.
• Little shrinkage.
• Preserve collagen.
• Fix proteins by making additive compounds not
precipitating.

36. Disadvantages
• Unpleasant irritating vapor to eyes, skin and
respiratory system.
• Formalin pigments. (NaCl hydrogen buffer).

37. Glutraldehyde
• Ultra structural studies and cytochemistry.
• E.M.
• Primary fixative followed by osmium tetroxide.
• Routine.

38. Advantages and dis.
• No advantages over formalin.
• Poor penetration in large pieces of tissue.
• Give +ve reaction with PAS tech.
• Expensive.

39. Mercuric chloride
• Corrosive sublimate.
• White crystalline soluble in water 7% and 33%
in alcohol.

40. Advantages.
• Powerful protein precipitant.
• Harden tissue easily.
• Used in mixture.
• Shrink but doesn’t distort tissue.
• Fix both nucleus and cytoplasm.

41. Disadvantages.
• Poisonous (acid fuchsin).
• Corrosive to metal.
• Produces mercury pigments.

42. Potassium dichromate
• Range in pH from 3.4-3.8.
• Less acidic fix cytoplasm and mitochondria.
• More acidic fix cytoplasm and nucleoproteins.
• Wash in running tap water after fixation.

43. Chromic acid
• Precipitate proteins and fix CHO.
• Powerful oxidizing agent.
• Wash in running water before treat with
alcohol (precipitate).

44. Picric acid
• Explosive.
• Precipitate nucleoproteins.
• Cause shrinkage.
• Component in Glycogen fixatives.
• After fixation transfer to alcohol.

45. Osmium tetroxide
• Very expensive.
• Vapor is irritant.
• Forms additive compounds.
• Has poor penetration.
• E.M.

46. Acetic acid
• Swells collagen fibers.
• Precipitate nucleoproteins.
• Solvent for cytoplasmic granules.

47. Ethyl Alcohol
• Cause shrinkage.
• Coagulate proteins.
• Precipitate Glycogen.
• Dissolve some but not all lipids.

48. Micro Anatomical fixatives
• 10% formalin and 10% buffered formalin.
• Mercuric chloride.
• Bouin’s fluid.
• Carnoy’s.
• Flemming’s
• Suza.
• Zinker.
• Sanfelice’s.

49. Cytological Fixatives
• Nuclear fixatives:
- Carnoy’s.
- Clark’s fluid.
- Flemming’s fluid.

50. Cytological Fixatives
• Cytoplasmic fixatives:
- Formal saline followed by Potassium
dichromate.
- Champy’s fluid.

51. Histochemical Fixatives
• Absolute alcohols.

52. Summary of Fixatives