This document provides an overview of tissue fixation. It begins with an introduction to fixation and its importance in histology. The main functions of fixatives are to prevent autolysis and putrefaction of tissue. Various fixation methods and types of fixatives are described, including aldehydes, metallic salts, picric acid, alcohols, chromates, and osmium tetroxide. The document discusses the reactions of fixatives with proteins, nucleic acids, lipids, and carbohydrates. Common fixatives like formaldehyde and glutaraldehyde are compared. Factors affecting fixation and the effects of fixation are also summarized.

1. Tissue fixation
Presented by
Dr. Shrikant Sonune
Guided by
Dr Ashok Patil,
Dr Shilpa Kandalgaonkar,
Dr Mayur Chaudhary,
Dr Suyog Tupsakhare,
Dr Mahesh Gabhane.

2. Content
• Introduction
• Function of fixative
• Methods of fixation
• Reaction of the cell(its component) with fixatives
• Simple aqueous fixatives or fixative ingredients
• Factors affecting fixation
• Effect of fixation
• References

3. Introduction

5. Introduction
• Fixation
• Tissue processing
• Sectioning
• Staining
• Staining
• Sectioning
• Tissue processing
• Fixation

6. Fixation : introduction
• Fixation is the complex series of chemical events and
differs for the different groups of chemical substances
found in tissues.
• It is most essential part of histology. Here where
everything starts.
• Why?

7. Introduction
• 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
bacterial attack will occur, the process is known as
putrefaction.

8. Definition :
• It is a process by which the constituents of the cells and
therefore of the tissues are fixed in a physical and partly
in a chemical state , so that they will withstand
subsequent treatment with various reagents with
minimum of loss, significant distortion or decomposition.
• The preservation and hardening of a tissue sample
to retain as nearly as possible the same relations
they had in the living body

9. Aims & objectives of fixation
• To prevent autolysis and putrefaction.
• Rapid and even penetration.
• To preserve cells and tissues in a life like manner as possible.
• Elements that are to be demonstrated must remain in
maximum concentration and precise localization.
• Stabilize labile elements.
• Must be rigid to allow sectioning.
• Must allow staining.
• Optical contrast must be induced for morphological
examination.
• Allow long storage of tissues

10. METHODS OF FIXATION
1. HEAT
2. CHEMICALS
- ADDITIVE
- NON ADDITIVE
- COAGULANT
- NON-COAGULANT
(Baker’s classification)
- COAGULANT
• ALCOHOL
• ZINC SALTS
• MERCURIC CHLORIDE
• CHROMIUM TRIOXIDE
• PICRIC ACID
- NON-COAGULANT
• FORMALDEHYDE
• GLUTARALDEHYDE
• OSMIUM TETROXIDE
• POTASSIUM
DICHROMATE
• ACETIC ACID

11. Classification of chemical fixatives
1. Aldehydes
Formaldehyde, glutaraldehyde
2. Oxidizing agents
Osmium tetroxide, potassium permanganate,
3. Proteins denaturing agents or coagulant
Acetic acid, methyl alcohol, ethyl alcohol.
4. Other cross linking agents
Carbodiimides
5. Miscellaneous
Mercuric sodium, picric acid , dye stuff.
(by bancroff)

12. Acc. to no. of fixatives used:
- Simple fixatives
- Compound fixatives
i) Micro anatomical fixatives
ii) Cytological
iii)Histochemical

13. Reaction of fixatives with Protein
Most important reactions which stabilizes proteins
by forming cross links between soluble protein &
structural protein. Ultimately providing some
mechanical strength.

14. Aldehydes
• Cross links are formed between protein molecules and
Aldehyde group of fixative.
• Aldehydes react with the basic amino acid residues of
proteins & there is an accompanying change in
isoeletric point of proteins.
• This may form the basis for the of the different staining
of tissues after different fixations.

15. • Process takes places in 2 step
1st step-small polymers are formed
2nd step small polymers cross-link
Formations of cross linkages between Aldehyde and
protein is measured by changes in viscosity, mechanical
strength and molecular size of protein.

