Microtechniques 1 - Fixation Flashcards
What are the 7 processing steps
Dissection Fixation Dehydration Infiltration/Embedding Sectioning Staining Slide
What are the 3 main reasons for fixing a specimen?
- Preserve it
- Allow subsequent processing
- Ensure its morphology remains as lifelike as possible
What are the 3 main methods used for fixation?
- Chemical (most common)
- Drying
- Physical
- HEAT: bacterial smears (destroys internal structures - cooked)
- FREEZING: cryoprotective agent used to reduce ice crystal damage, rapid result, poorer quality sections, special microtome, specialist application to retain lost substances
What are 8 desirable properties of a chemical fixative?
- Preserve tissue components (protein, DNA etc.)
- Prevent hardening
- Prevent volume changes
- Render tissue insoluble
- Rapid penetration and reaction
- Anti-bacterial
- Facilitate the type of staining required
- Facilitate later infiltration of wax/resins
Difference between a PRIMARY and MIXED fixative?
P: only one active ingredient
M: several ingredients mixed together (not all fixatives are suitable for this. Aims to combine all the pros and diminish the cons).
What 3 properties of a fixative do you have to consider when deciding what to use?
- CHEMICAL Features:
- Coagulant (separates water and protein) or non-coagulant (preserves organelles, greater chance of shrinkage)?
- Additive or non-additive? - BUFFERS and INDIFFERENT SALTS
- Maintain correct pH & adjust osmolarity (buffer)
- Adjust osmolarity (IS e.g. NaCl) - PHYSICAL features:
- Duration of exposure to fixative (too long = extraction of tissue parts, too short = underfixation)
- Temperature (too high = faster, can cook tissue, too low = v slow)
- Size of piece of tissue (too big = fixation gradient)
- Rate of penetration of fixative (depends on SA:V)
- Rate of reaction of fixative
- Method of exposing tissue to fixative (IMMERSION vs. PERFUSION)
- Special techniques
4 features of well preserved tissue vs. 4 features of poorly preserved tissue?
WELL
- Fine detail
- Minimal shrinkage
- Evenly preserved (no gradients)
- Tissue is adherent (no breaks)
POORLY (because of AUTOLYSIS or BACTERIAL DECAY)
- Shrunken nuclei that is poorly stained
- Fixation gradient
- Shrinkage
- Debris in lumen of hollow structures
PICRIC ACID details
- Coagulant
- Additive
- Explosive when dry (trinitrophenol) - avoid storing in metal containers/lids - forms PICRATE SALTS
- Little hardening
- Excessive shrinkage
- Fast penetration but slow reaction
- Fixes protein & DNA
- Fine coagulum
- Favours binding with acid dyes
- A mordant
What are 2 other examples (besides picric acid) of COAGULANT fixatives?
- Mercuric chloride
- Alcohols (ethyl, methyl) or acetone
How do you neutralise formalin solution & why do we need to? What is an alternative to neutralising that achieves the same result?
- Neutralise using an acid/bas indicator and weak NAOH
- Formalin may polymerise to inactive paraformaldehyde or convert to formic acid and become acidic. This makes it a less effective fixative and therefore neutralising it can prevent this.
- Alternatively make the formalin freshly from formaldehyde in a warm, slightly alkaline solution (prevents polymerising).
FORMALIN details
- Non-coagulant
- Additive
- Some hardening
- Some shrinkage
- Fast penetration, slow reaction
- Fixes protein, DNA
- Favours staining with basic dyes
- Precipitates: in acidic blood can form pigments - formalin haematin
Example of a MIXED fixative? What is it made up of?
Bouins = Formalin (issue with paraffin penetration) + Picric Acid (not much hardening) + Acetic Acid (swells and softens)
Example of an INCOMPATIBLE MIXED fixative? How is this overcome?
Hellys Fixative - dark and scum on surface.
- Make on the day of use
- Use POSTFIXATION (not usually done with Hellys but involves adding reagents and rinsing between each step)
What 3 things are considered when choosing the best fixative?
- Type of tissue being fixed
- What processing procedures will need to be done afterwards e.g. staining
- Fixation tests e.g. homogenous protein - egg white smears, gelatin smears, liver. Use this to check test specimen for: speed (colour changes), coagulum or not, volume changes, hardening, staining. Optional to alter: time, temperature, immersion/perfusion, partial vacuum or vapour fixation.
