Fixation CH 1 Flashcards
Fixation
(Definition)
- Alters tissue by stabilizing the protein so that it is resistant to further changes.
- Must change the soluble contents of the cell into insoluble substances so that they are not lost during subsequent processing.
- Can be chemical or physical
Functions of Fixatives
- To prevent putrefaction (bacterial attack during decay or
postmortem activity) - To prevent autolysis (enzyme attack during decay or
postmortem activity) - To help maintain the proper relationship between cells and
extracellular substances - Brings out differences in refractive indexes and to increase contrast between tissues for microscopic evaluation
- Enhance subsequent staining
Actions of Fixatives
1.) Chemical method – additive or non- additive, coagulant or non-coagulant
2.) Physical method – using heat to fixate tissue
- Microwave Processing
- Overheating alters nuclei staining
- Desiccation
Actions of Fixatives
- Additive fixation – protein combines chemically with a fixative molecule and protein then becomes insoluble. (Mercuric Chloride, chromium trioxide, picric acid, formaldehyde, glutaraldehyde, osmium tetroxide.)
- Non additive fixation – denaturation causes
protein to become less capable of maintaining an intimate relationship with water, but does NOT combine the protein.
(Acetone, Alcohols)
Actions of Fixatives
- Coagulant fixative – establishes a network in tissue that allows solutions to readily
penetrate or gain entry into the interior of the tissue
(Alcohol, zinc salts, mercuric chloride & picric acid) - Noncoagulant fixative – act by creating a gel that makes penetration by the subsequent solutions difficult.
(Formaldehyde, glutaraldehyde, osmium tetroxide, potassium dichromate and acetic acid)
Factors influencing Fixation
- Choice of fixative – staining procedure(s) for tissue must be considered.
- Penetration – fixatives penetrate at different
rates and fixate at different rates. (i.e. formalin penetrates fast but cross-links
slowly) - Tissue storage – certain fixatives do not allow for long term storage. NBF or 70% alcohol are acceptable for long term storage.
Factors influencing Fixation
1.) Temperature – increase in temperature increases the
rate of fixation (but also increases rate of autolysis &
diffusion of cellular elements).
2.) Size – tissue thickness should be no more than 3mm thick.
3.) Volume ratio – fixative volume should be at least 15
to 20 times greater than the tissue volume.
4.) Time – tissue should be placed in fixative immediately
after surgical removal duration in fixative is crucial; adequate fixation time is needed to complete the fixation process
Factors influencing Fixation
- pH – applies to aldehyde fixatives (i.e. pH below 5 can cause formation of formalin
pigment) and to immunohistochemisty techniques. - Osmolality – also must be considered for immunohistochemistry techniques.
Reactions of the cell with fixatives
- Nucleus – preferred fixatives for nucleic acids are acetic alcohol & Carnoy solution
- Proteins – fixation can affect the secondary or tertiary structures of proteins.
- Lipids – several fixatives will preserve but only two (osmium tetroxide & chromic acid) will fix so that they are not lost during subsequent processing
- Carbohydrates – can be lost during fixation
- Simple Aqueous Fixatives
1. Acetic acid
2. Formaldehyde
3. Glutaraldehyde
4. Mercuric chloride
5. Osmium tetroxide
6. Picric acid
7. Potassium dichromate
8. Zinc salts
- Acetic acid
- Formaldehyde
- Glutaraldehyde
- Mercuric chloride
- Osmium tetroxide
- Picric acid
- Potassium dichromate
- Zinc salts
Simple Aqueous Fixatives
- Acetic acid - Noncoagulant
- Acetic acid - Noncoagulant
- Concentrated is called glacial acetic acid due to freezing point of 16.6C
- Penetrates rapidly and leaves tissue soft
- Used in preservation of nucleoproteins
- Causes swelling of tissue
- Red blood cells (RBC) are lysed
- Precipitates DNA
- Causes Protein swelling more than any other
- Collagen greatly affected; absorbs water with contact at lower pH’s
Simple Aqueous Fixatives
- Formaldehyde
- Formaldehyde – Noncoagulant additive
- Gas that comes in 37% to 40% solution with water called formalin
- Commercial stock solutions of 37% to 40% formaldehyde are considered 100% formalin
- In preparing 10% formalin, 1 part commercial stock to 9 parts water (leaving a concentration of 3.7% to 4% formaldehyde)
Simple Aqueous Fixatives
- Formaldehyde
- Formaldehyde has a strong tendency to polymerize to dimers and trimers.
- Paraformaldehyde is a highly polymeric form of formaldehyle (has uses in EM).
- Commercial formalin contains 10% to 14% methanol to prevent polymerization
Simple Aqueous Fixatives
- Formaldehyde
- Reacts with the amino group (NH2) on the side chains of amino acids.
- Causes the formation of methylene bridges that cross-link the proteins together.
Simple Aqueous Fixatives
- Multiple Fomalin solutions
1.) 10% Aqueous Formalin
2.) 10% Formalin saline
3.) Calcium Formalin
- Recommended for phospholipids
4.) Formalin ammonium bromide
- Recommended for CNS
5.) Acetate Formalin
6.) 10% Neutralized & 10% NBF
7.) Modified Millonig Formalin
- Can be used for EM & Light microscopy
8.) Alcoholic Formalin
