Cells: Cell Fractionation

Question 1

What is the purpose of cell fractionation?

Answer 1

• Cell fractionation breaks cells and separates organelles so certain organelles can be observed under an electron microscope

Question 2

Process of Cell Fractionation

Answer 2

• Homogenisation - breaking up the cells
• Filtration - getting rid of the big bits
• Ultracentrifugation - separating the organelles

Question 3

Process of Cell Fractionation: Homogenisation

Answer 3

• Homogenisation - breaking up the cells

• Can be done by vibrating the cells or grinding the cells up in a blender
• This breaks up the plasma membrane and releases the organelles into solution
• Solution must be kept ice-cold, to reduce activity of enzymes that break down organelles
• Solution should be isotonic - have the same concentration of chemicals as cells being broken down - to prevent damage to organelles through osmosis
• Buffer solution should be added to maintain pH

Question 4

Process of Cell Fractionation: Filtration

Answer 4

• Filtration - getting rid of the big bits

• The homogenised cell solution is filtered through a gauze to separate any large debris
• The organelles are much smaller than the debris so they pass through the gauze

Question 5

Process of Cell Fractionation: Ultracentrifugation

Answer 5

• Ultracentrifugation - separating the organelles
- After filtration, you’re left with a solution containing a mixture of organelles; to separate a particular organelle you use ultracentrifugation

• Cell fragments are poured into a tube. The tube is put into a centrifuge (a machine that separates material by spinning) and is spun at a low speed
• The heaviest organelles get flung to the bottom of the tube by the centrifuge and form a thick sediment - the pellet
• The rest of the organelles stay suspended in the fluid above - the supernatant
• The supernatant is poured into another tube and spun in the centrifuge at a higher speed and pellet forms at bottom
• This is repeated until all organelles are separated out. Each time, the pellet at the bottom of the tube is made up of lighter and lighter organelles
• Organelles are separated in order of mass from heaviest to lightest

Question 6

Ultracentrifugation Order

Answer 6

• Nuclei
• Chloroplasts
• Mitochondria
• Lysosomes
• Endoplasmic reticulum
• Ribosomes
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