ISOLATION AND COMPOSITION OF NUCLEI AND NUCLEOLI 115 



ham and Thomas" somewhat variable from preparation to preparation. The writer 

 agrees with the latter statement. 



The first step in the separation is to remove as large a mass of cytoplasmic material 

 as possible without losing any appreciable quantities of nuclei. For this purpose the 

 ground tissue is suspended in a solvent mixture of rather low specific gravity com- 

 pared to that of the nuclei, such as 50% benzene, 50% carbon tetrachloride by volume 

 (Dounce et al.^), or 40% cyclohexane, 60% carbon tetrachloride (Mirsky et al.^^). 

 An opaque supernatant without too much crust should be obtained in this step, and, 

 if this is not found, the specific gravity of the suspending fluid must be changed 

 slightly. The supernatant is discarded, and the sediment can be subjected to the same 

 treatment again. 



In subsequent steps the aim is to adjust the specific gravity closer and closer to 

 that of the nuclei, while still sedimenting the nuclei, and then to raise the specific 

 gravity to the point where the nuclei come to the top of the centrifuge tube as a mat, 

 leaving heavy impurities in suspension and in the bottom of the centrifuge tube. It 

 is also on occasion desirable to adjust the specific gravity of the mixture as close as 

 possible to that of the nuclei so that the latter remain in suspension and impurities 

 pass both to the top and to the bottom of the centrifuge tube. 



In carrying out the centrifugation, it is advantageous to use relativel}^ large vol- 

 umes of the suspending medium, since entrapment of nuclei in other fractions is 

 thereby minimized. In all steps where specific gravity flotation is being used, high 

 centrifugal speeds (2000 to 3000 r.p.m.) are desirable, and times of centrifugation 

 from 25 min. to 1 hr. are required, depending upon the size of the centrifuge tubes 

 used. At the end of the preparation, it is advantageous to add a step or two of differ- 

 ential centrifugation in a solvent mixture of rather low specific gravity, wherein the 

 nuclei are sedimented by slow spinning for short periods of time, leaving fine material 

 in the supernatant. 



In making up the solvent mixtures- for the specific gravity flotations, the specific 

 gravity should be measured carefullj- with an accurate hydrometer reading to the 

 third decimal place. Such hydrometers should cover the range of specific gravities 

 that are useful in the flotations (about 1.250 to 1.450). The writer and collaborators 

 have always measured volumes and specific gravities of the solvents at room tempera- 

 ture, although this was unfortunately not stated in the description of the method.* 

 Subsequently our fractionations were carried out at 0°, so that all specific gravities 

 reported were presumably lower than the specific gravities obtaining during the 

 flotation procedures in the cold centrifuge. This may account in part for failure of 

 Mirsky et al.^^ to obtain good nuclei according to the reviewer's procedure, since 

 Mirsky (private communication) measures the specific gravities after cooling the 

 solvents. It is also implied by Mirsky et al.^^ that in their own procedure the specific 

 gravity measurements were made after cooling the solvents to 2 to 4°. 



According to Mirsky et al.,^^ the specific gravities of cell nuclei vary from tissue 

 to tissue and within the same tissue. For chicken erythrocytes the specific gravity 

 is said to be in the neighborhood of -1.34, and for calf thymus nuclei, about 1.37 to 

 1.41. This means that some heavy nuclei bearing cytoplasmic tabs will sediment to- 

 gether with light nuclei free from such tabs, and therefore it must be expected that 

 in most cases the yields cannot be very high and that some fractionation of the nuclei 

 themselves will have occurred. (The latter occurrence usually is also to be expected 

 in methods using aqueous solvents.) Methods for determining the specific gravities 

 of tissue components before isolation have been described by Behrens.^" 



" W. R.Kirkham and L.E.Thomas, J. BzoLC/iem. 200, 53 (1953). 

 6° M. Behrens, Z. physiol. Chem. 253, 185 (1938). 



