1. Differential Centrifugation
By Sophie Legg

2. Differential Centrifugation
This is the most common method of fractionating cells
Fractionation is the separation of the different organelles within the cell

3. Method:
1. Cut tissue in an ice-cold isotonic buffer. It is cold to stop enzyme reactions, isotonic to stop osmosis and a buffer to stop pH changes.
2. Grind tissue in a blender to break open cells.
Filter to remove insoluble tissue
                        

4. 4. Centrifuge filtrate at low speeds ( 1000 X g for 10mins )
This pellets the nuclei as this is the densest organelle

5. 5. Centrifuge at medium speeds ( 10 000 x g for 30 mins )
This pellets mitchondria which are the second densest organelle

6. 6. Centrifuge at high speeds ( 100 000 x g for 30 mins)
This pellets ER, golgi apparatus and other membrane fragments

7. 7 Centrifuge at very high speeds ( 300 000 x g for 3hrs)
This pellets ribosomes

8. Investigating Cell Function
Differential Centrifugation allows us to look at each organelle within the cell
We can look at the individual organelles and study them in detail
This helps to determine each organelles function within the cell

9. The Electron Microscope
Microscopes allow us to see living organisms which are too small to be seen by the naked eye
The electron microscope uses beams of electrons rather than light to illuminate the specimen
A beam of electrons has an effective wavelength of less than 1 nm so it can be used to resolve small sub-cellular ultra-structure
The development of the electron microscope allowed biologists to view the organelles within a cell for the first time

10. There are two types of electron microscope
The transmission microscope. (TEM)
Works like a light microscope, it transmits a beam of electrons through a thin specimen
Then focussing the electrons to form an image on a screen
This is the most common form of electron microscope and gives good resolution.
The scanning electron microscope (SEM)
This scans a fine beam of electron onto specimen and collects electrons scattered by surface
This has poor resolution but gives good 3-D images

11. Disadvantages of the Electron Microscope
The specimens must be fixed in plastice and viewed in a vacuum and so they must be dead
Sometimes specimens can be damaged by the electron beam and must be stained with an electron-dense chemical