Ultracentrifugation

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 lysed to break the cell membranes and mix up the cell contents. The lysate is then subjected to repeated centrifugations, each time removing the pellet and increasing the centrifugal force. Finally, purification may be done through equilibrium sedimentation, and the desired layer is extracted for further analysis.

Separation is based on size and density, with larger and denser particles pelleting at lower centrifugal forces. As an example, unbroken whole cells will pellet at low speeds and short intervals such as 1,000g for 5 minutes. Smaller cell fragments and organelles remain in the supernatant and require more force and greater times to pellet. In general, one can enrich for the following cell components, in the separating order in actual application:

High g-force makes sedimentation of small particles much faster than Brownian diffusion, even for very small particles. When a centrifuge is used, Stokes' law must be modified to account for the variation in g-force with distance from the center of rotation.

where

Before differential centrifugation can be carried out to separate different portions of a cell from one another, the tissue sample must first be lysed. In this process, a blender, usually a piece of porous porcelain of the same shape and dimension as the container, is used. The container is, in most cases, a glass boiling tube.

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