Cell Fractionation

Question 1

What would you need to do if you wanted to view organelles under an electron microscope ?

Answer 1

Separate them out from rest of the cell using cell fractionation

Question 2

Name the 3 steps to cell fractionation

Answer 2

1. Homogenisation - breaking up of cells.
2. Filtration - get rid of big bits.
3. Ultracentrifugation - Separating organelles

Question 3

What happens during homogenisation ?

Answer 3

Breaking up cells by vibrating cells/ grinding them in blender.
This breaks up cell surface membrane and released organelles into solution.

Solution kept ice cold / isotonic / add buffer

Question 4

Why ice cold?

Answer 4

Reduce enzyme activity which breaks down organelles

Question 5

Why isotonic ?

Answer 5

This means it should have the same conc of chemicals as the cells being broken down to prevent damage by osmosis

Question 6

Why buffer?

Answer 6

Maintain pH

Question 7

What’s does in the filtration step?

Answer 7

Gets rid of big bits separating cell debris from organelle

Filtered through a gauze to separate any large cell debris / tissue debris like connective tissue from organelles .

Organelles pass through gauze as smaller

Question 8

What happens during ultracentrifugation?

Answer 8

Separate out orgelles left in mixture.
Poured into tube to be spun in centrifuge at low speed. Heaviest organelles come out first like Nucleus.
Forms thick pellet at bottoms. And rest of organelles stay suspending in fluid at above sediment - supernatant.
Supernatant stained off poured into another tube and spun at higher speed. The next heaviest organelles form pellet at bottom.
Process repeated at higher/ higher speeds until all organelles are separated out .
Pellet each time consists of lighter/ lighter organelles.

Question 9

Order of mass starting with heaviest

Answer 9

Nucleus 
Chloroplast 
Mitochondria 
Lysosome 
Endoplastic reticulum 
Ribosomes