Lecture 4 microtomy and HE staining

Question 1

What is a cryostat?

Answer 1

A crostat is similar to a microtome in that it slices tissue into parts but it is kept very cold with substances like liquid nitrogen.

Question 2

What is cryotomy?

Answer 2

A method of sectioning fresh tissue, requiring a crypto stat and using OCT as an embedding medium for it.
OCT is optical coherence tomography, an imaging method that allows cross sectioning imaging.

Question 3

Cryotomy is used for?

Answer 3

• Rapid diagnosis
• Assessment of margins
• Immunofluorescence
• Enzyme Histochemistry
• Other staining methods

Question 4

Difference between progressive and regressive HE staining?

Answer 4

Progressive staining involves putting the slide through different stains to get the sample upon the slide darker and darker.
Regressive staining involves applying th stain upon the slide so it is dark to begin with before the slide is washed to become lighter and lighter. This is great for indicating nuclei.

Question 5

HE staining process in order:

Answer 5

1. Xylene - to get rid of the wax after the sample is embedded
2. Alcohol
3. Water
4. Haematoxylin
5. Water
6. Acid Alcohol
7. Water
8. Scott’s tap water
9. Water
10. Eosin - Is left on the slide for a while and checked under microscope.
11. Water
12. Alcohol
13. Xylene
14. Mounting on slide

Question 6

Ion exchange - charge attraction: What are the electrical interactions with Haematoxylin and Eosin and the Nucleus and cytoplasm?

Answer 6

Cytoplasmic content is positively charged and interacts with negatively charged Eosin.
Nuclear content is negatively charged and interacts with positively charged haematoxylin.

Question 7

What is an artefact and give examples

Answer 7

Something observed in a scientific investigation that isn’t normally present but occurs as a result of the procedure. In this process, they include:
- Creases (instead of straight sections layer out on a slide) - thicker staining as more stain has been taken on by some of the sample on the slide.
- Displacement
- Carry over
- Exploding sections - cells have been destroyed
- Incomplete sections - It is harder to make a diagnosis as there are more cells sitting on side of the sample