16. Formaldehyde
• Slow reaction
• Reversible*(in first 24 hr with
excess of water)
• Not good morphological
picture
• Less effective at cross
linking
• Loss of enzyme and
immunological activity is less
Glutaraldehyde
• Rapid
• Irreversible
• Good morphological
picture
• More effective at cross
linking
• Loss of enzyme and
immunological activity
more

17. Oxidizing agents
• React with protein
• Forms cross-links with proteins
• Reflected by rapid increase in viscosity
• After that decrease in viscosity , that phenomenon
is known as secondary liquefaction.
• Osmium tetroxide is more reactive towards protein.

18. Mercuric chloride
• It reacts with histidine residues in proteins.
• Also there is production of H+ ions making solution
more acidic more efficient.
• But after fixation ultra structural preservation is
poor.

19. Other fixatives
• Heat fixation /microwave fixation ------ reacts with
polar side chains of proteins. This increases their
thermal energy which cause denaturation of
proteins. This brings about tissue stabilization.

20. Reaction of fixative with nucleic acid
• Fixation brings about change in physical or
chemical state of DNA or RNA at room temperature.
• Few fixative react with nucleic acid chemically-
including mercury and chromium salts.
• Heating at 45 and 65 degrees with Aldehyde
fixatives, there is uncoiling of RNA and DNA
respectively.

21. • Ethanol, methanol and Carnoy’s fixative are
commonly used. DNA is largely collapsed in
methanol and ethanol.
• Presence of salts is known to be essential for the
maximum precipitation of nucleic acid from alcohol.

22. Reaction of fixative with lipids
Most of lipids are labile. So lost during routine
processing. To demonstrate them frozen section or
cryostat is used.
Aldehyde fixation:
Preservation of lipoproteins (fixation of protein
counterpart. )
Eg: phospholipids which contain amino group such as
phosphotidyl ethanolamine are fixed by aldehyde.
22

23. HgCl₂ react with highly unsaturated compound
which form complex. It also reacts with lipids known
as plasmalogen acetal phosphatides.
 Additives such as tannic acid may be used for
demonstration of lipid with light microscopy.

24. Ultrastructural demonstration---post fixation with
osmium tetroxide.
Cholesterol may be fixed with Digitonin for
Ultrastructural demonstration.
24

25. Reaction with Carbohydrates
 Single fixative not satisfactory.
 Alcoholic or picric acid fixatives
preservation of glycogen which appear coarse eg:
Alcoholic formaldehyde, Rossman’s solution.
 Ultra structural studies gluteraldehyde is
satisfactory while potassium permanganate
increase image contrast.
25

26.  Tanic acid and cetyl pyrimidium have been found
useful.
 Additives to vehicle like Alcian blue or ruthenium red
enhance glutaraldehyde fixation of glycogen and
mucins.
26

27. Reagents used in fixation
1. Aldehydes : formaldehyde, glutaraldehyde
2. Metallic: mercuric chloride, lead fixative
3. Picric acid fixative
4. Alcoholic fixative
5. Chromate fixative
6. Osmium tetra oxide fixative
7. Acetate fixative

28. Formaldehyde
• Powerful reducing agent.
• Most common fixative for routine fixation of biopsy
specimen.
• Formalin: 40%formaldehyde gas in water.
• Forms methelene bridges between protein
molecules.
• Method 4mm block - 8hrs at room temperature
4mm block - 2hrs at 45°C
28

29. • MOST COMMONLY IS USED FIXATIVE
10% formalin consist of
• Formalin (40% formaldehyde) 10 ml
• Water 90 ml

30. • Neutralization is necessary due to formation of formic
acid by addition of buffer to maintain pH of 7.
Buffer added: phosphate buffer
magnesium carbonate
• Protein groups involved in formation of cross links
amino, imino, peptide, hydroxyl, carboxyl and
sulphahydryl.
• Formaldehyde is also obtainable in a stable solid form
composed of high molecular weight polymers known as
paraformaldehyde .

