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
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