Fixation & Tissue Stabilisation

Question 1

What are the key aims of fixation?

Answer 1

> Preventing bacterial growth
Preventing cell autolysis
Allow for the visualisation of various cell/tissue components
Even and rapid penetration of the tissue
Acceptable cost and not too optically dense

Question 2

What are the 2 class of fixatives?

Answer 2

1. Coagulants (form visible coagulum & occupy space of tissue with fluid)
2. Non-coagulants (no visible coagulum formation)

Question 3

What is the most important cell/tissue component to be fixed, and how is that achieved?

Answer 3

Proteins. The formation of cross-links between soluble proteins and non-soluble, structural proteins renders the soluble ones insoluble. This process forms a gel-like substance.

Question 4

How do aldehydes fix tissue?

Answer 4

Through forming links between basic regions of neighbouring amino acids. Lipids are ‘contained’, but not necessarily fixed.

NB Aldehydes are non-coagulant fixatives

e.g. formaldehyde

Question 5

How do oxidising agents fix tissue?

Answer 5

Convert proteins to gel, whilst dissolving DNA too. They have little effect on carbohydrates/lipids, so are useful for lipid demonstration.

NB Oxidising agents tend to be non-coagulant

e.g. potassium dichromate

Question 6

How do protein denaturing reagents fix tissue?

Answer 6

By precipitating and dehydration. Proteins & carbohydrates are precipitated but lipids aren’t preserved/are dissolved.

This method is used for cytological samples due to the large morphological changes caused by dehydration (Morphology isn’t overly important for cytological samples).

e.g. ethanol, methanol

Question 7

How does temperature fixation work?

Answer 7

Heat - via protein denaturing (cooked)

Cold - freeze structures in place.

Question 8

What factors affect fixation, and how are they overcome?

Answer 8

pH - Tissues are best fixed when pH is adjusted to physiological range (6-8)

Penetration - Use of smaller, thinner specimens to allow a more even penetration of fixative

Osmolarity - Best results achieved with slightly hypertonic solution (so cells are slightly shrivelled)

Concentration & time - concentration of fixative, and time allowed for its action

Question 9

What are the 4 stages of tissue processing (after fixation)?

Answer 9

1. Dehydration (remove water from fixed tissue)
2. Clearing (remove dehydrating agent)
3. Impregnation (the addition of the embedding medium to the tissue)
4. Embedding (embed the impregnated tissue in its medium)

3/4 example - impregnate with paraffin, then embed in more paraffin (I think)

Question 10

What is the most common embedding medium, and why?

Answer 10

Paraffin wax

Cheap, low toxicity, easily removed from tissue section by solvents, various melting points