FIXATIVES

WHAT IS FIXATIVE?
It is defined as a substance that preserves the morphological and
chemical characteristics of cells and tissues and prevent autolytic and
putrefactive changes.
Fixation
It is the processes by which the constituents of the cells and tissues
are fixed in a physical and partly in a chemical state. This will result in
withstand of cells/tissue from subsequent treatment with various
reagents with a minimum loss, distortion or decomposition .
AIMS OF FIXATION
To prevent autolysis and putrefaction
 Preserve the tissue in life like manner
To prevent any change in size and shape of cells
To have better optical quality and clear staining of cells
To make the tissue hard and firm
TYPE OF FIXATION
CHEMICAL PHYSICAL
METHOD METHOD

• HEAT FIXATION
• COAGULANT • MICROWAVE
FIXATIVE FIXATION
• NON COAGULANT • FREEZE-DRYING
FIXATIVE AND
• COMPOUND
SUBLIMATION
FIXATIVE
CLASSIFICATION OF FIXATIVES

• Fixative classified in several ways:

 According to the components present
1. Simple fixative : contain single chemical
formaldehyde, glutaraldehyde, ethanol
2.Compound fixative: contain more than one chemical
Formalin based fixatives (10% BNF, formal calcium etc..)
Mercurial fixatives (Zenker's solution , helly’s solution,B5)
Picric acid fixatives(Bouin's fluid)
3.Alcohol containing fixatives: carnoy’s fluid, AAF
According to mode of action/type of fixation
1.Chemical method:
Aldehydes: formaldehyde, glutaraldehyde
Protein denaturing: acetic acid, methanol, ethanol
Oxidizing agents: osmium tetroxide, potassium permanganate
Other crosslinking agents: carbodiimides
2.physical method: heat ,microwave, freeze drying
Fixative based on nature of specimen/material used
1. Micro-anatomical:
formalin based fixatives, mercuric fixative, picric acid fixative
2.Histochemical fixatives:
NBF, cold acetone, absolute alcohol
3.Cytological fixatives:
Nueclear fixative: carnoy’s fixative, Clarke’s fluid, Flemings
Cytoplasmic fixative: champy’s fluid, Zenker's, formal saline
formal calcium ,ethanol, regaud’s fluid
Commonly used fixatives
Formaldehyde
Glutaraldehyde
Osmium tetroxide
Methanol/Ethanol
Bouin’s fixative
Mercury salt containing fixatives
• Zenker’s fluid
• Helly’s fluid
• B5 fixative
PREPARATION OF FIXATIVE
FORMALDIHYDE/FORMALIN:
Formaldehyde is a gas which commercially available as 40%solution in
water(formalin).
Mode of action : Formaldehyde reacts with proteins and forms cross-links
between the protein molecule and forms insoluble end product.
 ADVANTAGES DISADVANTAGES

• Cheap • Dermatitis
• Tissue penetration is good • Damage to nasal mucosa
• Best fixative for nervous system • Formalin pigment
• Cause less excessive hardness • It may be carcinogenic
• Can use for frozen section and fat
staining and enzyme studies
• Natural tissue color can restore
Formalin based fixative Preparation
composition quantity
1.10% formalin • Formalin 100ml
• DW 900ml
2.Formal saline • Formalin 100ml
• NaCl 9g
• DW 900ml
3.10%Neutral buffered formalin • Formalin 100ml
• Sodium phosphate monobasic 4g
• Anhydrous disodium phosphate 6.5g
• DW 900ml

4.Formal calcium acetate • Formalin 100ml

• calcium acetate 20g
• DW 900ml

5.Buffered formal sucrose • Formalin 10ml

• Sucrose 7.5g
• Phosphate buffer(pH7.4) 100ml
6.Alcoholic formalin • Formalin 10ml
• 70-95%alcohol 90ml
• Calcium acetate 0.5g

