74 SELECTION AND PREPARATION OF ORGANISMS 



High-frequency sound waves, often called "supersonic" or "ultrasonic" 

 vibrations, can be applied to some cells with reasonable success. An 

 oscillator serves as a source of sound waves which are transmitted 

 through oil to the container in which the biological material is placed. 



Separation of ParticMlates: Most of the grinding procedures result 

 in a thin paste or fluid suspension containing a mixture of cell parts 

 and other materials. Most such suspensions contain unbroken cells, cell 

 walls, pieces of connective tissue, and other odds and ends. Straining 

 through a coarse filter material removes much of this unwanted matter. 

 Filter materials commonly used are muslin, pads of glass wool, cheese- 

 cloth, and facial tissue supported by cheesecloth. 



Further separation of the particulates depends upon centrifugation. 

 Spinning at a low speed will cause settling of particles more dense than 

 those desired, leaving the desired particles in suspension. The liquid 

 is poured off and centrifuged at a higher speed. During this centrifuga- 

 tion the desired particles setde out, leaving any less dense particles sus- 

 pended in the liquid. The sediment is resuspended in a clean solution, 

 ready for use. Or it can be "washed" by recentrifuging and then is finally 

 resuspended in the desired solution. 



Preservation of Isolated Particulates: The procedures involved in the 

 preparation of subcellular particles are usually quite time consuming. 

 It is often impractical to prepare a new batch of particles for each experi- 

 ment, and yet, under ordinary conditions, the particles rnay not survive 

 from one day to the next. Fortunately, as many of these materials can 

 be preserved by freezing, large amounts may be prepared at one time. 

 Chloroplasts frozen and stored at —40° C have maintained their activity 

 until the supply was exhausted, a period of many months. 



The freeze-dying technique, sometimes called "lyophilization," freezes 

 and dries at the same time. The material to be treated is placed in a con- 

 tainer that can be evacuated. One wall of the chamber is in contact with 

 solid carbon dioxide at about —60° C. The reduced pressure causes the 

 evaporation of water from the biological material at the expense of heat 

 from the material. This water is then trapped by freezing against the 

 cold wall of the container. The resulting dry material can he stored 

 for long periods and then reconstituted by adding water. Some materials 

 preserved in this way retain most of their original activity. 



Assay Method: In most isolation procedures it is necessary to make 

 sure that the particles retain their chemical abilities by making meas- 

 urements of activity at various stages of the preparation. If it is suspected 

 that the particles will catalyze a certain chemical reaction, the assay 



