ISOLATION AND COMPOSITION OF NUCLEI AND NUCLEOLI 



113 



.Spherical 

 joint 



Special wide-bore 

 stopcock 



250 ml. 

 centrifuge bottles 



Fig. 4. Schematic construction of a second type of lyophilizing apparatus. Wide- 

 bore tubing and a stopcock with equally large bore should be used. The two traps 

 are cooled in Dewar flasks filled with acetone-dry ice or liquid nitrogen. The samples 

 in the 250-ml. centrifuge bottles are cooled with ice and water in a metal container. 

 A Dewar flask should not be used here, since it may shatter if a solid block of ice is 

 formed around the centrifuge bottles. The Dewar flasks should be put in large tin cans 

 and covered with heavy cloth as protection in case of accident. (0.2 times actual size.) 



them in large tin cans to be covered with heavy cloth during the lyophiHza- 

 tion. 



c. Description of Methods 



(1) Behrens' Original Procediire. In the procedure originally devised by Behrens, 

 the removal of water was accomplished in a rather crude way by drying over a desic- 

 cant in a vacuum desiccator. The dried tissue, after being ground by a mechanical 

 grinder and later a ball mill and then being sifted a number of times, was fractionated 

 bj' specific gravity flotations in the centrifuge, with mixtures of carbon tetrachloride 

 and benzene as suspending media. The aim was gradually to float off the whole cells 

 and much of the cytoplasmic material, leaving the nuclei as a sediment. Subsequently 

 the nuclei were floated off from a certain amount of heavy impurity which still fell to 

 the bottom of the tubes in a solvent mixture of sufficiently high specific gravity to 

 float the nuclei. Behrens and co-workers^"'" were able to obtain nuclei from a variety 

 of tissues, including heart muscle, brain, thymus, liver, pancreas, and wheat germ, 



" M. Behrens, Z. physiol. Chem. 209, 59 (1932). 



"» M. Behrens, Z. physiol. Chem. 232, 263 (1935). 



« M. Behrens, Z. physiol. Chem. 258, 27 (1939). 



« M. Behrens, Z. physiol. Chem. 220, 97 (1933). 



" R. Feulgen, M. Behrens, and S. Mahdihassan, Z. physiol. Chem. 246, 203 (1937). 



