Methods of studying cells

Question 1

Describe what is meant by cell fractionation

Answer 1

The process in which organelles are separated

Question 2

What are the 2 parts of cell fractionation called

Answer 2

• Homogenisation: cells are broken apart
• Centrifugation: the organelles are separated

Question 3

What 3 conditions must our tissue samples be kept under, when carrying out cell fractionation

Answer 3

• Buffered
• Isotonic
• Cold

Question 4

Why are tissue samples stored in a buffered solution

Answer 4

• Prevents PH changes, so that hydrogen and ionic bonds in the tertiary structure of proteins within organelles, remain unaffected

Question 5

Why are the tissue samples stored in an isotonic solution

Answer 5

• Equal water potential, therefore water doesn’t move into organelles via osmosis, preventing the lysis of organelles

Question 6

Why are tissue samples stored in a cold solution

Answer 6

• Reduces enzyme activity, which prevents the hydrolysis of proteins within organelles

Question 7

Describe how cells can be homogenised

Answer 7

• Physically using a mortar and pestle
• Chemically using a weak detergent
• The cell membranes are disrupted, to release the organelles within

Question 8

Describe what happens after homogenisation

Answer 8

The solution is filtered to remove unbroken cells and large pieces of debris, forming a mixture called a homogenate

Question 9

Describe what happens in centrifugation

Answer 9

• A centrifuge separates things according to their different densities
• The homogenate is placed in a centrifuge and spun
• The most dense organelle falls to the bottom (pellet) and is removed, and the liquid on top (supernatant) will be spun at a faster speed to cause the next most dense organelle to fall to the bottom

Question 10

Starting with some lettuce leaves, describe
how you would obtain a sample of undamaged
chloroplasts. Use your knowledge of cell
fractionation and ultracentrifugation to
answer this question.

Answer 10

• Chop up (accept any reference to crude breaking up);
• Cold;
• Buffer solution;
• Isotonic / same water potential;
• Filter and centrifuge filtrate;
• Centrifuge supernatant at higher speed;
• Chloroplasts in (second) pellet;