31. Advantages:
 Cheap, easy to prepare, relatively stable, staining without
preliminary procedures.
 Good preservation of cell morphology
 Good penetration properties.
 Do not cause excessive hardening.
 Best fixative for nervous system
31

32. Disadvantages-
Slow fixation reaction.
Morphological details less accurate than
glutaraldehyde.
Dermatitis of hand.
Fumes irritating to nostrils.
In tissue containing blood , dark brown artifact
pigment granules are formed.
32

33. Fixative Formula Advantages Disadvantages Uses
10%formal
saline
Water- 900ml
Nacl- 8.5gm
Formalin-100ml
•Less shrinkage
•Even fixation
•Easy sectioning
•Good staining
•Slow fixative •Hard tissues
•Neurological
tissues
•Gross specimen
fixation.
10% formalin 40%formaldehyd
e-100ml
Distilled water-
900ml
•Prevents pigments
•Good fixation
•Good penetration
•Preserves Enzymes
and organelles
•Longer time for
fixation
•Routine
specimen
•Used for IHC
10% buffered
neutral
formalin
Formalin -100ml
Water - 900ml
NaH₂Po₄-3.5gm
Na₂HPo₄-6.5gm
•Most routine
purpose
•Stops formation of
formalin pigment
•Fixes tissue rapidly
•Loss of basophillic
staining of the
cytoplasm and
nucleus
•Loss of reactivity
of myelin to
weigert iron
haematoxylin
method
33

34. Fixative Formula Advantage Disadvantage
Calcium acetate
formalin (formal
calcium)
Distilled water- 90ml
Calcium acetate
monohydrate- 2gm
Formalin - 10ml
•Buffered at pH7 by
acetate
•Preserves
phospholipids
•Less hardening or
damage
•Sectioned easily
•Artifacts due to
calcium
Alcoholic
formaldehyde
Formalin- 100ml
95%alcohol- 900ml
Calcium acetate-0.5gm
•Rapid Fixation
•Glycogen is better
preserved
•RBC are lysed
Formol calcium Formalin- 100ml
Distilled water- 900ml
10%calcium chloride-
100ml
•Preservation of lipids •Artifacts due to
calcium
Neutral buffered
phenol formalin
Neutral buffered
formaldehyde-100ml
Phenol- 20gm
•Stops formation of
formalin pigment
•Fixes tissue rapidly
34

35. Glutaraldehyde
• Introduced by Sabatini, Bensch and Barrett
• It is a dialdehyde.
• Stable in acid solution: in pH 3 to 5
at 0 ° to 4° C
• Used in electron microscopy with osmium
tetraoxide.
35

36. • Fixation of small tissue: 2.5% solution for 2-4 hrs at
room temperature
• Fixation of large tissue: 4% solution
for minimum 6-8hrs
fully fixed for 24hrs
36

37. Advantages:
 Better preservation of cellular and fluid proteins than
formaldehyde
 More stable cross linkages
 More rapid fixing action than formalin.
 Less shrinkages than formalin
 Give better section of blood clot and brain
 Does not corrode metal
 More pleasant and less irritating
37

38. Disadvantages
 More expensive
 Less stable
 Penetrates tissue more slowly than formalin
 Inferior to formalin for PAS technique.
38

39. Metallic fixative
mercury
• Mercuric ions act chiefly by combining with the
acidic group of proteins and strong combination with
sulfur thiol radicles.
Advantages:
 Better staining of nuclei and connective tissue.
 Give best results with metachromatic staining
 Routine fixative of choice for preservation of detail
of photography.
39

40. Disadvantages
 Corrode all metal except nickel alloy.
 Solution deteriorates rapidly.
 Reduce amount of demonstrable glycogen.
 Penetration is slow.
 Long time fixation results in unduly hard and brittle tissue.
 Diffuse black granules are seen in tissue fixed with HgCl₂.
40

41. Picric acid fixative
• It reacts with histone and basic proteins and forms
crystalline picrates with amino acid.
• It preserves glycogen well.
• Disadvantage:
 Considerable shrinkage of tissue.
 It dyes the tissue - yellow colour.
44

42. Alcoholic fixative
• Mechanism of action: alcohol denatures and
precipitate protein, possibly by disrupting hydrogen
and other bonds.
47

