LM: Preparation Of Material (Fixation) (L7) Flashcards
LM: Preparation of material attributes?
• Investigation of live material is not always possible.
• Examination & analyses of dead tissues & cells is therefore often more practical.
• Use of dead material allows permanent preparations to be made.
Why can we not use live specimens? (4)
● Cutting thin sections of live tissues/cells damages cells.
● Few stains & histochemical tests can be used on live material.
● Live cells do not always stain well.
● One can’t make permanent preparations of live materials.
Steps/Processes for preparation of “dead” material? (4)
• Fixation.
• Embedding.
• Sectioning.
• Staining.
Fixation?
= kills & “fixes” the cells & tissues.
Embedding attributes? (2)
• If sections are to be cut (provides support when cutting sections).
• In paraffin wax.
Sectioning?
= done if required.
Why do we stain tissues? (2)
● To improve contrast.
● To discriminate amongst tissue & cell components.
Fixation aims/Aims of Fixation/Why fix specimens? (4)
● Kill the living protoplasm quickly to minimize autolysis & degenerative changes.
● Convert the mobile, dynamic vacillated protoplasm of living cells into an immobile, firm gel & stabilize it in as natural a state as possible.
● Stabilize cells & tissues so that the original structure & distribution of cells in tissues & tissues in organs is maintained.
● Make tissue & cell components more reactive to stains.
Results of fixation? (4)
● Cell contents, cells & tissues are preserved in as close to their original state as possible.
● Cell contents, cells & tissues are made resistant to distortion do that they can withstand further processing (dehydration, embedding, sectioning, etc).
● Refractive indices of certain components of the tissue are increased so that they can be distinguished more easily from others.
● The material is prepared for effective staining.
Things to note when dealing with fixatives? (2)
• Wear gloves when dealing with fixatives as they are carcinogenic!!
• Entire sample must be in fixative.
Fixative process?
= chemical fixatives are classified in several different ways according to how they act.
Types of chemical fixatives? (6)
• Additive fixatives.
• Non-additive fixatives.
• Coagulant fixatives.
• Non-coagulant fixatives.
• Coagulant non-additive fixatives.
• Non-coagulant Additive fixatives.
Additive fixatives?
= incorporated into & cross link protein molecules.
Non-additive fixatives?
= not incorporated into protein molecules & don’t cross link them.
Coagulant fixatives?
= coagulate proteins.
Non-coagulant fixatives?
= do not coagulate proteins.
Explain Coagulant non-additive fixation diagram before and after?
● Before
= just loose strands.
● After
= strands are now mushed together.
Explain Non-coagulant additive fixation diagram before and after fixation?
● Before
= loose strands.
● After
= strands are now connected/linked to each other.
Coagulant fixatives attributes? (3)
• Coagulate & denatured proteins: form a precipitate of protein clungs & strands.
• Make tissues more opaque (coagulation of protein).
• Distort fine structures of the protoplasm (acceptable at LM level, but not for EM).
Egs of Coagulant fixatives? (8)
• Methanol.
• Ethanol.
• Acetone.
• Nitric acid.
• HCl.
• Pitric acid.
• Mercuric chloride.
• Chromium trioxide.
Non-coagulant fixatives attributes? (4)
• Transform proteins into a transparent gel.
• No clumping of proteins (suitable for both LM & EM).
• Aldehydes leave protein structures & enzymatic activity intact.
• Formalin & glutaraldehyde don’t alter membrane permeability.
Egs of Non-coagulant fixatives? (5)
• Aldehydes (eg, formaldehyde & glutaraldehyde).
• Acrolein.
• Asonium tetroxide.
• Potassium dichromate.
• Acetic acid.
Chemical fixatives attributes? (3)
● Often used in combination with properties of different fixatives being complementary.
● Optimum fixing conditions can vary according to the tissue being fixed.
● In addition to the type & concentration of fixatives, effective fixation is dependent on a number of factors.
Factors affecting chemical fixation? (6)
• pH.
• Osmolarity.
• Temperature.
• Duration of fixation.
• Method of application.
• Size & density of the tissue.
pH attributes? (3)
• Some fixatives (eg OsO4) can drop the pH of tissues drastically.
• Buffe solutions are therefore required to try to keep the pH optimal & prevent distortions.
• Common buffers.
Egs of common buffers? (2)
• Cacodylate buffer.
• Phosphate buffer (Pbs).
How to adjust the pH?
We use HCl (to make pH more acidic) or NaOH (to make pH more alkaline).
Eg of how chemical fixatives are used in combination with properties of different fixatives being complementary?
In light microscopy, a mixture of formalin, acetic acid & ethanol is used (FAA).
Osmolarity attributes? (2)
• If the fixative is isotonic with cells, the cells will neither shrink nor swell.
• If not, severe distortion (swelling or shrinking) may occur.
How to adjust osmolarity?
Add electrolytes (NaCl or CaCl2) or non-electrolytes (PVP).
PVP stands for?
Polyvinylpyrrolidone.
Temperature attribute?
Optimal temperature is specific for different fixatives (eg, room temperature mostly, or 3⁰C rarely).
Fixative penetration rate attributes? (2)
• Ideally, fixatives should penetrate rapidly & kill and fix specimens quickly.
• Rate is influenced by several factors.
Factors that influence the rate of penetration of fixatives? (5)
• Type of fixative.
• Molecular size of fixative.
• Solubility in lipids.
• Polarity of the molecules.
• Size & density of tissue block.
Eg of the Penetrative rate of fixative?
Formalin > Glutaraldehyde or OsO4, but glutaraldehyde fixes better than formalin.
Size of tissue block attributes? (4)
• To achieve uniform fixation, a small specimen is required.
• If specimen is too thick or large, outer layers maybe overfixed & may stain more darkly later, while the core remains unfixed (even fixation is ideal).
• What is small?
• Specimen shape is important.
What is a small tissue block for LM:
For histology?
May be several cm³, especially for plant material.
What is a small tissue block for LM:
For cytology?
A few mm³.
What is a small tissue block for TEM?
1mm³ or less.
Why is specimen shape important?
It’s because a thin or flat specimen can be larger in other dimensions.
After fixation process goes two ways, what are those two ways?
• Material doesn’t require sectioning.
• Material requires sectioning.
Layout of the 1st outcome after fixation process? (3)
Material doesn’t require sectioning
|
Washing
|
Staining
Layout of the 2nd outcome after fixation process? (4)
Material requires sectioning
|
Washing
|
Dehydration
|
Embedding (either in wax or resin)
Eg of Material that doesn’t require sectioning?
Plant material.
Eg of Material that does require sectioning?
Animal material.
