ISOLATION OF CYTOPLASMIC PARTICULATES 465 



for the portion at the very bottom which consists of nuclei, red cells, 

 and debris, in a small amount of the "supernate" which has been left 

 in the tube after decanting the bulk of it. Centrifuge this suspension 

 for 30 min. at 2000 times gravity and withdraw the "supernate" by 

 suction with a capillary pipette. Take up the sediment in enough 

 alkaline saline solution to bring the total volume to one-twelfth that 

 of the liver extract ; hence the volume at this point is usually 20-25 

 ml. Leave the small disc of packed debris and nuclei in the tube and 

 do not suspend it with the rest of the sediment. Finally dilute 15-20 

 ml. portions of the suspension which contains the "large granules" to 

 35-45 ml. with alkaline saline and centrifuge for 30 min. at 2000 

 times gravity. The sediment of "large granules" may be resuspended 

 in alkaline saline and recentrifuged in the same manner for an 

 additional washing, and the washing process may be repeated several 

 times. The "large granule" fraction represents about 10-15% by dry 

 weight of the total solids in the liver extract. Suspensions of "large 

 granules" become increasingly acid on standing and may require 

 additijn of alkali to maintain neutrality. 



Instead of the use of 2000 times gravity to effect separation of 

 "large granules" from the extract a force of 18,000 times gravity for 

 3-5 min. periods will give better fractionation. Celluloid tubes of 14 

 ml. capacity were used in the high-speed attachment of the Inter- 

 national centrifuge (page 448) for this purpose. 



B. SUBMICROSCOPIC PARTICULATES 



Claude (1940) discovered and separated submicroscopic particu- 

 lates from saline extracts of embryonic chick tissue, and subse- 

 quently separated these bodies from a wide variety of other tissues 

 of various species as well, Claude (1943b). Originally, Claude con- 

 sidered that these cellular units might be mitochondria or their 

 fragments, but has since agreed that this is unlikely because the par- 

 ticles are much too small. Claude (1943a) proposed that the term 

 "microsome" be applied to this cellular entity; it has also been re- 

 ferred to by others as the submicroscopic lipoprotein complex. A 

 second submicroscopic particulate, composed of glycogen, was 

 demonstrated by Lazarow (1942, 1943) in the guinea pig liver cell. 



