SELECTION AND PREPARATION OF ORGANISMS 75 



consists of measuring the rate of that reaction. After a routine procedure 

 has been developed, the assay can be carried out at less frequent inter- 

 vals or delayed until the end of the preparative steps. 



Preparation of chloroplast fragments— an example 



The following example is only one of many methods of preparing 

 chloroplasts or chloroplast fragments. These chloroplasts are prepared 

 by Dr. J. D. Spikes and his colleagues at the University of Utah for the 

 principal purpose of studying the light-absorbing phase of photosyn- 

 thesis. Much of the other activity normally carried on in chloroplasts 

 does not occur in these fragments. Although this inactivity would be 

 unfortunate in some circumstances, it is advantageous here because it 

 simplifies the experimental setup. 



Other chloroplast preparations can perform other activities, but the 

 preparative steps, of course, are slightly different. If the chloroplasts are 

 prepared according to one set of prescribed steps, they can carry on phos- 

 phorylation reactions in the light and may even fix some carbon dioxide. 



In Dr. Spikes' method, sugar beet leaves are the usual source of chloro- 

 plasts. The sugar beet plants are raised in a special chamber under con- 

 trolled conditions of light and temperature. The leaves are harvested and 

 washed in cold water. In the cold room, approximately 50 g of washed 

 leaves are placed in a Waring Blendor with 60 ml of a solution 0.5 m in 

 sucrose and 0.01 m in KCl. The mixture is blended for two minutes, 

 resulting in a thin paste which is filtered through four layers of cheese- 

 cloth. The green liquid is centrifuged for five minutes at 1000 X g. The 

 supernatant fluid, consisting of a mixture of chloroplast fragments and 

 other cytoplasmic materials, is separated from the sediment of whole 

 cells, nuclei, and cell walls. The fluid is then centrifuged in a Spinco 

 Model L Preparative Ultracentrifuge for fifteen minutes at 144,000 X g. 

 The supernatant fluid is discarded, and the sedimented chloroplast frag- 

 ments are resuspended in the sucrose-KCl solution and centrifuged again. 

 Several such washing steps are carried out, each of which removes much 

 of any remaining cytoplasmic material other than chloroplast fragments. 

 After the final centrifugation the chloroplast fragments are suspended in 

 sucrose-KCl solution. 



Chlorophyll in the chloroplast fragments is determined spectrophoto- 

 metrically by comparison with a standard curve. The suspensions are 

 diluted with sucrose-KCl solution until they have approximately 500 mg 



