LM: Preparation Of Material (Fixation) (L7)

Question 1

LM: Preparation of material attributes?

Answer 1

• 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.

Question 2

Why can we not use live specimens? (4)

Answer 2

● 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.

Question 3

Steps/Processes for preparation of “dead” material? (4)

Answer 3

• Fixation.
• Embedding.
• Sectioning.
• Staining.

Question 4

Fixation?

Answer 4

= kills & “fixes” the cells & tissues.

Question 5

Embedding attributes? (2)

Answer 5

• If sections are to be cut (provides support when cutting sections).

• In paraffin wax.

Question 6

Sectioning?

Answer 6

= done if required.

Question 7

Why do we stain tissues? (2)

Answer 7

● To improve contrast.

● To discriminate amongst tissue & cell components.

Question 8

Fixation aims/Aims of Fixation/Why fix specimens? (4)

Answer 8

● 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.

Question 9

Results of fixation? (4)

Answer 9

● 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.

Question 10

Things to note when dealing with fixatives? (2)

Answer 10

• Wear gloves when dealing with fixatives as they are carcinogenic!!

• Entire sample must be in fixative.

Question 11

Fixative process?

Answer 11

= chemical fixatives are classified in several different ways according to how they act.

Question 12

Types of chemical fixatives? (6)

Answer 12

• Additive fixatives.
• Non-additive fixatives.
• Coagulant fixatives.
• Non-coagulant fixatives.
• Coagulant non-additive fixatives.
• Non-coagulant Additive fixatives.

Question 13

Additive fixatives?

Answer 13

= incorporated into & cross link protein molecules.

Question 14

Non-additive fixatives?

Answer 14

= not incorporated into protein molecules & don’t cross link them.

Question 15

Coagulant fixatives?

Answer 15

= coagulate proteins.

Question 16

Non-coagulant fixatives?

Answer 16

= do not coagulate proteins.

Question 17

Explain Coagulant non-additive fixation diagram before and after?

Answer 17

● Before
= just loose strands.

● After
= strands are now mushed together.

Question 18

Explain Non-coagulant additive fixation diagram before and after fixation?

Answer 18

● Before
= loose strands.

● After
= strands are now connected/linked to each other.

Question 19

Coagulant fixatives attributes? (3)

Answer 19

• 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).

Question 20

Egs of Coagulant fixatives? (8)

Answer 20

• Methanol.
• Ethanol.
• Acetone.
• Nitric acid.
• HCl.
• Pitric acid.
• Mercuric chloride.
• Chromium trioxide.

Question 21

Non-coagulant fixatives attributes? (4)

Answer 21

• 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.

Question 22

Egs of Non-coagulant fixatives? (5)

Answer 22

• Aldehydes (eg, formaldehyde & glutaraldehyde).

• Acrolein.
• Asonium tetroxide.
• Potassium dichromate.
• Acetic acid.

Question 23

Chemical fixatives attributes? (3)

Answer 23

● 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.

Question 24

Factors affecting chemical fixation? (6)

Answer 24

• pH.
• Osmolarity.
• Temperature.
• Duration of fixation.
• Method of application.
• Size & density of the tissue.

Question 25

pH attributes? (3)

Answer 25

• 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.

Question 26

Egs of common buffers? (2)

Answer 26

• Cacodylate buffer.
• Phosphate buffer (Pbs).

Question 27

How to adjust the pH?

Answer 27

We use HCl (to make pH more acidic) or NaOH (to make pH more alkaline).

Question 28

Eg of how chemical fixatives are used in combination with properties of different fixatives being complementary?

Answer 28

In light microscopy, a mixture of formalin, acetic acid & ethanol is used (FAA).

Question 29

Osmolarity attributes? (2)

Answer 29

• If the fixative is isotonic with cells, the cells will neither shrink nor swell.

• If not, severe distortion (swelling or shrinking) may occur.

Question 30

How to adjust osmolarity?

Answer 30

Add electrolytes (NaCl or CaCl2) or non-electrolytes (PVP).

Question 31

PVP stands for?

Answer 31

Polyvinylpyrrolidone.

Question 32

Temperature attribute?

Answer 32

Optimal temperature is specific for different fixatives (eg, room temperature mostly, or 3⁰C rarely).

Question 33

Fixative penetration rate attributes? (2)

Answer 33

• Ideally, fixatives should penetrate rapidly & kill and fix specimens quickly.

• Rate is influenced by several factors.

Question 34

Factors that influence the rate of penetration of fixatives? (5)

Answer 34

• Type of fixative.
• Molecular size of fixative.
• Solubility in lipids.
• Polarity of the molecules.
• Size & density of tissue block.

Question 35

Eg of the Penetrative rate of fixative?

Answer 35

Formalin > Glutaraldehyde or OsO4, but glutaraldehyde fixes better than formalin.

Question 36

Size of tissue block attributes? (4)

Answer 36

• 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.

Question 37

What is a small tissue block for LM:

For histology?

Answer 37

May be several cm³, especially for plant material.

Question 38

What is a small tissue block for LM:

For cytology?

Answer 38

A few mm³.

Question 39

What is a small tissue block for TEM?

Answer 39

1mm³ or less.

Question 40

Why is specimen shape important?

Answer 40

It’s because a thin or flat specimen can be larger in other dimensions.

Question 41

After fixation process goes two ways, what are those two ways?

Answer 41

• Material doesn’t require sectioning.
• Material requires sectioning.

Question 42

Layout of the 1st outcome after fixation process? (3)

Answer 42

Material doesn’t require sectioning
|
Washing
|
Staining

Question 43

Layout of the 2nd outcome after fixation process? (4)

Answer 43

Material requires sectioning
|
Washing
|
Dehydration
|
Embedding (either in wax or resin)

Question 44

Eg of Material that doesn’t require sectioning?

Answer 44

Plant material.

Question 45

Eg of Material that does require sectioning?

Answer 45

Animal material.