ISOLATION AND COMPOSITION OF NUCLEI AND NUCLEOLI 109 



Schneider'^ and the use of ethylene glycol by Mazia." Dallam and Thomas'^ have pub- 

 lished a method for isolating cell nuclei with glycerol. The combined use of the Waring 

 Blendor and the colloid mill or the new ball -type homogenizer can be employed for 

 homogenization. If the Waring Blendor and colloid mill are used, mitochondria are 

 disintegrated. 



Complete descriptions of methods for isolating cell nuclei with ethjlene glycol or 

 glycerol are lacking and the reviewer has not worked out isolation procedures in great 

 detail. The general procedure is to homogenize one part by weight of fresh liver with 

 5 parts by volume of 80% ethylene glycol or glycerol. The final concentration of sol- 

 vent in such a homogenate is about 70%. Filtration through cheesecloth is difficult and 

 centrifugation of the nuclei requires considerably longer than with the methods pre- 

 viously described. The once-centrifuged nuclei are then washed three or four times 

 with 70% ethylene glycol or glycerol, care being taken to centrifuge long and hard 

 enough to avoid excessive losses of nuclei. 



The nuclei thus obtained are capable of forming gels with alkali and hence have 

 presumably been protected from autolj'sis by the high concentration of organic sol- 

 vent. They are rather swollen and of a reddish color which is due to the presence of 

 some adsorbed hemoglobin. Some whole cells are apt to be present, since it is difficult 

 to break all of the cells in ethylene glycol or glycerol without excessively lowering the 

 yield of nuclei. The yield as a rule tends to be low, but with some practice it is possible 

 to obtain reasonably good quantities of nuclei with the large- or small-scale variation 

 of the method. The reviewer prefers ethylene glycol to glycerol, although either sol- 

 vent can be used. 



Polyethylene glycol has been recommended by McClendon and Blinks'* for use 

 in the isolation of plastids from plant cells. Although this material does not appear to 

 have been tried in the isolation of cell nuclei, it might well furnish a good medium. 



Pectin has been used in the isolation of plant cell nuclei by Brown.'* This is pre- 

 sumably another illustration of the use of a high-molecular-weight colloid for the pur- 

 pose of increasing the viscosity of the solution so as to allow better separation of the 

 nuclei. Other materials that might possibly be useful are dextran and polyvinyl- 

 pyrrolidine. 



(5) Methods Involving the Use of Buffered or Unbuffered Salt Solutions. It is possible 

 under certain conditions to isolate cell nuclei in buffer or saline solutions. In general 

 this cannot be done in a satisfactory manner with the Waring Blendor or colloid mill, 

 but in certain cases it can be achieved with ground-glass hand-homogenizers. A 

 serious drawback to the use of saline or buffers is that the mitochondria tend to ag- 

 glutinate, and hence, if any purification is to be achieved by differential centrifuga- 

 tion, very dilute homogenates must be used and the homogenizer must be used to re- 

 suspend the nuclei after each centrifugation. Dilute buffers are sometimes used in 

 conjunction with sucrose solutions, as advocated by Wilbur and Anderson.'^ 



Using 0.9% NaCl solution in homogenization and in all subsequent steps, the re- 

 viewer has been able to isolate fairly pure nuclei from normal rat liver. The ball-type 



'2R. M. Schneider, Federation Proc. 11, 140 (1952). 



" D. Mazia, in "Trends in Physiology and Biochemistry" (Barron, ed.), p. 99. Aca- 

 demic Press, New York, 1952. 

 '^ R. D. Dallam and L. Thomas, Federation Proc. 12, 193 (1953). 

 '6 J. H. McClendon and L. R. Blinke, Nature 170, 577 (1952). 

 »« R. Brown, Nature 168, 941 (1951). 

 " K. M. Wilbur and N. G. Anderson, Exptl. Cell Research 2, 47 (1951). 



