1. SEMINAR

2. TOPIC:
FIXATION

3. SUBMITTED BY
RINSEENA M. GAFOOR

4. SUBMITTED TO
Mr. REJO JACOB JOSEPH
&
Mrs.ARCHANA MADAM

5. FIXATION

6. DEFINITION:- It is the process by which the morphology and
microscopic anatomy of the tissue is preserve as
life like .Fixation prevents autolysis and putrefaction
changes in the tissue.
Putrefaction is caused by bacterial invasion and
subsequent destruction of the tissue.

7. AUTOLYSIS
It is a self destruction due to the action
of enzymes which are liberated as soon as
cell dies.
Autolysis leads to the disappearance of
nuclei, cloudiness of the cytoplasm and cell
loses its staining property. so these changes
in the cell can be prevented either by freezing
or by adding some chemical substance
called fixative to the tissue .

8. Most of the fixative act by denaturing
or precipitating protein which will form a
network , which make all other cell components

9. Aim And Functions of Fixative
Fixative should prevent autolysis and putrefaction
by rapid coagulation of the cytoplasm.
Minimize the risk of infection.
It should not shrink or swell, nor destroy the
structure of the tissue.
It should rapidly and evenly penetrate the tissue.
It will provide a condition to allow clear staining
of section.
It will not alter any chemical changes of the cell.
To keep the tissue to the living state without loss
of rearrangement.

10. FACTORS INVOLVING FIXATION
pH and Buffer
Generally satisfactory fixation occurs at
the pH 6.8. For some purpose fixative
at specific Ph is chosen
e.g.: For the fixation of gastric mucosa,
pH is 5.5.
Acetic acid is used for lowering the pH
Few buffers can also be used
e.g.: phosphate buffer, bicarbonate
buffer

11. Temperature
Lower temperature slows down autolysis
and higher temperature enhances the
rapidity of fixation
Routinely fixation carried out by room
temperature
For electro microscopy and histoscopy
temperature range chosen is 0.4 c.
Formalin heated at 60 c is used to fix the
urgent biopsy specimen, but higher
temperature may destroy the cells or tissue..

12. Penetration
Depth of the penetration
D α √t
D=k √t d=mm
t=hrs
k=diffusion coefficient

13. Osmolarity
Best results are obtained by using slightly
hypertonic solution is used.
Concentration
It is determined by solubility, staining pattern
and its effectiveness.
10% formalin is used routinely for brain
fixation.
15% formalin is used for friezing the time of
3 % gluteraldehyde. Is used for
electron microscopy.

14. Duration
Prolong fixation in formaldehyde will cause
shrinkage and hardening and will cause
destruction of
some enzyme, but in gluteraldehyde prolong
fixation is advantage than formaldehyde.

15. THEORETICAL ASPECTS OF FIXATION
Reaction of fixative with protein
Most of the important reaction during fixation
is to stabilize proteins
Aldehydes
It cause cross links with protein.
The reaction is pH dependent _ i.e. reaction
is more rapid at high pH.
Reaction of formaldehyde is reversible with
in 24 hrs but with neutraldehyde it is rapid
and irreversible.

16. Oxidizing Agents
Osmium tetroxide form cross links with
proteins where as potassium permanganate
and potassium dichromate unless reactive
towards protein.

17. Non chemical fixative
Heat and microwave fixative are
non-chemical type of fixative. Direct flaming
leads to the destruction of tissue.
Using microwave fixative heating is controlled.
Using microwave fixative tissue molecules
can be oscillated and high frequency leads
to generate internal heating which fixed the
protein by denaturation. it is very rapid
method and it is used when rapid diagnosis
is needed

18. Reaction of fixation with lipids
Fixative used for lipid demonstration is
aldehyde. Following aldehyde fixation,
most lipids are not stable but it removes
during conventional histopathological
process.
Reaction of fixative with carbohydrate
60_80% glycogen will lose in aqueous
solution, so alcoholic fixatives are
recommended for glycogen fixation. .

