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Tissue fixation
Presented by
Dr. Shrikant Sonune
Guided by
Dr Ashok Patil,
Dr Shilpa Kandalgaonkar,
Dr Mayur Chaudhary,
Dr...
Content
• Introduction
• Function of fixative
• Methods of fixation
• Reaction of the cell(its component) with fixatives
•...
Introduction
Introduction
• Fixation
• Tissue processing
• Sectioning
• Staining
• Staining
• Sectioning
• Tissue processing
• Fixation
Fixation : introduction
• Fixation is the complex series of chemical events and
differs for the different groups of chemic...
Introduction
• Once the tissue is removed from the body it will go
through a process of self-destruction. This process
is ...
Definition :
• It is a process by which the constituents of the cells and
therefore of the tissues are fixed in a physical...
Aims & objectives of fixation
• To prevent autolysis and putrefaction.
• Rapid and even penetration.
• To preserve cells a...
METHODS OF FIXATION
1. HEAT
2. CHEMICALS
- ADDITIVE
- NON ADDITIVE
- COAGULANT
- NON-COAGULANT
(Baker’s classification)
- ...
Classification of chemical fixatives
1. Aldehydes
Formaldehyde, glutaraldehyde
2. Oxidizing agents
Osmium tetroxide, potas...
Acc. to no. of fixatives used:
- Simple fixatives
- Compound fixatives
i) Micro anatomical fixatives
ii) Cytological
iii)H...
Reaction of fixatives with Protein
Most important reactions which stabilizes proteins
by forming cross links between solub...
Aldehydes
• Cross links are formed between protein molecules and
Aldehyde group of fixative.
• Aldehydes react with the ba...
• Process takes places in 2 step
1st step-small polymers are formed
2nd step small polymers cross-link
Formations of cross...
Formaldehyde
• Slow reaction
• Reversible*(in first 24 hr with
excess of water)
• Not good morphological
picture
• Less ef...
Oxidizing agents
• React with protein
• Forms cross-links with proteins
• Reflected by rapid increase in viscosity
• After...
Mercuric chloride
• It reacts with histidine residues in proteins.
• Also there is production of H+ ions making solution
m...
Other fixatives
• Heat fixation /microwave fixation ------ reacts with
polar side chains of proteins. This increases their...
Reaction of fixative with nucleic acid
• Fixation brings about change in physical or
chemical state of DNA or RNA at room ...
• Ethanol, methanol and Carnoy’s fixative are
commonly used. DNA is largely collapsed in
methanol and ethanol.
• Presence ...
Reaction of fixative with lipids
Most of lipids are labile. So lost during routine
processing. To demonstrate them frozen ...
HgCl₂ react with highly unsaturated compound
which form complex. It also reacts with lipids known
as plasmalogen acetal p...
Ultrastructural demonstration---post fixation with
osmium tetroxide.
Cholesterol may be fixed with Digitonin for
Ultrast...
Reaction with Carbohydrates
 Single fixative not satisfactory.
 Alcoholic or picric acid fixatives
preservation of glyco...
 Tanic acid and cetyl pyrimidium have been found
useful.
 Additives to vehicle like Alcian blue or ruthenium red
enhance...
Reagents used in fixation
1. Aldehydes : formaldehyde, glutaraldehyde
2. Metallic: mercuric chloride, lead fixative
3. Pic...
Formaldehyde
• Powerful reducing agent.
• Most common fixative for routine fixation of biopsy
specimen.
• Formalin: 40%for...
• MOST COMMONLY IS USED FIXATIVE
10% formalin consist of
• Formalin (40% formaldehyde) 10 ml
• Water 90 ml
• Neutralization is necessary due to formation of formic
acid by addition of buffer to maintain pH of 7.
Buffer added: pho...
Advantages:
 Cheap, easy to prepare, relatively stable, staining without
preliminary procedures.
 Good preservation of c...
Disadvantages-
Slow fixation reaction.
Morphological details less accurate than
glutaraldehyde.
Dermatitis of hand.
Fu...
Fixative Formula Advantages Disadvantages Uses
10%formal
saline
Water- 900ml
Nacl- 8.5gm
Formalin-100ml
•Less shrinkage
•E...
Fixative Formula Advantage Disadvantage
Calcium acetate
formalin (formal
calcium)
Distilled water- 90ml
Calcium acetate
mo...
Glutaraldehyde
• Introduced by Sabatini, Bensch and Barrett
