Introduction to Histology

Question 1

What are the 10 stages of histological preparation/ examination?

Answer 1

1. Obtain the tissue
2. Fixation
3. ‘Cut up’ into blocks
4. Tissue processing
5. Section cutting and Mounting
6. Staining
7. Section scanning
8. Microscopy
9. Diagnosis
10. Prognosis prediction

Question 2

Why is tissue fixation important?

Answer 2

This stops the process of degradation - it therefore preserves the tissue. It stops intrinsic enzymes breaking down the tissue and prevents bacterial contamination. It also increases mechanical strength to preserve structure.

Question 3

What chemicals are used in fixation? How do they work?

Answer 3

Formaldehyde- the most common. The proteins are linked together using covalent bonding. Formaldehyde is however not useful in DNA analysis. If you want to test DNA you would need a fresh sample. Glutaraldehyde is similar to formaldehyde but is a larger molecule. It is used in electron microscopy. Ethanol fixes by precipitation. It denatures proteins and causes aggregation.

Question 4

How the tissue processed? Why is it processed?

Answer 4

The tissue must the processed as it is filled with water - wax is hydrophobic. The tissue will therefore repel the wax. The water is removed from the tissue using alcohol. When put in the wax, the tissue is then stiff and resistant to mechanical trauma and so can be sliced thinly,
1. Use alcohol to remove water
2. Replace water with xylene
3. Xylene is replaced with paraffin wax
4. Orientate tissue to form a block.
The end products is known as a formalin-embedded paraffin wax tissue

Question 5

What do we use to stain tissue?

Answer 5

Haematoxylin and eosin (H&E). The most common. Haematoxylin is a basic dye - it stains acidic structures purple, hence nuclei acids. Eosin is an acidic dye. Stains basic structures pink, hence proteins in the cytoplasm are pink.
PAS is also useful. Combined with diastase (enzyme that removes glycogen). Gram stain. Gram positive bacteria = red, gram negative bacteria = blue. Oil red O for fat. Can only be used on frozen samples.

Question 6

What is immunochemistry? how is it used?

Answer 6

A specific technique used to identify specific proteins. It is similar to the ELIZA test in which it utilises antibodies. The primary antibody binds to the antigen in the sample. Another antibody then binds to the primary antibody. Can be used to provide specific information on protein expression - used in diagnosis, prognosis and prediction of response to therapy.

Question 7

What is the basic difference between gram positive and gram negative bacteria?

Answer 7

The differences between Gram positive vs Gram negative bacteria are primarily related to their cell wall composition. Gram positive bacteria have cell walls composed mostly of a substance unique to bacteria known as peptidoglycan, or murein. These bacteria stain purple after Gram staining. Gram negative bacteria have cell walls with only a thin layer of peptidoglycan and an outer membrane with a lipopolysaccharide component not found in Gram positive bacteria. Gram negative bacteria stain red or pink after Gram staining.