FIXATION Flashcards
• The first and most critical step in histotechnology. prevents degeneration ,decomposition, putrefaction and distortion of tissues after removal from the body.
Fixation
• Primary aim of fixation:
– to preserve the morphologic and chemical integrity of the cell in as life- like manner as possible
• Secondary goal of fixation:
– to harden and protect the tissue from the trauma of further handling, – so it is easier to cut during gross examination
• To preserve the tissue
– _ stops all cellular activities
• To prevent breakdown of cellular elements
– prevents autolysis by inactivating the __ or by chemically altering, stabilizing and making the tissue components insoluble
– protects the tissue from further decomposition after death due to bacterial or fungal colonization
- • To coagulate or precipitate __ substances
– Fixation renders insoluble certain tissue components that may otherwise leak out during subsequent histologic handling
Fixation
Fixatives
lysosomal enzymes
protoplasmic
2 Basic Mechanisms
Additive fixation
Non-additive fixation
chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links or molecular complexes and giving stability to the protein.
Ex. ___,___,__
Additive fixation
Formalin, mercury, osmium tetroxide
fixative is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water within the protein molecule.
Ex. ____
Non-additive fixation
Alcoholic fixatives
Effects of Fixatives
• Preserve the morphologic and chemical integrity of the cell • Harden soft & friable tissues
• Inhibit bacterial decomposition
• Act as mordants or accentuators
• Osmolality
– Slightly hypertonic solutions 400-450 mOsm
- Isotonic – 340 mOsm
• Concentration
– Formaldehyde – 10%
– Glutaraldehyde – 3%
• 0.25% ideal conc in Immuno EM
Duration of fixation
– Usually for 2-6 hours
– Formalin can be washed after fixation for 24 hours
Hydrogen ion concentration –
pH 6and 8 satisfactory
• Temperature
– Room temp
– T. Processors: 400C,
– EM & Histochemistry: 0-40C
– Formalin Heated to 600C:rapid fixation
– Formalin at 1000C - fix tissues with tuberculosis
• Thickness of section
– 1-2 mm2 for electron microscopy – 2 cm2 for light microscopy
– Large solid tissue
• Uterus
• Brain (suspended whole in 10% buffered formalin for 2-3 weeks)
Practical Considerations of Fixation
• Speed
– prevent autolysis and putrefaction
• Penetration
– Formalin diffuses at 1 mm/hr
• Volume
– Amount of fixative – 10-25x the volume of tissue to be fixed
• DurationofFixation
– Fibrous organs take longer to fix than biopsies or scrapings
– Can be cut down using heat, vacuum, agitation or microwave
Characteristics of fixation
• ____,___,__
• Must be ___
• Inhibit ___
• Must permit ___ and ___
• Must make cellular components insoluble to __ solutions.
- Cheap, stable, safe to handle
- isotonic
- bacterial decomposition
- rapid and even penetration of tissues
- hypotonic
Types of Fixatives
According to COMPOSITION
1. Simple Fixatives: one component
2. Compound Fixatives: 2 or more components
• According to ACTION
– Microanatomical fixatives
– Cytological fixatives
– Histochemical fixatives
Aldehyde
Formaldehyde, Glutaraldehyde
Metallic Fixatives
Ø Mercuric chloride
Ø Chromate fixatives
> Potassium dichromate
>Chromic acid
Lead fixatives
Ø Acetone
Ø Alcohol
ØPicric acid
ØAcetic acid
ØOsmium tetroxide (Osmic acid)
Simple Fixatives
- Aldehydes
- Metallic Fixatives
3 Lead Fixative
4 Heat
made up of two or more fixatives which have been added.
Compound Fixatives
- Permit general microscopic study of tissue structures
Microanatomical Fixatives
- Preserve specific parts
Cytological Fixatives
Nuclear
Cytoplasmic
- Preserve the chemical constituents of cells and tissues
Histochemical Fixatives
Microanatomical Fixatives
- 10% Formol Saline
- 10% Neutral Buffered Formalin 3. Heidenhein’s susa
- Zenker’s solution
- Zenker’s formol (Helly’s solution) 6. Bouin’s solution
- Brasil soln
– preserve nuclear structure (chromosomes). Contain glacial aa, pH 4.6 or less
Nuclear fixatives
1. Flemming’sFluid
2. Carnoy’sFluid
3. Bouin’sFluid
4. Newcomer’sFluid
5. Heidenhainsusa
– preserve cytoplasmic structure, no glacial aa, pH > 4.6
Cytoplasmic Fixatives
1. Flemming’s Fluid without acetic acid
2. Helly’s Fluid
3. Regaud’s Fluid (Muller’s fluid)
4. Orth’s Fluid
- Formol Saline 10%
- Absolute Ethyl Alcohol
- Acetone
- Newcomer’s Fluid
Histochemical Fixatives
• 10% - most widely used
• A gas produced by the oxidation of __
• ___ - unsatisfactory for routine fixation
• Dilution – _
• Usual fixation time – __
• Buffered to
Formaldehyde (formalin)
- methyl alcohol
- Pure stock solution of 40%
- 1:10 or 1:20
- 24 hours
- ph 7 with phosphate buffer
Formaldehyde (formalin)
ADVANTAGES
- Cheap, readily available, easy to prepare, relatively stable
- Compatible with most stains
- Preserves fats, glycogen and mucin
- Allows tissue enzymes to be studied because it does not precipitate proteins
- Recommended for nervous tissue preservation
- Allows natural tissue colors to be restored; recommended for colored tissue photography
- Tolerant fixative used for mailing specimen
DISADVANTAGES of formaldehyde ( Formalin)
- May cause sinusitis, allergic rhinitis, excessive lacrimation or allergic dermatitis
- May produce considerable shrinkage of tissues
- A soft fixative and does not harden some cytoplasmic structures adequately enough for paraffin embedding
Ø Microanatomical fixative
Ø Recommended for fixation of central nervous tissues and general postmortem tissues for histochemical examination
Ø Fixation time: ___
Ø 48 hours at __
Ø Preserves __ and __
Ø Demonstrates ___ and __
10% Formol Saline
- 24 hours at 35C / 95F
- 20-25 °C / 65-77 °C
- enzymes and nucleoproteins
- fats and mucins
Slow fixative
Metachromatic reaction of amyloid is reduced
10% Formol Saline
• Recommended for preservation and storage of surgical, post-mortem and research specimen
• Fixation time: _
• Prevents precipitation of __
• Best fixative for tissues containing __ and__
10% Neutral buffered formalin or Phosphate-buffered formalin
4 -24 hours
acid formalin pigments
iron pigments and for elastic fibers
• Longer to prepare
• Positivity of mucin to __ is reduced
• Reactivity of myelin to ___ is reduced
• Inert towards lipids, especially ___
10% Neutral buffered formalin or Phosphate-buffered formalin disadv
PAS
Weigert’s iron hematoxylin
neutral fats and phospholipids
