FIXATIVE

Awal Mir Khattak

Demonstrator MLT

B.Sc. MLT Baqai Medical University Karachi

M.Sc. Hematology Baqai Medical University Karachi
M.Phil. Medical Lab Sciences, The University of Haripur
 FIXATIVE
• A chemical compound that fixed the constituent of
the cells physically and partly also in chemical state.
• Fixative prevent the tissue from putrefaction and
autolysis or decomposition.
• Autolysis: Lysis or dissolution of cells by its own
enzyme action probably as a result of rupture of
lysosomes.
• Putrefaction: The breakdown of tissue by external
agents i-e bacterial decomposition.
 AIM/PURPOSE OF FIXATIVE
1. To preserve the tissue in as life like manner as
possible.

2. To prevent postmortem changes like autolysis and

putrefaction
 MECHANISM OF ACTION
• Most fixatives act by denaturing or precipitating
proteins, which then form a sponge or meshwork,
tending to hold the other cell constituents.
• Good fixation is the most important factor for the
production of satisfactory results in histopathology.
Following factors are important.
• Fresh tissue
• Proper penetration of tissue by fixative
• Correct choice of fixative
 REACTIONS OF FIXATIVE
1. Proteins:
 Cross links are formed between proteins.
 Soluble proteins are fixed to structural proteins-
insoluble-mechanical strength-allowing subsequent
manipulations on tissues
 Formaldehyde -reversible.
 Glutaraldehyde -rapid & irreversible.
 React with basic amino-acid residues
 REACTIONS OF FIXATIVE
2. Nucleic acid:

 Fixation brings a change in the physical & chemical

state of RNA & DNA.

 Uncoiling of DNA & RNA occurs with formalin when

heated to 45˚C & 65˚C respectively.
 REACTIONS OF FIXATIVE
3. Carbohydrates
• Carbohydrates are more water soluble- difficulty in
total preservation
• They bind with fixed protein
• So the fixatives which are used for proteins, can be
used for carbohydrate preservation.
• Fixed protein traps carbohydrates.
• Glycogen not bound to protein- fixed protein form
lattice around glycogen to preserve it
• Glycogen are more demonstrable in liver cells
 REACTIONS OF FIXATIVE
4. Lipids:
 Phospholipids are fixed by formaldehyde.
 Formaldehyde reacts with unsaturated fatty acids
hence less lipid can be demonstrated in tissue stored
in it for a long time.
 Mercuric chloride reacts with lipids to form
complexes.
 Ultrastructural demonstration of lipids – post fixing in
imidazole-osmium tetroxide.
 QUALITY OF GOOD FIXATIVE
1. Prevents autolysis and bacterial decomposition.
2. Preserves tissues in their natural state and fixes all
components (protein, carbohydrates, fats).
3. Makes the cellular components insoluble to reagents
used in tissue processing.
4. Preserves tissue volume.
5. Avoids excessive hardness of fixed tissue.
6. Allows enhanced staining of tissues.
7. Is be non-toxic and non-allergic for user.
8. Is not be very expensive.
 CLASSIFICATION OF FIXATIVE
1. On the bases of specimen structure
(A) Micro-anatomical fixatives (Tissue):
When anatomy of tissues with correct relationship of
tissue layers & large aggregate of cells is to be
preserved.
i. Buffered formal saline
ii. Buffered glutaraldehyde
iii. Zenker's formal saline
iv. Bouin's fluid
 CLASSIFICATION OF FIXATIVE
1. On the bases of specimen structure
(B) Cytological fixatives:
To preserve constituent elements of cells.
i. Ethanol
ii. Methanol
iii. Ether
 CLASSIFICATION OF FIXATIVE
1. On the bases of specimen structure
(C) Histochemical fixatives:
Used when histochemical tests are to be applied ( IHC).
i. Formal saline
ii. Cold acetone
iii. Absolute alcohol
 CLASSIFICATION OF FIXATIVE
2. Baker Classification of fixative
Coagulant fixatives includes:
1. Formaldehyde
2. Gluteraldehyde
3. Osmium Tetroxide
4. Potassium Dichromate
5. Acetic Acid
Non-Coagulant fixatives includes:
1. Alcohol
2. Zinc salts
3. Mercuric chloride
4. Chromium trioxide
5. Picric Acid
 CLASSIFICATION OF FIXATIVE
3. General classification of fixative
a. Chemical Fixative:
1. Aldehydes: formaldehyde, glutaraldehyde, acrolein
2. Oxidizing agents: osmium tetroxide, potassium
permanganate, potassium dichromate
3. Protein denaturing agents: acetic acid, methyl
alcohol, ethyl alcohol
4. Other cross linking agents: carbodiimides
 CLASSIFICATION OF FIXATIVE
2. General classification of fixative
5. Unknown mechanism: mercuric chloride, picric acid

