SELECTION AND PREPARATION OF ORGANISMS 73 



complex chemistry. Mitochondria have been isolated and continue to 

 metabolize. Even the ribosomes, which are below the limits of visibility 

 of the light microscope, continue to perform chemical reactions if prop- 

 erly treated. Several hundred enzymes have been purified from biological 

 materials. Many of these purified enzymes can be handled like any other 

 chemical materials. 



A set of routine steps has evolved for the separation of almost any 

 of these subcellular particulates (particles of cellular material below the 

 cellular level of organization). The procedure used for the preparation 

 of one particle obviously will not work for the preparation of another. 

 Even the steps involved in the preparation of one entity, such as mito- 

 chondria, are different from one laboratory to another. As you might 

 expect, particulates prepared by one method do not show exactly the 

 same kinds of behavior as particulates prepared by another method. If 

 experiments are to be repeated, the preparation of the parts of cells must 

 follow the same routine as nearly as possible. 



Nevertheless, all of the various procedures involve many of the same 

 steps, including grinding and differential centrifugation. The differences 

 between methods are differences in composition of solutions, in the time 

 and speed of centrifugation, and in the order in which operations are 

 performed. It seems reasonable to present a general discussion of the 

 various steps, followed by one specific technique as an example. Refer- 

 ences at the end of the chapter should make it possible to find techniques 

 for preparing other subcellular particulates. 



Temperature: Most of the preparative steps are conducted at low tem- 

 peratures. The parts of cells are unstable anyway, breaking down very 

 rapidly at room temperature. The most convenient means of maintaining 

 a temperature of to 4° C is to work in a walk-in refrigerator. Most 

 laboratories conducting cellular physiology arfd biochemistry experiments 

 now have at least one such cold room. 



Grinding: The tissue is usually broken up first by some mechanical 

 method. A mortar and pestle offers a gentle means of grinding. The tissue 

 is ground together with some water, buffer solution, or a complex solu- 

 tion of sugars, salts, etc. Sometimes sharp sand is added to speed the 

 grinding. Another grinding device, almost as gentle, is the homogenizer, 

 constructed of a hollow glass cylinder with a closely-fitted cylindrical 

 pestle. Several models are on the market. Larger quantities of material 

 are chopped finely in the Waring Blendor or its equivalent. The high- 

 speed blades can macerate almost any plant or animal material (includ- 

 ing fingers) in a short time. 



