Histological Techniques: Section 2: Fixation of tissues

1. Histological Techniques
Section 2
Fixation of Tissue
Dr Mathew Joseph
MD Anatomy (3rd year)
All India Institute of Medical Sciences, Rishikesh

2. Contents
Fixation of Tissue:
• 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

4. Section 2
Fixation of Tissue

5. Introduction
Fixation is the complex series of chemical events and
differs for the different groups of chemical substances
found in tissues.
Definition
“A process by which the constituents ofthe cells or
tissues are fixed in a physical and chemical state so
that they will withstand subsequent treatment with
various reagents with a minimum loss, distortion or
decomposition”

6. • Once the tissue is removed from the body:
Self-destruction / Autolysis.
• If tissue is left without any preservation:
Bacterial attack / Putrefaction.

8. Aim & Objectives
• Toprevent autolysis and putrefaction.
• Rapid and even penetration.
• Topreserve cells and tissues in a life like manner as
possible.
• Elements that are to be demonstrated must
remain in maximum concentration and precise
localization.

9. Aim & Objectives
• 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. Method of Fixation
1. Physical:
2. Chemical:

12. Methods of Fixation
1. HEAT
2. CHEMICALS
- ADDITIVE
- NONADDITIVE
- 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

14. Classification of chemical fixatives
1. Aldehydes : Formaldehyde
Glutaraldehyde
2. Oxidizing agents :
Osmium tetroxide
Potassium permanganate
3.Proteins denaturing agents:
Coagulant Acetic acid
Methyl alcohol
Ethyl alcohol
4.Other cross linking agents :
Carbodiimides
5. Miscellaneous: Mercuric sodium, Picric acid , Dye stuff.
(by bancroff)

15. Acc. to no. of fixatives used:
1. Simple fixatives
2. Compound fixatives
i) Micro anatomical fixatives
ii)Cytological
iii)Histochemical

16. 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.

17. 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.

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

19. 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

20. 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.

21. 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.

22. 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.

23. Reaction of fixative with nucleic acid
• Fixation brings about change in physical or chemical
state of DNA or RNA at roomtemperature.
• 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.

24. • 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.

25. Reaction of fixative with lipids
Most of lipids are labile. So lost during routine processing.
Todemonstrate 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

26. • 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.

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

28. 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

29. • 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

30. 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

31. 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

32. • Most commonly is used fixative
•10% formalin consist of
Formalin (40% formaldehyde): 10 ml
Water :90 ml

33. • 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 .

34. 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

35. 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

36. Fixative Formula Advantages Uses
10%formal
saline
Water-
Nacl-
900ml
8.5gm
Formalin-100ml
•Less shrinkage
•Even fixation
•Easy sectioning
•Good staining
Disadvantages
•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

37. 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

38. 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

39. • 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

40. 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

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

42. 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

43. 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

44. 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

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

46. Fixative Type Formula Advantage Disvantage Use
Ethanol and
methanol
Cytological
Cytoplasmic
Ethyl alcohol
and
Rapid
penetration
•Inflammable
•Causes
•Smears
Glycogen
fixative Methyl
alcohol
Shrinkage
and hardens
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

47. 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

48. 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

49. 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

50. 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.
• Rapid fixing agent
• Stains tissue structure in a additive way as a
grey black deposit.
52

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

52. 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 WAYTHEY DO UNDER THE MICROSCOPE.

53. • 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

54. 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

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

56. 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

57. 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

58. Temperature
High temperature:
Rapid fixation reactions favors fixation.
Fixation should be carried out atgradually 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

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

60. 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

61. • 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

62. 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

63. • 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

64. 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

65. Duration
1. Long duration
Aldehyde :
• inhibit enzyme activity and immunological
reactions
• 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

66. 2. 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

67. 3. 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.

68. 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.

69. References
1. John D. Bancroft: Theory & Practice Of Histological
Techniques.

70. Thank You