ISOLATION AND COMPOSITION OF NUCLEI AND NUCLEOLI 107 



second washing is carried out with 1% gum arabic solution. If desired the nuclei can 

 be suspended in gum arabic solution, sucrose solution, or distilled water. Two or three 

 quick washings with water or sucrose solution are necessary to remove gum arabic, 

 but it is not possible to remove all traces in this way. Gum arabic subsequentlj^ inter- 

 feres in the colorimetric determination of PNA. 



In these procedures all solutions used are ice cold, and all centrifugations are car- 

 ried out in a refrigerated centrifuge. Care is taken not to allow much warming of the 

 homogenate during filtrations. It is desirable to work in the cold room if possible. T^e 

 adjustment of the pH to 6.2 with citric acid without the addition of more CaCl2 pre- 

 vents the clumping of mitochondria and nuclei which occurs in the procedure of 

 Hogeboom and Schneider. Treatment with gum arabic solution removes erythrocytes 

 (by laking) . 



The method just described maj^ also be carried out on a small scale, using the im- 

 proved homogenizer already mentioned, with the particular advantage that mitochon- 

 dria can be removed while still intact, so that contamination of the nuclei by ad- 

 sorbed mitochondrial fragments is avoided. The nuclei appear spherical or slightly 

 deformed, still possess the nuclear membrane, and are of a hyaline appearance. They 

 are quite clean microscopically and almost free from the cell membranes mentioned 

 by Hogeboom et al.^^ They are also obtainable in reasonabl}- good yield, but it is 

 unlikely that any method at present known is capable of producing clean nuclei in 

 very high yield, despite claims to the contrary-. 



The adaptation of the above procedure to small-scale work furnishes a method for 

 isolating cell nuclei which is believed to be superior even to the latest method of 

 Hogeboom et al.^^ The chief drawback to the method of Hogeboom et al. is that, 

 if calcium chloride is added during the washing, enough agglutination of the mitochon- 

 dria and cell membranes is caused to make complete separation of these contaminants 

 very difficult or impossible, at least by the technique of differential centrifugation. 

 Lowering of the pH to 6.2 after the first homogenization in the procedure described in 

 this chapter obviates this difficulty and at the same time prevents the agglutination 

 of the nuclei themselves, but the procedure admittedly incoqDorates certain steps 

 used by Hogeboom et al.^^ and is based on the discovery by R. Schneider and Peter- 

 man^* of the beneficial effects of CaCla ■ 



The details of the procedure as adapted to small-scale work with rat liver are as 

 follows. Two young or medium-sized rats are killed by decapitation and the livers are 

 dissected out. After removal of as much as possible of the connective tissue and large 

 veins by further dissection, the livers are weighed and then gently pounded to a pulp 

 in an ice-cold mortar. This pulp is mixed with a volume of homogenizing liquid (0.25 M 

 sucrose containing 0.005 M CaCU) equal to five times the weight of the livers. The 

 pulped liver is suspended as well as possible and is homogenized in the ball-type 

 homogenizer (Fig. 2) with a rather loosely fitting pestle (0.1- to 0.2-thousandths of an 

 inch clearance). In this step the plunger is raised and lowered six times. The bottom 

 of the homogenizer rests in a large rubber stopper with a hole bored part way through 

 to receive the end of the homogenizer tube. The homogenizer and rubber stopper are 

 immersed in a large beaker containing a mixture of ice and water for cooling. 



After this preliminary treatment, the homogenate is filtered once through curity 

 grade 90 cheesecloth and twice through 120-grade cheesecloth or other similar fabric. 

 The filtered homogenate is then homogenized in the ball-type homogenizer using a 

 well-fitting ball (0.5-thousandths of an inch or slightly less in clearance). About 18 

 to 24 strokes are generally required to break a sufficiently high proportion of the cells, 

 since the calcium chloride renders the cells somewhat resistant to breaking. The prog- 

 ress of the homogenization can be followed by means of the naicroscope, using the 4- 



