Fixation 2

Microscopical examination
of autolysed tissues
• The nucleus of
poorly fixed autolysed tissue
shows
• Pyknosis
:Condensation
• Karyorrhexis :
Fragmentation
• Karyolysis: lysis and
complete loss
• The cytoplasm
• May becomes swollen
and glanular
 Classification
• Types of fixatives according to their nature:
• Physical fixatives:
• Heat, microwave.
• Chemical fixatives:
• Which classified according to their:
• Components:
• . Simple Compound
• Action:
• Coagulant ..additive
• Non-coagulant..non additive
• c. Uses:
• Micro-anatomical fixatives Cytological
• d. Chemical nature:
• Precipitating (Protein-denaturizing) fixatives
• Cross-linking fixatives..
• .
• Chemical fixatives according to Components:
• Simple Compound
• 95% Ethyl Alcohol • Pcric acid +formalin
Bouin’s • +acitic acid
• fixative
10% Formalin

Carnoy’s • Ethyl alcohol +

• Chloroform + Acitic acid
Acetic acid
fixative
• . According to their Action:
• Coagulant & Non-Coagulant
• 1. Coagulation – will allow the solutions to penetrate into
the interior of the tissue very easily.
• 2. Non-coagulant - they act by creating a gel barrier that
makes solutions more difficult to penetrate to the interior
of the tissue.
• Coagulant Fixatives Reagents includes:
• • Alcohol • Zinc salts • Mercuric chloride •
Chromium trioxide • Picric Acid
• Non-Coagulant Fixatives Reagents includes:
• • Formaldehyde • Gluteraldehyde • Osmium
Tetroxide • Potassium Dichromate • Acetic
Acid
Chemical nature: how to stabilise the
protein:

• Cross linking: act by creating chemical bonds

between proteins in tissue in order to stabilise
the protein.

• Precipitating (or denaturing) fixatives act by

reducing the solubility of protein molecules
• Another classification
• 1. Aldehydes (formaldehyde, glutaraldehyde).
• 2. alcoholic fixatives :methanol, ethanol,
Carnoy’s fluid
• 3. Oxidizing agents: potassium permengenate
potasium dichromate ,osmium tetroxide,
chromic acid.
• 4. Miscellaneous: mercuric chloride, picric
acid, vapour fixation, liquid nitrogen
Factors affect the quality of
fixation
 FACTORS AFFECTING FIXATION
• Temperature
• Duration and Size
• Penetration
• Volume Ratio
• Concentration
• Osmolality
• pH
• Time
 Formalin pigments
• At the acidic pH of unbuffered formaldehyde, the
hemoglobin metabolic products are chemically
modified to form a brown-black, insoluble
pigment.

• It is removed easily with an alcoholic solution of

picric acid.
• To avoid the formation of formalin pigment,
neutral buffered formalin is used as the preferred
formaldehyde-based fixative.
Formalin pigment in tissue
 Choice of Fixative
The main problem with fixatives used in histological
staining is the loss of molecules that are targets of
specific histochemical methods by fixative

• Typically, some molecules are soluble in aqueous

fixatives (e.g. glycogen),

• while others are soluble in organic-based fixatives

(e.g. lipids).
 • The fixative should be selected with caution
avoiding fixatives that may affect future
histochemical studies.

• . If future studies include a diagnostic for Gout a fixative

containing water will not work since the water will dissolve
the Urate crystals.

• If Enzyme studies are required, so the fixative of choice is

absolute alcohol. frozen sections are preferred.
 REACTIONS OF THE CELL WITH
FIXATIVES
• Glycogen
• Can show different degrees of polymerization.
• When fixed with formaldehyde fixatives for
long time in room temp.
• lipids
• are largely lost from tissues during processing
and could be will fixed by osmium tetroxide
and chromic acid

• Lipids can be demonstrated in cryostat

sections fixed with reagents containing
mercuric chloride and potassium dichromate
• Cross-linkage fixatives (Aldehydes)
• Formaldehyde (formalin) :
• It’s gas soluble in water in 40%.
• Formalin penetrates tissue well, but is
relatively slow.
• The storage of formalin leads to formation of
formic acid.
• The formation of formic acid can be avoided by
neutralization using buffered solution.
• The standard solution is 10% neutral buffered
formalin (4% formaldehyde).
• A buffer prevents acidity cause of formol-heme
pigment in the tissues.(formalin pigment)
• 10% formal-saline:
• Formalin 1ooml
• Sodium chloride 8.5 g
• Tap water 900 ml
• Buffered 10% formalin (pH 7.0):
• Formalin 100ml
• Tap water 900ml
• Acid Na-phosphate monohydrate 4g
• Anhydrous disodium phosphate 6.5g
• Mercuric chloride:
• These fixatives are widely used as
secondary fixatives.
• lead to formation of Mercury pigment.
• This removed by alcoholic iodine or Na-
thiosulphate and prevented when use
buffered fixative.
• E.g. Susa fixative – Zenker’s fluid
• Susa fixative:
• Mercuric chloride 45g
• Na CL 5g
• TCA 20g
• Acetic acid 40ml
• Formalin 200ml
• D.W 800ml
• Zenker’s fluid
• Mercuric chloride 50g
• Potasium di chromate 25g
• Acetic acid 50ml
• D.W 950ml
• Bouin’s fixative:
• Saturated picric acid 75 ml
• Formalin 25ml
• Acetic acid 5ml
 Artifact
• Structures or features in tissue that interfere
with normal histological examination.
• And not related to the normal histology or
pathology of the tissue and come from
outside sources

• Fixation artifacts
• Formalin pigment
• Streaming artifact
• Mercury Pigments
WHAT IS AFTER FIXATION