ISOLATION AND COMPOSITION OF NUCLEI AND NUCLEOLI 111 



can be studied in nuclei obtained by nonaqueous procedures. However, these 

 authors have not yet pubhshed figures showing the extent of damage, if any, 

 to enzymes which they have studied in nuclei obtained by the nonaqueous 

 procedure. In the reviewer's laboratory it has been found that aldolase is 

 severely damaged in liver cell nuclei isolated by a modified Behrens tech- 

 nique,* whereas Stern and Mirsky^^ find that aldolase in wheat germ nuclei 

 is not damaged to any extent by a similar procedure. In all studies on en- 

 zymes present in nuclei obtained by nonaqueous procedures, the extent of 

 damage caused by the method should be ascertained and reported, or else it 

 should be demonstrated that no such damage occurs. 



A second obvious hmitation to the isolation of cell nuclei in nonaqueous 

 media is that studies of lipids cannot be made with such nuclei. 



A third limitation, which is not so obvious, is that for several reasons 

 the degree of purity of nuclei isolated in nonaqueous media is not easy to 

 determine. In the first place, many of the nuclei become broken in the 

 grinding procedures, and these broken pieces concentrate with the whole 

 nuclei. Also, small pieces of cytoplasm can often be observed sticking to a 

 given nucleus. Finally, the nuclei tend to swell and agglutinate when placed 

 in aqueous solution prior to the application of stains for determining purity. 

 Such agglutination makes an estimation of the amount of impurity by mi- 

 croscopic observation quite difficult. In the dry state or when suspended in 

 nonaqueous solvents, the nuclei are so misshapen and angular that they 

 are only with difficulty recognizable as nuclei. 



b. Description of Apparatus for Lyophilizing Tissue 



The first step in the isolation of nuclei in nonaqueous media is the lyo- 

 philization of the tissue. In this process, the temperature of the material 

 should be kept as low as possible to insure minimal damage to the cells and 

 minimal translocations of diffusible substrates. The manipulations, more- 

 over, do not involve small blocks of tissue to be sectioned with the micro- 

 tome, but, instead, relatively large masses of material, in the neighborhood 

 of 50 to 200 g. or more. With the type of apparatus commonly available, 

 it is therefore necessary to lyophilize at a higher temperature than when 

 small blocks of tissue are used. In the reviewer's laboratory a bath of ice 

 and water has been used to insure a temperature at the worst no higher 

 than 0°, but, since a solid block of ice is formed around the flasks during the 

 lyophilization, the operating temperature is actually lower than this. If 

 the material is lyophiUzed in the air, the temperature is also lower than 0° 

 until the end of the procedure when the rate of evaporation becomes low. At 

 this point the temperature rises abruptly before the tissue is quite anhy- 

 drous, and damage may result. To illustrate this point, we have found that 



58 H. Stern and A. E. Mirsky, /. Gen. Physiol. 36, 181 (1952). 