• It is a dialdehyde.
• Stable in acid solution: in pH 3 to 5
...
• Fixation of small tissue: 2.5% solution for 2-4 hrs at
room temperature
• Fixation of large tissue: 4% solution
for mini...
Advantages:
 Better preservation of cellular and fluid proteins than
formaldehyde
 More stable cross linkages
 More rap...
Disadvantages
 More expensive
 Less stable
 Penetrates tissue more slowly than formalin
 Inferior to formalin for PAS ...
Metallic fixative
mercury
• Mercuric ions act chiefly by combining with the
acidic group of proteins and strong combinatio...
Disadvantages
 Corrode all metal except nickel alloy.
 Solution deteriorates rapidly.
 Reduce amount of demonstrable gl...
Picric acid fixative
• It reacts with histone and basic proteins and forms
crystalline picrates with amino acid.
• It pres...
Alcoholic fixative
• Mechanism of action: alcohol denatures and
precipitate protein, possibly by disrupting hydrogen
and o...
Fixative Type Formula Advantage Disvantage Use
Ethanol and
methanol
Cytological
Cytoplasmic
fixative
Ethyl alcohol
and
Met...
Fixative Formula Type Advantages Use
Clarke’s
Fixative
•Abs.alcohol-
75ml
•Glacial Acetic acid-
25ml
Cytological
Nuclear
f...
Chromate fixative
• Chromium salts in water form Cr-O-Cr complexes
which have affinity for acidic and hydroxyl group of
pr...
Fixative Formula Type Advantage and use Disadvantage
Orth’s
fluid
2.5%potassium
dichromate-
100ml
Sodium
sulphate-1gm
Just...
Osmium Tetraoxide
• It is highly reactive substance , being easily
reduced.
• It gels protein probably by a process of bri...
Osmium tetraoxide
• Rapid fixing agent
• Stains tissue structure in a additive way as a grey
black deposit.
Fixative Formula Type Advantages Disadvantage Use
Flemming’s
fixative:
1%aquaous
chromic acid-15ml
2%aquaous
osmiumtetraox...
EFFECT OF FIXATION
RULE #1 IS THAT FIXATIVES DENATURE
MACROMOLECULES; FIXATION CHANGES THE
SHAPE OF LARGE MOLECULES. THIS ...
• RULE #2 IS THAT DIFFERENT FIXATIVES
PRODUCE THEIR OWN MORPHOLOGICAL
PATTERNS. THAT IS AN OBJECTIVE FACT THAT
DOES NOT IM...
RULE #3 IS THAT FIXATION IS A CHEMICAL
REACTION THAT IS NOT INSTANTANEOUS. ITS
RATE IS DEPENDENT UPON THE CHEMICAL
NATURE ...
Factors affecting fixation.
Hydrogen ion concentration
Temperature
Penetration
Osmolality
Concentration duration
Othe...
Hydrogen ion concentration
• Satisfactory fixation occurs between pH 6 to 8.
• Stabilization of tertiary and quaternary st...
Commonly used buffer system are : Phosphate, s-
collidine, veronal acetate, Tris and cacodylate.
•pH chosen must be as nea...
Temperature
High temperature
Rapid fixation reactions favors fixation.
Fixation should be carried out at gradually increas...
Low temperature
Low temp. Slows down Autolysis more accurate
details.
Ultra structure and enzyme histochemistry and electr...
Penetration
• Fixation depends on diffusion of fixative into the
tissue.
• Penetration of fixatives is a slow process.
• S...
• Slow rate of diffusion and reaction give rise to various
zones of tissue fixed to different degrees.
• d=k √t (d-depth p...
Osmolality
• Hypertonic solutions - cell shrinkage.
• Isotonic and hypotonic solutions - cell swelling
• In general fixati...
• By varying the Osmolality, structure of membrane system
within various cells can be altered.
Thus , additives to fixativ...
Concentration
• Low concentration of fixative with neutral pH favors
fixation.
• Glutaraldehyde solution is used as 3% sol...
Duration
• Long duration
- In Aldehyde :
- a) inhibit enzyme activity and immunological reactions
- b) shrinkage and tissu...
i) Changes in volume-
Ideally, changes in processing and fixation cancel each other
giving no net change.
Formalin fixed t...
ii) Substances added to vehicle:
Fixative
fixative agent + Buffer + water.
Salts added have denaturing and stabilizing eff...
Plastination
A technique or process used to preserve
bodies or body parts, tissue. The water and fat
are replaced by certa...
References
 John D. Bancroft: Theory & Practice Of
Histological Techniques.
 Culling’s: Histological Techniques
 Fixati...
Fixation
Fixation
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Fixation

