Cell Fractionation

Question 1

What is Cell Fractionation?

Answer 1

It is the process where cells are broken up and the different organelles they contain are separated out

Question 2

What must happen before cell fractionation can occur?

Answer 2

The tissue is placed in a cold, buffered solution of the same water potential as the tissue

Question 3

Why is the solution cold?

Answer 3

To reduce enzyme activity that might break down the organelles

Question 4

Why is the solution of the same water potential as the tissue?

Answer 4

To prevent organelles bursting or shrinking as a result of osmotic gain or loss of water

Question 5

Why is the solution buffered?

Answer 5

So that the pH does not fluctuate. Any change in pH could alter the structure of the organelles or affect the functioning of the enzymes

Question 6

What are the 2 stages of cell fractionation?

Answer 6

Homogenisation and Ultracentrifugation

Question 7

What is Homogenisation?

Answer 7

• Cells are broken up by a homogeniser (blender)
• This releases the organelles from the cell
• The resultant fluid, known as the homogenate, is filtered to remove any complete cells or large pieces of debris

Question 8

What is Ultracentrifugation?

Answer 8

• It is the process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge
• This spins tubes of homogenate at very high speeds to create a centrifugal force

Question 9

What is the first stage of Ultracentrifugation?

Answer 9

• The tube of filtrate is placed in the centrifuge and spun at a slow speed
• The heaviest organelles, the nuclei, are forced to the bottom of the tube, where they form a sediment or pellet

Question 10

What is the 2nd stage of Ultracentrifugation?

Answer 10

The fluid at the top of the tube (supernatant) is removed, leaving just the sediment of nuclei

Question 11

What is the 3rd stage of Ultracentrifugation?

Answer 11

• The supernatant is transferred to another tube and spun in the centrifuge at a faster speed than before
• The next heaviest organelles, the mitochondria/chloroplast are forced to the bottom of the tube

Question 12

What is the 4th stage of Ultracentrifugation?

Answer 12

The process is continued in this way so that, at each increase in speed, the next heaviest organelle is sedimented and separated out

Question 13

What is the order in the weight of the organelles?

Answer 13

Nuclei - 1000
Chloroplasts - 3,500
Mitochondria
Lysosomes - 16,500
Endoplasmic Reticulum
Ribosomes - 100,000