Differential Centrifugation

By Sophie Legg

Differential Centrifugation

• This is the most common method of fractionating cells

• Fractionation is the separation of the different organelles within the cell

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. Centrifuge filtrate at low speeds ( 1000 X g for 10mins )

• This pellets the nuclei as this is the densest organelle

• 5. Centrifuge at medium speeds ( 10 000 x g for 30 mins )

• This pellets mitchondria which are the second densest organelle

• 6. Centrifuge at high speeds ( 100 000 x g for 30 mins)

• This pellets ER, golgi apparatus and other membrane fragments

• 7 Centrifuge at very high speeds ( 300 000 x g for 3hrs)

• This pellets ribosomes

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

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

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

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