ISOLATION OF CYTOPLASMIC PARTICULATES 463 



chloride soln. of pH 6.0 to 6.2. Grind the tissue gently in a mortar 

 and knead through bolting silk. Suspend the liver emulsion (about 

 30 g.) from the liver of a 400-500 g. guinea pig in no more than 200 

 ml. of the physiological saline soln. and centrifuge for 30 min. at 

 not over 600 R.P.jNI. Centrifuge the supernatant fluid once or twice 

 at 1500-2000 R.P.M. for not over 1 min. Centrifuge the supernatant 

 for 10 min. at 2000 R.P.M. and suspend the sediment in 200-300 ml. 

 of the saline soln. Repeat this washing process until the supernatant 

 is free of soluble protein; it usually requires four or five washings. 



Follow the degree of isolation at various steps by fixing a smear 

 of the material on a slide wdth osmic acid and staining with aniline- 

 acid fuchsin and methyl green. The distinct yellowish color of the 

 unstained mitochondria can also be employed as something of a 

 check on the separations. 



The lower tip of the cake in the centrifuge tube may be contami- 

 nated with cell fragments ; if so, cut off and discard this portion after 

 drying. Finally wash the mitochondria with distilled water contain- 

 ing 1 drop 1 N acetic acid in 200 ml.; centrifuge, and dry the 

 sediment in the centrifuge tubes in vacuo over phosphorus pentoxide. 



During the preceding treatments the mitochondria undergo some 

 swelling and change from rods to round granules. The granules do 

 not coalesce and clump unless the centrifugation is at too high a 

 speed, which results in fragmentation and consequent agglutination. 



Claude Procedure for Certain Neoplastic Cells of the Rat 



The cells used as the source of mitochondria were obtained from 

 10-15 g. tumors that develop within 10-12 days after subcutaneous 

 inoculation of leukemic cells into rats. The advantage of this source 

 of material is the uniformity of the cell type, the relative lack of 

 connective tissue, the scant blood supply, and the absence of ap- 

 preciable necrosis. Furthermore, the mitochondria compose the major 

 portion of the large cytoplasmic granules. 



The following process should be carried out in the cold (2-8°). 

 Chill freshly removed tumors, pass through a 1 mm. mesh masher, 

 and grind the pulp for 3-5 min. in a mortar. Add, very slowly at first, 

 a 0.85% sodium chloride soln., buffered to pH 7.2 with phosphate 

 having a final concentration of 0.005 M, to a total volume equiva- 

 lent to five times the weight of tissue pulp. (It is important to main- 