43. Fixative Type Formula Advantage Disvantage Use
Ethanol and
methanol
Cytological
Cytoplasmic
fixative
Ethyl alcohol
and
Methyl
alcohol
Rapid
penetration
•Inflammable
•Causes
Shrinkage
and hardens
•Smears
Glycogen
Carnoy’s
fixative
Cytological
Nuclear
fixative
Abs.Alcohol-
60ml
Chloroform-
30ml
Glacial acetic
acid-10ml
•Excellent
Nuclear
fixation and
•Rapid
penetration
•Destroys
Cytoplasmic
Elements &
lipids
• Glycogen
Preservation
48

44. Fixative Formula Type Advantages Use
Clarke’s
Fixative
•Abs.alcohol-
75ml
•Glacial Acetic acid-
25ml
Cytological
Nuclear
fixative
•Good penetration
and nuclear
details
•preserves
cytoplasmic
elements
Smears and
chromosome
study.
Alcohol
formalin
95%ethanol
Formalin-10ml
Useful fixative
for sputum
49

45. Chromate fixative
• Chromium salts in water form Cr-O-Cr complexes
which have affinity for acidic and hydroxyl group of
proteins so that complexes between adjacent
protein molecules are formed.
• This leads to disruption of internal salt linkages of
protein ,thereby increasing the reactive basic
groups and enhancing acidophilia in staining.
50

46. Fixative Formula Type Advantage and use Disadvantage
Orth’s
fluid
2.5%potassium
dichromate-
100ml
Sodium
sulphate-1gm
Just before
using,formalin-
10ml
Cytoplasmic
fixative
Regauds
fluid
Potassium
dichromate
Just before
use,formalin-
20ml
Cytoplasmic
fixative
Demonstration
of,RBC colloid
containing tissue,
preserve
phospholipid
•Solution darken on
standing
•Prolonged fixation
tend to bleach all
tissue pigment.
•Contraindicated in
carbohydrates
•Decrease intensity
of PAS reaction.
51

47. Osmium Tetraoxide
• It is highly reactive substance , being easily
reduced.
• It gels protein probably by a process of bridge
formation between compounds.
• With lipid it forms mono and diester linkages which
are then rendered insoluble and non extractable by
fat solvent such as alcohol and xylene.
52

48. Osmium tetraoxide
• Rapid fixing agent
• Stains tissue structure in a additive way as a grey
black deposit.

49. Fixative Formula Type Advantages Disadvantage Use
Flemming’s
fixative:
1%aquaous
chromic acid-15ml
2%aquaous
osmiumtetraoxide
-4ml
Acetic acid -1ml
Nuclear
fixative
•It is expensive.
Penetration is
slow.
•Difficult to
counterstain.
•Cause reversal
of tissue
acidophilia
•In electron
microscopy
Champy’s
fluid
3% Potassium
dichromate-
7ml
1% Chromic acid -
7ml
2% Osmium
tetraoxide -
4ml
Cytoplas
mic
fixative
Preserves
mitochondria,
fat, yolk, lipids
•Needs to be
freshly prepared
•Preferred for
mitochondria
54

50. EFFECT OF FIXATION
RULE #1 IS THAT FIXATIVES DENATURE
MACROMOLECULES; FIXATION CHANGES THE
SHAPE OF LARGE MOLECULES. THIS RULE IS
THE BASIS FOR THE VARIED FUNCTIONS OF
FIXATION AND WHY FIXED SPECIMENS LOOK
THE WAY THEY DO UNDER THE MICROSCOPE.

51. • RULE #2 IS THAT DIFFERENT FIXATIVES
PRODUCE THEIR OWN MORPHOLOGICAL
PATTERNS. THAT IS AN OBJECTIVE FACT THAT
DOES NOT IMPLY GOOD OR BAD. WHETHER WE
LIKE WHAT WE SEE IS A SUBJECTIVE MATTER
PREDOMINANTLY BASED ON OUR INDIVIDUAL
TRAINING. MANY CHEMICALS ACT AS
FIXATIVES IN THAT THEY DENATURE
MACROMOLECULES, BUT FEW PRODUCE

52. RULE #3 IS THAT FIXATION IS A CHEMICAL
REACTION THAT IS NOT INSTANTANEOUS. ITS
RATE IS DEPENDENT UPON THE CHEMICAL
NATURE OF THE FIXATIVE SOLUTION AND ITS
TEMPERATURE.
Freida L. Carson

