Differential Centrifugation - Sample Preparation

Sample Preparation

Before differential centrifugation can be carried out to separate different portions of a cell from one another, the tissue sample must first be homogenised. 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.

The tissue sample is first crushed and a buffer solution is added to it, forming a liquid suspension of crushed tissue sample. The buffer solution is a dense, inert, aqueous solution which is designed to suspend the sample in a liquid medium without damaging it through chemical reactions or osmosis. In most cases, the solution used is sucrose solution; in certain cases brine will be used. Then, the blender, connected to a high-speed rotor, is inserted into the container holding the sample, pressing the crushed sample against the wall of the container.

With the rotator turned on, the tissue sample is ground by the porcelain pores and the container wall into tiny fragments. This grinding process will break the cell membranes of the sample's cells, leaving individual organelles suspended in the solution. This process is called homogenization. A portion of cells will remain intact after grinding and some organelles will be damaged, and these will be catered for in the later stages of centrifugation.