Fixatives are chemicals used to preserve biological tissues from decay. They terminate biochemical reactions and may increase mechanical strength. Fixatives disable enzymes and protect samples from damage. Common fixatives include formaldehyde, glutaraldehyde, osmium tetroxide, alcohols, and picric acid. Fixation aims to inhibit autolysis, preserve tissues, harden them, and improve staining. Factors like temperature, concentration, and duration impact fixation quality. Different tissues require specific fixatives for optimal preservation. An ideal fixative kills cells quickly without damage, penetrates rapidly, prevents decay, hardens tissues, and allows long-term storage.

1. FIXATIVESFIXATIVES
Dr.Ishwarya.S
II yr post graduate

2. Definition of FixationDefinition of Fixation
 A chemical process by which biological tissues are
preserved from decay, either through autolysis or
putrefaction
It terminates any ongoing biochemical reactions,
and may also increases the mechanical strength or
stability of the treated tissues.

3. Aims and Effects of fixationAims and Effects of fixation
 Inhibition of Autolysis and putrefaction
 Preservation
 Hardening
 Solidification of colloid material
 Effects on staining

4. Features of fixativesFeatures of fixatives
 Disable intrinsic biomolecules – particularly
proteolytic enzymes
 Protect a sample from extrinsic damage.
 Increase their mechanical strength & rigidity.

5. Heat fixation
Perfusion
Immersion
Methods of FixationMethods of Fixation

6. Types of fixationTypes of fixation
Physical methods of fixation
Chemical methods of fixation

7. Physical MethodsPhysical Methods
Heat Fixation
Microwave fixation
- Glyoxal based fixatives
Freeze drying and freeze substitution

8. Chemical FixationChemical Fixation
 Coagulant Fixatives
 Non - coagulant cross linking fixatives

9. Coagulant FixativesCoagulant Fixatives
 Alcohols (Ethanol, Methanol)
 Acetone
 Zinc Salts
 Mercuric Chloride
 Chromium Trioxide
 Picric acid

10. Non Coagulant FixativesNon Coagulant Fixatives
 Formaldehyde
 Gluteraldehyde
 Osmium Tetroxide
 Potassium Dichromate
 Acetic acid

11. FormaldehydeFormaldehyde
 10% Buffered Formalin
- 100 ml of 40% Formaldehyde
- 900 ml of distilled water
- 4g Sodium phospatase (Monobasic)
- 6.5g of sodium phosphate (dibasic)
 Methylene Glycol
 Cross links between protein end groups
 Amino acid Lysine
 Pigment Formation

12. Gluteraldehyde
Osmium Tetroxide
Picric Acid
Mercuric Chloride
Metallic ions as a fixative supplement
Compound fixatives

13. Removal of fixation pigmentsRemoval of fixation pigments
Formalin 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.

14. Mercury Pigments
 Immerse the sections in Gram or Lugol’s Iodine for 10
minutes.
 Then place the section in a 5% solution of Sodium
thiosulfate for 3 minutes.
 Wash slides well in running water for 10 minutes.

15. Melanin Pigments
 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

16. 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
 Additives

17. 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
2.5% Potassium
bichromate
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

18. Choice of FixativeChoice of Fixative

22. Fixation for individual tissuesFixation for individual tissues
 Eyes
- NBF, 48 hours
 Brain
 Breast
- 10% NBF for minimum of 6-8 hrs
 Lungs

23.  Lymphoid tissue
 Testis
 Muscle Biopsies
 Renal biopsies

24. Characteristics of a good fixativeCharacteristics of a good fixative
1. It must kill the cell quickly without shrinkage or
swelling
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
use

25. Thank youThank you