464 MECHANICAL SEPARATION OF CELLULAR COMPONENTS 



tain the slightly alkaline reaction to prevent clumping of cyto- 

 plasmic components.) Centrifuge the cellular suspension at 1500 

 times gravity for 4 min., and follow by a 10 min. run, in order to 

 throw down most of the debris, unbroken cells, and nuclei. Separate 

 the mitochondria from the supernatant fluid by centrifuging at 2400 

 times gravity for 25 min., or else at 18,000 times gravity for 4 min. 

 Discard the supernatant liquid and resuspend all but the bottom 

 portion of the sediment in buffered saline soln. Wash the mitochon- 

 dria by two to three sedimentations and resuspensions in buffered 

 saline soln. discarding the bottom layer after each centrifugation to 

 eliminate erythrocytes or nuclei that may have escaped earlier 

 separation. The final mitochondria suspension consists of granules 

 0.5-1.5 fx in diameter. The centrifugations were conducted in the type 

 SB, size 1, centrifuge of the International Equipment Co. (page 448) . 



Claude Procedure for Isolation of "Large Granules" from Liver 



Rat livers are used which have been depleted of blood by bleeding, 

 as well as guinea pig livers which have been perfused from the portal 

 vein or aorta with physiological saline solution. In the latter in- 

 stance the animals are prepared by placing them under ether anes- 

 thesia and injecting heparin intravenously, or directly into the heart, 

 in a dose of 1-2 mg. per 100 g. body weight. All the operations in the 

 preparation of the extract and the separation of the "large granules" 

 are carried out in a cold room at around 0°, and all the solutions are 

 employed at this temperature. 



Preparation of Extract. Chill livers immediately after removal 

 and force through a tissue masher fitted with a 1 mm. mesh screen. 

 Grind 60-80 g. of the pulp in a mortar about 5 min. and add drop- 

 wise 0.85% sodium chloride (made slightly alkaline to prevent ag- 

 glutination by adding 0.2 ml. 1 N sodium hydroxide/1.) until 20-30 

 ml. have been introduced. Then add the solution more rapidly until 

 a final volume is obtained equivalent to five times the weight of the 

 liver pulp used. Centrifuge the suspension at 1500 times gi-avity for 

 3 min. and discard all the sediment. Centrifuge the supernatant 

 twice more for 3 min. at 1500 times gravity and discard the deposit 

 each time. The resulting supernatant liver extract is used for sub- 

 sequent separations. 



Separation of "Large Granules." Centrifuge the liver extract 

 for 25 min. at 2000 times gravity. Resuspend the sediment, except 