19. Reaction of fixative with nucleic acid
Ethanol, methanol, & carnoy’s fixative are used for demonstration of nucleic acid. These fixatives bring about the physical and chemical changes of nucleic acid

20. CLASSIFICATION OF FIXATIVE
Fixatives are classified into SIMPLE and COMPOUND
Based on chemical action fixatives are further classified into
ALDEHYDE
Formaldehyde
Gluteraldehyde
METALLIC FIXATIVE
Mercuric chloride
Lead fixative
OXIDIZING AGENTS
Osmium tetroxide
K+ permanganate
K+ dichromate
PROTIEN DENATURING AGENTS
Acetic acid
Methyl alcohol
Ethyl alcohol
MISCELANEOUS
Picric acid
Non aldehyde

21. FORMALDEHYDE
Formalin is a routine fixative.
Commercially available formaldehyde is called formalin.
40% formaldehyde is considered as 100% formalin.
This is a saturation of formaldehyde gas.
10% formalin_10ml dissolved in90ml of water.
Action
Formaldehyde reacts with proteins to form cross links
between the molecules and form insoluble product.
b) Formaldehyde reacts with lipids cause degradation.
c) Formalin becomes cloudy in all atmospheres or in
long storage due to formation of Para formaldehyde.
It is prevented by adding methanol.
d) Formalin is usually acid due to the formation of acid
content which may cause the formalin

22. Advantage
It is cheap and easy to prepare.
It penetrates rapidly and never hardens the tissue.
Natural color can be restored, so it can be used in
museum mounting.
It allows most of the staining property without any
chemical changes.
Disadvantages
It will cause irritation to nose, eyes, and skin. Hence
adequate ventilation and the use of gloves are essential.
It is not suitable in electron microscopy.
It forms white precipitate or Para formaldehyde to form
black color formalin pigment due to the formic acid and
will cause misinterpretation.

23. Gluteraldehyde
It is mainly used for electron microscopy and
usually used in combination with osmium tetroxide.
It has more rapid action than formaldehyde.
Disadvantage
It is expensive.
Its penetration is poor.
It over hardens the tissue.

24. It is a protein precipitate which rapidly penetrates and
hardens the tissue.
Disadvantage
Formation of black or brown color pigment.
This can be removed by treatment of tissue in 5% iodine
prepared in 80% alcohol.
Keep for 5 min.
Then rinse in running tap water.
Keep in aqueous sodiumthiosulphate for 30min.
Then again wash in running tap water.
Mercuric chloride

25. Osmiumtetroxide
It demonstrates lipids. It gives excellent
preservation of cells. So it is used in electron
microscopy.
Disadvantage
It is expensive.
It penetrates poorly.
It is a very irritating chemical and will cause
conjunctivitis.
It will be very easily reduced and it should be
stored in dark place.
It react lipids and form blackening.

26. Potassium dichromate
It has binding effect on protein, similar to that of
formalin.
It gives fixation of cytoplasm without precipitation
of proteins.
It preserves phosphotides and it is used for the
fixation for the fixation of mitochondria.
Following fixation in potassium dichromate, tissue
must be washed in running tap water before dehydration, otherwise will cause formation of insoluble lower oxide.

27. Chromic Acid
It is prepared by dissolving anhydrous chromium tetroxide
in distilled water.
It precipitates protein and preserve carbohydrate.
It is a powerful oxidizing agent.
Acetone
It is used for the histochemical demonstration of tissues
and enzymes.
Disadvantage
Glycogen is not preserved by using acetone.

28. Picric Acid
It precipitates protein.
It gives a brilliant contrast for trichrome stain.
Disadvantage
Formation of insoluble picrate.
It is explosive, causes lyses of red cells and remove iron
from the tissue.
Ethyl alcohol
It denatures protein and precipitates them.
It also precipitates glycogen.
It is used for glycogen demonstration.
When it is fixed with other reagents such as carnoy’s,
fixation is rapid.
It dissolves fats and lipids.
It penetrates slowly and makes to harden the tissue.

29. Acetic Acid
It is not usually using alone as it will cause swelling.
Trichloro acetic acid
It is used for compound fixative.
It is a protein precipitates and has a slight decalcifying
property.

