Introduction to Clinical Sciences and Introduction to Cellular Pathology

Question 1

List the stages of tissue preparation for microscopy.

What is the purpose of each stage?

What does each stage involve?

Answer 1

Stages of tissue preparation for microscopy:

1 - Fixation.

• The purpose of fixation is to preserve tissues in a lifelike condition by inhibiting enzyme activity and metabolism.
• Fixation may involve dehydration (e.g. by air drying or application of salt), or more commonly cooling / freezing or application of a chemical fixative.

2 - Tissue support.

• The purpose of this stage is to modify the structure of the tissue so as to prevent it from collapsing when it is cut into thin slices.
• This firstly involves dehydration (if not already done in the fixation step) by application of ethanol. The ethanol is subsequently replaced by a clearing agent, such as xylene, which, unlike water, is miscible with paraffin wax. The tissue is finally submerged in the molten wax, providing the tissue with structural support.

3 - Slicing.

• The purpose of slicing is to enable passage of light through the tissue for microscopic analysis of a single layer of cells.
• This involves slicing the tissue into sections of ~4 microns using a microtome.

4 - Staining.

• The purpose of this stage is to provide structures in the tissue section with colour.
• This involves application of chromophore-containing compounds onto the tissue section, which may bind directly to tissues or via an intermediate compound known as a mordant. Common stains include H&E, PAS, silver stain and Martius scarlet blue stain.

Question 2

List 4 common chemical fixatives.

Answer 2

4 common chemical fixatives include:

1 - Formaldehyde.

2 - Glutaraldehyde.

3 - Methanol.

4 - Ethanol.

Question 3

What are the relative charges of haematoxylin and eosin?

To which cellular structures do these substances bind?

What colours do these substances show under microscopy?

Answer 3

Oxidised haematoxylin-Al3+:

1 - Is positively charged.

2 - Binds to chromatin and RNA, which carry a negative charge.

3 - Is purple.

Eosin:

1 - Is negatively charged.

2 - Binds to positively charged proteins in the cytoplasm and extracellular matrix.

3 - Is pink.

Question 4

What additional step is required for preparing bone for microscopy?

What is the purpose of this step?

Answer 4

• Bone must be decalcified through application of an acid before microscopy.
• This softens the tissue, allowing it to be cut into thin slices.