7.Acetic alcoholic formalin • Formalin 5ml

• Glacial acetic acid 5ml
• 70% alcohol 90ml
Glutaraldehyde
ADVANTAGES DISADVANTAGES
 Used for electron microscopy
Commercially - 25%Glutaraldehyde
• Rapid • Not ideal for IHC
Action: cross-linking • Preserve Good • Non specific PAS
morphology staining
• Less shrinkage • Poor penetration
Preparation: • Protein preservation • costly
Buffered glutaraldehyde
25%Glutaraldehyde - 16ml
Phosphate buffer pH7.4 - 84ml
Mercury fixative
Mode of action: fixation by precipitating protein

ADVANTAGES DISADVANTAGES

• Quick fixation • Rate of penetration of fixative

• Best result with metachromatic reduces after the few millimeters
staining • Intolerant fixative
• Best photomicrography • Radio opaque
• Good cytoplasmic , • Mercury pigment
nuclei ,connective tissue staining
Preparation of mercury fixative:
Fixative Advantage Disadvantage Use
Zenker’s fluid • good for bloody specimen • Mercuric pigment Very highly vascular
and trichrome staining • Dichromate pigments organs(spleen)
• Rapid and even penetration

Helly’s fixative • Good cytoplasmic and micro • Mercuric pigment Bone marrow, spleen
anatomical fixative • Dichromate pigments
FMA • Rapid and even penetration • Intolerant fixative Skin biopsies
• Brilliant staining • Mercuric pigment
Potassium dichromate
Mode of action: similar to formalin and causes fixation cytoplasm without
precipitation

ADVANTAGES DISADVANTAGES
Preparation:
• Preserves • Unsuitable for IHC
phospholipid
• Fixation of
mitochondria
PICRIC ACID FIXATIVE
Mode of action: It precipitate protein and forms picrates with amino acid and
nucleoprotein.

ADVANTAGES DISADVANTAGES

• Brilliant result with trichrome • Causes yellow discoloration

staining.
• Penetrate well
• Good fixative for connective
tissue,glycogen and lipids
Preparation of picric acid containing fixative:
Alcohol fixatives
Most commonly used are ethanol and methanol
Mode of action: (COAGULATIVE FIXATIVES) Removes water, Destabilize
hydrogen bonds and disrupts tertiary structure of protein.
Uses: blood smear fixation, cytology smear fixation, frozen section

ADVANTAGES DISADVANTAGES

• 80%-100% methanol-fixative for • Shrinkage of tissue

smear • Hardens the tissue
• Ethanol –preservation of enzyme • Contraindication to lipid study
• Good for demonstration of
glycogen ,enzyme
Preparation of alcohol containing fixative:
Osmium tetroxide
Preparation:
2-4% osmium tetroxide in buffer
Advantage:
Good for lipids/mitochondria/Golgi apparatus
Disadvantage: DNA clumping ,Toxic
Use: Electron microscopy
Trouble shooting in fixation
Problem Cause Remedies
Nuclear margin is indistinct and • Incomplete fixation • Check the concentration of
nuclei are fuzzy with bubbling formalin
• Keep more time in formalin
• Do not put more cassettes
together
Tissue shrinkage with large • Poor fixation • Proper fixation time
artefactual spaces • Prolonged fixation • Check fixative concentration
• Immediately fix the tissue after
biopsy

Insoluble brownish- black granular • Formalin pigment due to acid • Use buffered formalin
pigment hematin formation
(formaldehyde reacts with
blood)
Intra epithelial Cleft formation • Formalin may evaporate, • Keep formalin in closely caped
Calcium carbonate is bottle
precipitated
 Fixatives of choice
Tissue Fixative Time Target Fixative
substance
Day to day routine 10%formalin • 12to 24h (large
sample 10%NBF tissue) Protein 10% buffered
• 6h (small tissue) formalin
Lipid Osmium
Lymph node B5 18h tetroxide/frozen
GIT specimen 10%formalin 6h
Glycogen Alcohol
Testis Bouin’s/formalin 6h
Mucco- Frozen
Bone marrow AZF/Bouin's 3h polysacharide
Spleen Zenker’s 6h Enzyme Frozen
Eye 10%formalin 48h DNA/RNA Alcohol
Iron Alcohol
THANK YOU