53. Factors affecting fixation.
Hydrogen ion concentration
Temperature
Penetration
Osmolality
Concentration duration
Other factors:
Volume changes
Substances added to vehicle
58

54. Hydrogen ion concentration
• Satisfactory fixation occurs between pH 6 to 8.
• Stabilization of tertiary and quaternary structure of
proteins
• By addition of acids pH decreases
destruction of proteins and cause precipitation.
• Hence, fixatives must be neutralized by adding buffer.
59

55. Commonly used buffer system are : Phosphate, s-
collidine, veronal acetate, Tris and cacodylate.
•pH chosen must be as near the biochemical optimum
as possible.
•For electron microscopy , tissue must be fixed with a
gradually increasing pH
60

56. Temperature
High temperature
Rapid fixation reactions favors fixation.
Fixation should be carried out at gradually increasing
temperatures
Disadvantages : 1. Risk of tissue distortion
2. Deleterious effect on
certain antigen.
Use : 1.Rapid fixation of urgent biopsy
specimen.
2.To fix tissue with tuberculosis formaldehyde at
100°C is used.
61

57. Low temperature
Low temp. Slows down Autolysis more accurate
details.
Ultra structure and enzyme histochemistry and electron
microscopy , temp. range of 0 – 4 degrees is required.
62

58. Penetration
• Fixation depends on diffusion of fixative into the
tissue.
• Penetration of fixatives is a slow process.
• Size of specimen is important to ensure complete
penetration of fixatives.
• Small or thin slices of blocks - satisfactory fixation
• Large blocks of specimen - slow fixation 63

59. • Slow rate of diffusion and reaction give rise to various
zones of tissue fixed to different degrees.
• d=k √t (d-depth penetrated , t-time , k-coefficient of
infusibility.
• Fixed tissue acts as a barrier to subsequent inward
diffusion of fixatives.
64

60. Osmolality
• Hypertonic solutions - cell shrinkage.
• Isotonic and hypotonic solutions - cell swelling
• In general fixatives that act mainly on protein
precipitants cause shrinkage irrespective of what
the osmotic pressure is and for non protein
precipitants, reverse is true.
65

61. • By varying the Osmolality, structure of membrane system
within various cells can be altered.
Thus , additives to fixatives can alter extracellular space in
tissues.
• Sucrose is commonly added to osmium tetroxide for ultra
structural studies
• Fixative solutions must be preferably isotonic, thus
cell swelling is compensated by processing and wax
impregnation.
66

62. Concentration
• Low concentration of fixative with neutral pH favors
fixation.
• Glutaraldehyde solution is used as 3% solution but it is
effective even at concentration as low as 0.05% with
correct pH of fixative.
• Presence of buffer causes polymerization of Aldehyde
with a consequent decrease in effective concentration.
• Staining of tissue is altered with the concentration of
fixative employed.
67

63. Duration
• Long duration
- In Aldehyde :
- a) inhibit enzyme activity and immunological reactions
- b) shrinkage and tissue hardening.
• Glutaraldehyde
• longer duration of fixation
• effective polymer formation
• advantageous.
- In oxidizing fixatives : degrade the tissue by oxidative
cleavage of proteins and loss of peptides.
68

64. i) Changes in volume-
Ideally, changes in processing and fixation cancel each other
giving no net change.
Formalin fixed tissues along with paraffin embedding causes
33% shrinkage .
69

65. ii) Substances added to vehicle:
Fixative
fixative agent + Buffer + water.
Salts added have denaturing and stabilizing effect on
proteins.
Eg : NaCl2 + HgCl2 , Tannic acid, Alcian blue for
ultra structure.

66. Plastination
A technique or process used to preserve
bodies or body parts, tissue. The water and fat
are replaced by certain plastics(or resin),
yielding specimens that can be touched, do
not smell or decay, and even retain most
properties(especially morphology) of the
original sample.

67. References
 John D. Bancroft: Theory & Practice Of
Histological Techniques.
 Culling’s: Histological Techniques
 Fixation and Processing-Freida L. Carson