To understand the structure of the smallest unit of life the cells and their components we need a good microscope. However, other techniques are also needed for studying the functions of cell organelles. For study of function of a particular sale organelle, it is to be isolated from the other components of the cell and it should be allowed to perform its normal functions outside the body, may be in a test tube. For this, only used processes are the Cell fractionation andTracer techniques.
The meaning of Cell fractionation is the separation and isolation of individual components of a cell. It involves the techniques namely the homogenisation and differential centrifugation.
Homogenisation: -
Homoginiser
It means breaking of cells by rupturing the membrane of the cell of a specimen of tissue taken in a suitable medium. The medium must have correct pH, ionic composition and temperature. In general this is done by grinding the cells in a glass homogeniser consisting of a tube and a close fitting, beaded piston which is attached to a motor. In the tube the tissue is taken w3ith a suitable medium and the piston is rotated by the motor at very high speed. By applying osmotic shock or ultrasonic vibrations to the tissue specimen the homogenisation also may be done. From homogenisation of a tissue the fluid obtained, which is known as tissue or cell homogenate that contains all the components of the tissue suspended in the fluid medium.
Differential Centrifugation: -
To separate the cell components present in homogenate is than subjected to differential centrifugation to complete the process of cell fractionation. Centrifugation is a process in which the particles suspended in a fluid; tend to move outward when they are put into the centrifugal force.
Differential Centrifugation
It is done by motorised instrument which is called centrifuge. The centrifuges have suspended test tubes that can be rotated horizontally at different speed to exert centrifugal force. When different homogenates are taken in the test tube and the centrifuge is rotated, the cell components tend to move outward and settle down. The process of settling down of the cell components depends upon their size and density. When the rotation of the centrifuge is faster, the particles will be smaller that will be sediment.
As we know that the cell fractionation is the process of separation and isolation of individual components of cell, to complete the separation and isolation process the centrifuge is rotated at series of increasing speed for some increasing duration. After each speed, the supernatant is decanted. The meaning of supernatant is the liquid above the pellet. The decanted supernatants then re-centrifuged at a higher speed. In this way a series of pallets containing cell organelles of successively smaller and smaller sizes are obtained. This is known as differential centrifugation.
For the cell fractionation a special type of centrifuge is used and the name of this special type is ultracentrifuge. This can rotate as a very high speed and operates at a low temperature usually and must in a vacuum condition.
Next we shall go on details about the Tracer techniques. Click on