ISOLATION AND COMPOSITION OF NUCLEI AND NUCLEOLI 97 



such as grades 60, 90, and 120. With small amounts of material, the 60-mesh 

 cheesecloth can be dispensed with. Since 120-grade cheesecloth is only 

 occasionally available, it is usually necessary to substitute some other kind 

 of cloth of the same degree of fineness. Bolting silk is advantageous for the 

 final filtration but is very expensive. Cotton flannel has also been used in 

 our laboratory and elsewhere with considerable advantage, especially in 

 stubborn cases of contamination with fiber and whole cells. 



When simple filtrations are used to remove the fiber, it is best not to com- 

 plete the homogenization until most of the fiber has been filtered off. A 

 preliminary homogenization is effected which is just sufficient to produce 

 a resonably smooth homogenate, free from lumps. However, if bolting silk 

 or cotton flannel is used, the homogenization must be completed before 

 filtration through either of these materials. 



Should the organ under examination contain a great deal of fiber, it is 

 necessary to remove the bulk of the fiber before homogenization is begun. 

 To do this, the whole organ, or as large a piece of it as can be used at one 

 time, is placed in a stainless steel cylinder fitted with a piston of the same 

 material and a perforated stainless steel disk through which the tissue can 

 be forced by the application of sufficient pressure. The piston and cylinder 

 fit on the top of a heavy walled stainless steel container designed to catch the 

 tissue as it is forced through the perforated plate. The whole apparatus 

 (see Fig. 1) is placed in a hydraulic press such as the Carver press, and the 

 tissue is forced through the perforated plate at the lower end of the cylinder, 

 leaving most of the fiber as a mat on the upper side of the plate. It is possible 

 to place a stainless steel wire screen on top of the perforated plate to get 

 rid of even more fiber, but in this case the pressure required is very high 

 and some damage may be done to the nuclei. It has been claimed by Hoge- 

 boom and Schneider^ that the use of a device such as has just been de- 

 scribed causes some damage to nuclei of liver cells, but nevertheless in work 

 with organs containing a great deal of fiber, it is at least very worthwhile 

 and possibly essential to use the device, since fiber is not readily removed 

 once homogenization has been completed and since too much fiber may 

 even prevent satisfactory homogenization. 



The next point to be considered is the technique of homogenization. 

 Much of the early work on the isolation of cell nuclei from mammaUan 

 tissues involved the use of the Waring Blendor, with relatively large-scale 

 samples of tissue. The Waring Blendor, or a similar homogenizer, is very 

 useful in preparing relatively large quantities of nuclei for certain purposes, 

 but it has certain serious drawbacks. In the first place, something must be 

 done to reduce the fragility of the nuclei. For this purpose the pH can be 



' G. H. Hogeboom and W. C. Schneider, J. Biol. Chem. 197, 611 (1952). 