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After biopsy tissue fixation is considered as the most important process. here in this presentation various points have been elaborated.

Published in: Health & Medicine

Fixation

  1. 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. 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. 3. Introduction
  4. 4. Introduction • Fixation • Tissue processing • Sectioning • Staining • Staining • Sectioning • Tissue processing • Fixation
  5. 5. 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?
  6. 6. 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.
  7. 7. 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
  8. 8. 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
  9. 9. 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
  10. 10. 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)
  11. 11. Acc. to no. of fixatives used: - Simple fixatives - Compound fixatives i) Micro anatomical fixatives ii) Cytological iii)Histochemical
  12. 12. 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.
  13. 13. 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.
  14. 14. • 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.
  15. 15. 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
  16. 16. 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.
  17. 17. 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.
  18. 18. 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.
  19. 19. 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.
  20. 20. • 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.
  21. 21. 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
  22. 22. 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.
  23. 23. Ultrastructural demonstration---post fixation with osmium tetroxide. Cholesterol may be fixed with Digitonin for Ultrastructural demonstration. 24
  24. 24. 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
  25. 25.  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
  26. 26. 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
  27. 27. 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
  28. 28. • MOST COMMONLY IS USED FIXATIVE 10% formalin consist of • Formalin (40% formaldehyde) 10 ml • Water 90 ml
  29. 29. • 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 .
  30. 30. 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
  31. 31. 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
  32. 32. 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
  33. 33. 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
  34. 34. 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
  35. 35. • 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
  36. 36. 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
  37. 37. Disadvantages  More expensive  Less stable  Penetrates tissue more slowly than formalin  Inferior to formalin for PAS technique. 38
  38. 38. 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
  39. 39. 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
  40. 40. 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
  41. 41. Alcoholic fixative • Mechanism of action: alcohol denatures and precipitate protein, possibly by disrupting hydrogen and other bonds. 47
  42. 42. 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
  43. 43. 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
  44. 44. 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
  45. 45. 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
  46. 46. 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
  47. 47. Osmium tetraoxide • Rapid fixing agent • Stains tissue structure in a additive way as a grey black deposit.
  48. 48. 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
  49. 49. 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.
  50. 50. • 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
  51. 51. 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
  52. 52. Factors affecting fixation. Hydrogen ion concentration Temperature Penetration Osmolality Concentration duration Other factors: Volume changes Substances added to vehicle 58
  53. 53. 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
  54. 54. 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
  55. 55. 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
  56. 56. 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
  57. 57. 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
  58. 58. • 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
  59. 59. 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
  60. 60. • 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
  61. 61. 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
  62. 62. 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
  63. 63. 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
  64. 64. 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.
  65. 65. 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.
  66. 66. References  John D. Bancroft: Theory & Practice Of Histological Techniques.  Culling’s: Histological Techniques  Fixation and Processing-Freida L. Carson

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