6. HOPE fixatives: Hepes-glutamic acid buffer mediated

Organic solvent Protection Effect)

formalin like morphology, good protein antigenicity

for enzyme histochemistry, good for RNA & DNA
yields.

b. Physical agents: heat, microwave, Freeze drying

FACTORS EFFECTING ON FIXATION
1. Size and thickness of the piece of tissue
2. Tissues covered by large amounts of mucus or blood,
or organs containing very large amount of blood fix
slowly.
3. Fatty and lipomatous tissues fix slowly.
4. Fixation is accelerated by agitation.
5. Fixation is accelerated by maintaining temperature
around 60°C.
6. Hydrogen ion concentration (Buffer)
Best fixation occurs between pH 6-8
 PREPARATION OF FIXATIVE
• Formal (10% formalin) calcium acetate
Tap water 900 ml
Formaldehyde (37%) 100 ml
Calcium acetate 20 g
Note: This is a good fixative for preservation of lipids.
• Formal (10% formalin) saline
Tap water 900 ml
Formaldehyde (37%) 100 ml
Sodium chloride 9 g
 PREPARATION OF FIXATIVE
• Formal (10% formalin) buffered saline
Tap water 900 ml
Formaldehyde (37%) 100 ml
Sodium chloride 9 g
Sodium phosphate, dibasic 12 g
• Formalin buffered zinc
10% neutral buffered formalin 1000 ml
Zinc chloride 1.6 g
 PREPARATION OF FIXATIVE
• Zenker’s solution
Distilled water 250 ml
Mercuric chloride 12.5 g
Potassium dichromate 6.3 g
Sodium sulfate 2.5 g
Note: Just before use, add 5 ml of glacial acetic acid to
95 ml of above solution. This is a good fixative for bloody
(congested) specimens and trichrome stains.
 PREPARATION OF FIXATIVE
• Bouin’s solution
Saturated aqueous solution of picric acid 1500 ml
Formaldehyde (37%) 500 ml
Glacial acetic acid 100 ml
Note: Fixation time should not be more than 5 days.
• Alcoholic formalin
Ethanol (95%) 895 ml
Formaldehyde (37%) 105 ml
 PREPARATION OF FIXATIVE
• Fixative for metabolic bone disease
Phosphate buffer (page 528) 900 ml
Formaldehyde (37%) 100 ml
Adjust pH to 7.35.
• Davidson’s fixative for eyes
95% ethyl alcohol 300 ml
Formaldehyde (37%) 200 ml
Glacial acetic acid 100 ml
Distilled water 300 ml
Note: This fixative gives good nuclear details and may also be
used to fix hematopoietic organs as well as rodent testes (fix
for 24 hours).
 PREPARATION OF FIXATIVE
• Miller’s or Möller’s solution
Potassium dichromate 2.5 g
Sodium sulfate 1 g
Distilled water 100 ml
• B5 fixative
Mercuric chloride 12 g
Sodium acetate 2.5 g
Distilled water 200 ml
Note: Add 2 ml of formaldehyde (37%) to 20 ml of above
solution just before use. It is frequently used for bone
marrow, lymph nodes, spleen, and other hematopoietic
tissues.
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