30. COMPOUND FIXATIVES
These are the product of two or more simple fixative
mixed together to get combined effect of their properties.
It is classified into three groups.
Micro Anatomical Fixatives
Cytological Fixatives
Histochemical Fixatives

31. MICROANATOMICAL FIXATIVES
It is used to preserve the anatomy of the tissue with correct relationship
of tissue layers and large aggregates of cell.
Formal calcium
Formalin- 10ml
Calcium chloride- 2gm
Water- 90ml
Advantage
It is used for the fixation of lipids.
It prevents the formation of formalin pigment
Formal saline
Nacl- 9gm,
Formalin pigment will form.
It is ideal for fixing the brain.

32. Buffered formalin
Formalin – 10ml
NaH2Po4 – 0.4gm
Na2HPo4 – 0.65gm
Water – 90ml
Advantage
Formalin pigment will not form.
Sucrose – 7.5gm
Phosphate buffer – 100ml s

33. Advantage
This fixative gives excellent preservation of fine structure, phospholipids and some enzymes.
It is recommended for cytochemistry and electron microscopical studies.
For best results it is used at 4
Mitochondria and endoplasmic reticulum is well preserved in electron microscopical pictures by these fixatives.

34. Alcoholic formalin
Formalin – 10ml
95% formalin – 90ml
Calcium acetate – 0.5gm
Advantage
Rapid fixation and it is a good glycogen fixative
Disadvantage
It will cause shrinkage
Acetic Alcoholic Formalin
Formalin – 5ml
Glacial acetic acid – 5ml
70% alcohol – 90ml
Excellent glycogen preservation
Rapid fixation
It is not ideal for routine fixative

35. Buffered gluteraldehyde
Gluteraldehyde stock solution – 16ml
Phosphate buffer – 84ml
Mercuric Chloride Containing Fixatives
Tissue fixed with mercuric chloride containing fixative
cause the formation of black precipitate of mercuric
pigment.
This pigment has to remove from the deparaffinised
section before staining.
This is done by treating the section in 0.5% iodine and
prepared in 80% alcohol for 5-10min and washed in
running water and decolorize with sodium thiosulphate
and after that wash again in running tap water.

36. Heidenhan’s susa
Mercuric chloride – 4.5gm
Nacl -0.5gm
TCA – 2gm
GAA – 4ml
Formalin – 20ml
Distilled water – 80ml
Advantage
It is excellent fixative for routine biopsy
It gives brilliant staining with good cytological details.
Rapid penetration with minimum shrinkage.
It is having slight decalcifying power because it is
incorporated with TCA.
Disadvantages
Formation of mercuric pigment and it should be
removed by treating with iodine.

37. Zenker’s Fluid
Mercuric chloride- 5gm
Potassium dichromate- 2.5gm
Sodium sulphate – 1gm
Distilled water – 100ml
Add glacial acetic acid immediately better use – 5ml
Advantages
Good routine fixative and excellent staining for
connective tissue fibers.
Disadvantages
Its penetration is poor.
After fixation wash the fixed tissue in running tap water
to remove excess dichromate

38. Helly’s fluid
Mercuric Chloride – 5gm
Potassium dichromate – 2.5gm
Sodium sulphate – 1gm
Distilled water – 100ml
Add formalin before use – 5ml
Advantage
It is an excellent micro anatomical fixative.
It is a very good fixative for bone marrow, spleen and
blood containing tissue.
Disadvantages
It is slow in action than zenker fluid.

