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II yr post graduate
Definition of FixationDefinition of Fixation
A chemical process by which biological tissues are
preserved from decay, either through autolysis or
It terminates any ongoing biochemical reactions,
and may also increases the mechanical strength or
stability of the treated tissues.
Aims and Effects of fixationAims and Effects of fixation
Inhibition of Autolysis and putrefaction
Solidification of colloid material
Effects on staining
Features of fixativesFeatures of fixatives
Disable intrinsic biomolecules – particularly
Protect a sample from extrinsic damage.
Increase their mechanical strength & rigidity.
Methods of FixationMethods of Fixation
Types of fixationTypes of fixation
Physical methods of fixation
Chemical methods of fixation
Physical MethodsPhysical Methods
- Glyoxal based fixatives
Freeze drying and freeze substitution
Chemical FixationChemical Fixation
Non - coagulant cross linking fixatives
10% Buffered Formalin
- 100 ml of 40% Formaldehyde
- 900 ml of distilled water
- 4g Sodium phospatase (Monobasic)
- 6.5g of sodium phosphate (dibasic)
Cross links between protein end groups
Amino acid Lysine
Metallic ions as a fixative supplement
Removal of fixation pigmentsRemoval of fixation pigments
Immerse the sections in saturated absolute alcohol with
picric acid for 10 mins to 3 hrs.
Then wash with water.
Also, a solution of 70% alcohol containing 3 mL of
ammonium hydroxide for 30
minutes to 3 hours.
Wash sections well in 1% acetic acid.
Immerse the sections in Gram or Lugol’s Iodine for 10
Then place the section in a 5% solution of Sodium
thiosulfate for 3 minutes.
Wash slides well in running water for 10 minutes.
Place the section in a solution of Potassium
permanganate for 20 mins
Followed by a solution of Oxalic acid to
remove the excess of potassium permanganate
Factors affecting fixationFactors affecting fixation
Buffers and PH
Size of the specimens
Duration of Fixation
Temperature of Fixation
Concentration of Fixative
Osmolality of Fixatives & Ionic composition
Rate of PenetrationRate of Penetration
Fixative 4 Hrs 8 hrs 12 hrs
10% Acetic acid 3.8 mm 5 mm 5 mm
10% Formalin 2.7 mm 4.7 mm 5 mm
95% Ethanol 1.7 mm 3.5mm 5mm
7.5% Mercuric chloride 2 mm 3 mm 3.5 mm
Sat. aqueous Picric acid 1 mm 1.5 mm 1.75 mm
1 mm 1.5 mm 1.75 mm
0.7% Chromic acid 0.6 mm 1 mm 1.2 mm
4% Osmium Tetroxide 0.3 mm 0.5 mm 0.7 mm
Characteristics of a good fixativeCharacteristics of a good fixative
1. It must kill the cell quickly without shrinkage or
2. It must penetrate the tissue rapidly
3. It must inhibit bacterial decay and autolysis
4. Harden the tissue and render it insensitive to
subsequent treatment as staining
5. It should allow tissue to be stored for long time
6. It should be simple to prepare and economical in