ISOLATION OF CELL NUCLEI 459 



tions of this medium by successive centrifugations at 1500 R.P.M., 

 carrying out all the operations in a cold room at 0°. Follow the de- 

 gree of purification by making wet smears of both supernatant and 

 sediment after each centrifugation. Smears may be stained after fix- 

 ing in osmic acid vapor. 



The principle of separating cellular components by centrifugation in non- 

 aqueous media (benzene-carbon tetrachloride mixtures) of controlled specific 

 gravity was first applied by Behrens (1932) to the isolation of nuclei from 

 calf heart, muscle, and thymus (Behrens, 193S). Fuelgen, Behrens, and 

 Mahdihassan (1937) used this technique to obtain nuclei from rye germ cells, 

 and later Behrens (1939) employed the same procedure for the separation of 

 nuclei from liver cells. In this country Mayer and Gulick (1942) used a 

 modified Behrens technique to isolate nuclei from bovine thymus cells, and 

 Williamson and Gulick (1944) applied the method to other cells having a 

 large proportion of nucleus such as those of human tonsil and bovine super- 

 mammary lymph gland. The limitation should be borne in mind that the 

 benzene-carbon tetrachloride mixtures will extract lipids from the nuclei. It 

 is claimed that proteins are not significantly affected. In general the technique 

 seems to involve a great deal of time and manipulation, and it is too drastic 

 a process for many purposes. 



Behrens Procedure for Isolation of Nuclei from Thymus 

 and Lymph Cells (as Modified by Gulick et al.) 



Cut the tissue into pea-size bits and freeze in liquid air as soon as 

 possible after removing from the animal. Dehydrate by treatment 

 with a number of changes of 10 vol. portions of dry acetone cooled 

 below — 20°. Remove remaining fat by continuous extraction with 

 ether that has been dried over anhydrous sodium sulfate; after the 

 extraction, remove the residual ether in a vacuum desiccator over 

 cone, sulfuric acid. Grind the dry material in a power mill until 

 it can pass a 40 mesh sieve. After suspending the powder in a ben- 

 zene-carbon tetrachloride mixture of specific gravity 1.25, com- 

 minute further in a ball mill with glass beads. It usually takes 4-8 

 weeks at 30-50 R.P.M. to effect separation of the nuclear and cyto- 

 plasmic particles. Test for the completeness of this separation from 

 time to time by staining a drop of the suspension with hematoxylin 

 and eosin in the following manner: 



Dry a smear of the suspension on a glass slide, and place for 1 min. 

 in a filtered fresh mixture of equal vol. of 1% yellowish eosin and 

 Harris ripened alum hematoxylin. Transfer to citric acid-sodium 



