ISOLATION AND COMPOSITION OF NUCLEI ANT) NUCLEOLI 99 



and render the preparation worthless. Finally, when nuclei are isolated at 

 pH values higher than 4, the Blender does destroy a considerable proportion 

 of nuclei even under the best conditions obtainable, so that the yields are 

 low. In spite of these drawbacks, nuclei can be isolated from Uver tissue 

 at pH 4 or slightly lower in good yield and in a condition remarkably free 

 from microscopically visible impurities. Such nuclei are quite suitable for 

 studies of nuclear lipid, DNA, nuclear PNA, and histone, or for analytical 

 studies of the nonhistone nuclear protein, much of which may however be 

 denatured. These nuclei are not in general suitable for enzyme studies 

 mainly because the pH obtaining during the isolation is so low that many 

 or most enzymes are rendered inactive. 



One of the worst drawbacks in the use of the Waring Blendor for prepar- 

 ing nuclei at pH 6.0 or above for use in enzyme work is that the mito- 

 chondria are broken, and adsorption of microscopically invisible mito- 

 chondrial fragments on the nuclear surfaces can cause serious contamination 

 of the nuclei with mitochondrial enzymes. The somewhat low yields of 

 nuclei obtained when the Waring Blendor is used are not particularly 

 troublesome since, if a sufficient amount (50 g.) of tissue is used, enough nu- 

 clei are obtained for many enzyme analyses. A final drawback to the use of 

 the Waring Blendor is that it must be rim for a considerable length of time 

 to achieve proper breaking of the cells, and temperature control therefore 

 becomes a problem. 



Another device that can be used in the preparation of cell nuclei on a 

 large scale is the colloid mill,^'*'' such as that sold by the Premier Mill Com- 

 pany of Geneva, New York. (Models of several sizes are available.) The 

 colloid mill consists essentially of a short truncated stainless steel cone 

 which can be rotated at very high speed inside a second fixed stainless steel 

 cone. These cones are machined to fit each other very exactly. The material 

 to be homogenized is fed into the mill by gravity through a stainless steel 

 funnel and, since the cones are placed with axes vertical and bases down- 

 ward, the centrifugal force which is developed in the liquid layer between 

 the cones causes the liquid to be fed downward at a reasonably high speed, 

 which depends upon the spacing between the cones. The latter may be ad- 

 justed over a ^vide range, but clearance of 1- to 2-thousandths of an inch or 

 under certain circumstances even less are generally used. A medium or 

 high speed (attainable by changing pulleys) is in general preferable to a 

 low speed, but in the writer's laboratory a medium speed (10,000 r.p.m.) 

 has been found to give best over all results, with a cone clearance of 1.5- 

 thousandths of an inch. The funnel of the mill can be chilled to 0° before use 

 by being filled with crushed ice or by being placed in the cold room. 



'''"' The colloid mill was introduced in cytochemistry by Mirsky for the isolation of 

 chromosomes. 



