Differential Centrifugation

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

•The specimens must be fixed 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.
Industrial centrifuges
Tubular bowl centrifuge
• Simplest type and can provide very high centrifugal force.

• Centrifuge can be cooled and hence it is advantageous in

protein and other thermally labile bioproduct separation.

• Mostly used in pilot plant level.

• I t consist s of a long narrow cylindrical bowl suspended from

the top rotating at high speed, in an outer stationary casing.

• Bowl dimensions range from 8 to 15 cm in diameter and upto

150 cm in height.
The feed is introduced at the bottom of the
bowl and discharge of the supernatant occurs
through an annular opening at the top.

The feed liquid moves upward at a uniform

velocity carrying with it the solid particles.

The solid deposit on the bowl's inner wall as a

thick paste.

Bowl must be dismantled and cleaned, once the

efficiency drops.
Multiple chamber bowl
centrifuge
• Contains a number of concentric tubes connected
in such a way that a zigzag flow of the feed
suspension through the chamber is achieved.

• Solids are deposited on the outermost chamber

wall.

• Solid discharge is done manually.

• Employed in fractionation of human blood

plasma.
Disc bowl centrifuge
• Consists of shallow wide cylindrical bottom
driven bowl rotating at moderate speed in a
stationary casing.

• Bowl is about 30 to 100cm in diameter

contains a number of closely spaced metal
discs, located one above the other.

• Disc have one or more set of matching

holes, which forms a channel through which
the feed material flows.
Feed is introduced through a centrally located
feed pipe from above.

The clarified liquid flows out through an annular

slit near the neck of the bowl.

Under the influence of centrifugal force, the dense

phase of the feed travels towards the bowl wall.

While the lighter phase displaced towards the

centre.

The solids may be removed intermittently or

continuously from the sides.
Properties of industrial centrifuges

• Tubular bowl
• High centrifugal force •Limited solids capacity
• Good dewatering • Foams
• Easy to clean • Difficult to recover protein

• Chamber
• Large solids capacity • No solids discharge
• Good dewatering • Cleaning difficult
• Bowl cooling possible • Solids recovery difficult

• Disc type
• Solids discharge • Poor dewatering
• No foaming • Difficult to clean
• Bowl cooling possible