Differential Centrifugation

Question 1

What is centrifugation and, what Is It useful for?

Answer 1

• Centrifugation is used to separate organelles according to their density (heaviest organelle separates first).
• It is useful to see how drugs effect cells.

Question 2

What are the 3 main steps to preparing tissues to be broken open?

Answer 2

1. Ice-cold
   
   • to slow down enzyme action which prevents damage to the organelles.
2. Isotonic Solution
   
   • this prevents osmosis so, prevents organelles from rupturing.
3. Buffer Solution
   
   • this prevents ph from changing which, in turn, prevents proteins from denaturing.

Question 3

Describe the differential centrifugation of a leaf.

Answer 3

1. Places some leaves into a solution that is ice-cold, isotonic and buffered.
2. Break open the cells of the leaf using a grinder/ blender.
3. Filter solution to remove debris.
4. Centrifuge at a low speed for a short time. This will cause the densest organelle to be separated from the mixture (the nucleus).
5. Pour of the supernatant into another tube and, centrifuge for a longer time on a faster speed. This will remove the next densest organelle (the chloroplasts).
6. Pour off the supernatant and centrifuge even faster for an even longer time. This will remove the next densest organelle (the mitochondria).