39. ADVANTAGE: IT PENETRATE RAPIDLY IT IS USED FOR THE PRESERVATION OF CHROMOSOME. IT PRESERVE CHROMATIN BETTER THAN CARNOY’S FLUID IT ALSO PRESERVE POLYSACCHARIDE

40. FLEMINGS FLUID 1% aq CHROMIC ACID - 15ml 1% OSMIUM TETROXIDE -4ml GLACIAL ACETIC ACID – 1ml FOLLOWING FIXATION BY USING TH FLEMMING’S FLUID TISSUE SHOULD BE WASHEDOVER NIGHT. IT IS RARELY USED BECAUSE OF POOR PENERATION
FLEMINGS FLUID 1% aq CHROMIC ACID - 15ml 1% OSMIUM TETROXIDE -4ml GLACIAL ACETIC ACID – 1ml FOLLOWING FIXATION BY USING TH FLEMMING’S FLUID TISSUE SHOULD BE WASHEDOVER NIGHT. IT IS RARELY USED BECAUSE OF POOR PENERATION

41. CYTOPLASMIC FIXATIVE CHAMPY’S FLUID REGAURD’S FLUID SCHAUDINN’S FLUID
HELLY’S FLUID
MULLER’S FLUID
FORMAL SALINE

42. CHAMPY’S FLUID IT PREVNTS MITOCHONDRIUM, FATS AND LIPIDS
IT SHOULD PREPARE FRESHLY BEFORE USE
IT PENETRATES POORLY
THIN PIECES OF TISSUES HAVE TO BE TREATED FOR THESE FIXATIVES

43. REGAURD’S FLUID IT SHOULD PREPARE FRESHLY BEFORE USE RAPID PENETRATION
IT WILL CAUSE OVER HARDENING OF THE TISSUE
IT CAN BE USED AS ROUTINE FIXATIVE
IT PRESERVE MITOCHONDRIA VERY WELL

44. SCHAUDINN’S FLUID IT IS A VERY GOOD CYTOPLASMIC FIXATIVE
IT WILL CAUSE EXCESSIVE SHRINKAGE
IT IS NOT RECCOMMENDED FOR
TISSUE BIOPSY
TO REMOVE THE MERCURIC PIGMENT
TISSUES OR SMEARS SHOULD BE TREATD WITH IODINE, ALCOHOL AND SODIUM THIOSULPATE
WASH IN RUNNIG TAP WATER

45. HELLY’S FLUID (ZENKER’S-FORMAL)
IT CAN BE USED AS A CYTOPLASMIC FIXATIVE
PARTICULARLY FOR BONE MARROW AND BLOOD FORMING ORGANS

46. MULLER’S FLUID
IT’S A VERY RARELY USED FIXATIVE

47. FORMAL SALINE
AFTER THE FIXATION USING THE POST CHROMIUM GIVES GOOD CYTOPLASMIC FIXATION.

48. HISTOCHEMICAL FIXATIVS
After using histochemical fixatives cryostat and frozen’s are used for cutting of the section
A good histochemical fixative should preserve the constituent to be demonstrated, preferably preserving its morphological relationship
Bind or preserve a specific tissue constituent without effecting the relative groups to be used in its visualization.

49. BUFFERED FORMALIN ADVANTAGE
Formalin pigment is not formed by using this fixative’s.

50. FORMAL SALINE
ADVANTAGE
Formalin pigment will not form.

51. COLD ACETONE These fixatives is used in 0-4oc.
Its used for the study of enzymes particularly for the phosphates.

52. ABSOLUTE ALCOHOL 95 % alcohol is called absolute alcohol.
It needed 24 hrs fixation.
Its occasionally recommended as a basic fixatives , but in most histochemical techniques formalin can be used as an alternative with a consequent improvement in micro anatomical and cytological preservation.

53. VAPOUR FIXATION
Vapour fixatives may be used to fix cryostat –cut section of fresh tissues and sections or blocks of frozen dried tissue.
Formaldehyde vapour generated from heated paraformaldehyde is a very high reactivity.
Cryostat sections mounted on slides may be placed in a closed vessel above the formaldehyde and the vessel placed in an oven at 60-70˚c for 2 hrs.
Using this method we have produced sections showing excellent preservation of glycogen with a very good morphological details

54. Fixatives which have been used for vapour fixation usually at a temperature of 60- 80˚c.
Formaldehyde ˚c.
Acetaldehyde ˚c-1-4hrs.
Gluteraldehyde ˚c.
Osmium tetroxide
Diacetyl
Acetic acid
Glycoxylic acid
Glyoxal