FIXATIVESFIXATIVES

Dr.Ishwarya.SII yr post graduate

Definition of Fixation Definition 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.

Aims and Effects of fixationAims and Effects of fixation

Inhibition of Autolysis and putrefaction

Preservation

Hardening

Solidification of colloid material

Effects on staining

Features of fixativesFeatures of fixatives

Disable intrinsic biomolecules – particularly

proteolytic enzymes

Protect a sample from extrinsic damage.

Increase their mechanical strength & rigidity.

Heat fixation

Perfusion

Immersion

Methods of FixationMethods of Fixation

Types of fixationTypes of fixation

Physical methods of fixation

Chemical methods of fixation

Physical MethodsPhysical Methods

Heat Fixation

Microwave fixation - Glyoxal based fixatives

Freeze drying and freeze substitution

Chemical FixationChemical Fixation

Coagulant Fixatives

Non - coagulant cross linking fixatives

Coagulant FixativesCoagulant Fixatives

Alcohols (Ethanol, Methanol)

Acetone

Zinc Salts

Mercuric Chloride

Chromium Trioxide

Picric acid

Non Coagulant FixativesNon Coagulant Fixatives

Formaldehyde

Gluteraldehyde

Osmium Tetroxide

Potassium Dichromate

Acetic acid

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

Gluteraldehyde

Osmium Tetroxide

Picric Acid

Mercuric Chloride

Metallic ions as a fixative supplement

Compound fixatives

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.

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.

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

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

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

Choice of FixativeChoice of Fixative

Fixation for individual tissuesFixation for individual tissues

Eyes - NBF, 48 hours

Brain

Breast - 10% NBF for minimum of 6-8 hrs

Lungs

Lymphoid tissue

Testis

Muscle Biopsies

Renal biopsies

Characteristics of a good Characteristics of a good fixativefixative

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

Thank youThank you