460 MECHANICAL SEPARATION OF CELLULAR COMPONENTS 



phosphate buffer (pH 3.9) and, after 3 min., dry, clear with immer- 

 sion oil, and examine microscopically. Eosin stains the cytoplasmic 

 particles, and hematoxylin the nuclear material. 



Before separating the suspended cellular components, remove con- 

 nective tissue by several sedimentations in pure benzene. Discard the 

 benzene carrying the connective tissue fragments each time. Then 

 centrifuge the cellular powder from progressively denser mixtures of 

 benzene and carbon tetrachloride, retaining the sediment in the tube 

 each time. When the proportion of carbon tetrachloride has been in- 

 creased to the point where no more powder settles, add benzene until 

 the denser nuclear fraction begins to come down under centrifuga- 

 tion. At this final separation, the specific gravity of the suspension 

 medium was between 1.345 and 1.350 at 28° when bovine thymus 

 cells were used. Nuclear concentrates prepared in this manner were 

 found to possess less than 5% contamination. The product is a fine 

 granular light tan dust that is quite hydroscopic, and it is obtained 

 in 4.3 to 6.7% yield on the basis of the dry weight of the original 

 tissue. Highest yields are given by thymus material. 



