What is differential centrifugation and how is it done?

Differential centrifugation is a common procedure in microbiology and cytology used to separate certain organelles from whole cells for further analysis of specific parts of cells. In the process, a tissue sample is first homogenised to break the cell membranes and mix up the cell contents.

How does centrifugation machine works?

A centrifuge is a device, generally driven by an electric motor, that puts an object, e.g., a rotor, in a rotational movement around a fixed axis. A centrifuge works by using the principle of sedimentation: Under the influence of gravitational force (g-force), substances separate according to their density.

Why we use differential centrifugation for mitochondrial separation?

The advantages of the differential centrifugation method to isolate mitochondria are that: i) the method is fast and mitochondria can be isolated within 1-1.5 h (respiratory experiments should be performed as quick as possible); ii) it is inexpensive; and iii) it is very efficient and the mitochondria obtained by …

What is the principle of differential centrifugation?

Principle of Differential centrifugation Differential centrifugation is based upon the differences in the sedimentation rate of biological particles of different size and density. As the increasing centrifugal force is applied, initial sedimentation of the larger molecules takes place.

How does the process of differential centrifugation work?

Differential centrifugation works by a stepwise increase in the centrifugation speed. Lower speeds at the beginning are used to eliminate the heavier food particles from the sample, and the speed is then increased until the targets themselves are pelleted.

What happens to the supernatant in differential centrifugation?

After each centrifugation, the supernatant (non-pelleted solution) is removed from the tube and re-centrifuged at an increased centrifugal force and/or time.

How are particles affected by density gradient centrifugation?

Under centrifugal force, the particles move until their density is similar to the density gradient. Thus, the isopycnic centrifugation is also known as buoyant or equilibrium centrifugation. Biological particles depend on the osmotic pressure of the density gradient.

How are particles separated in a centrifugation process?

Ideally centrifugation is continued enough to pellet all the largest class of particles, the resulting supernatant then being centrifuged at a higher speed to separate medium-sized particles and so on.