ISOLATION A NT) COMPOSITION OF NUCLEI AND NUCLEOLI 145 



for example, in mammalian liver cells conform to the concept of nucleoli 

 as enunciated by Caspersson and Schultz. It is unlikely that this is so, and 

 at the present time it looks as though the amphibian egg-cell nucleoli may 

 be the exception rather than the rule. 



2. Electron Microscopy of Nucleoli 



Nucleoh are usually so small that an intranucleolar morphology cannot 

 easily be made out by use of the Hght-microscope, Moreover, the usual 

 fixatives of histologists seem to obliterate internal structure, as can be seen 

 by comparing electron micrographs made after using the ordinary fixatives 

 with those made after using a superior fixative such as the buffered osmic 

 acid of Pallade.^^ 



Good electron micrographs of nucleoh within cells often show a vermi- 

 form structure. ^^^'^^^ Nucleoli in isolated nuclei, or isolated nucleoli them- 

 selves, are likely to show a vesicular structure. This may well be due to 

 alterations of structure during the isolation procedure, although vacuo- 

 lated nucleoli have also been reported by Lewis^*^ in normal and malignant 

 fibroblasts. 



3. Isolation of Nucleoli 



Three reports are available on the isolation of nucleoli. Krakauer^^^.iss 

 was the first to report a concentration of nucleoli from homogenates of liver 

 cells made in very strong sucrose solutions. Photographs were not given and 

 the degree of purity of these nucleolar fractions cannot be stated with any 

 certainty. 



Vincent reported the isolation of nucleoh from the eggs of starfish. ^^ 

 Photographs of the isolated material indicate a very high degree of purity 

 of the material (see Fig. 5). The cells were ruptured at pH 6.0 by being 

 passed rapidly through a No. 18 needle of a hypodermic syringe, at a tem- 

 perature of 2 to 4°. The nuclei were not previously isolated, but instead 

 the nucleoh were isolated directly from the homogenate by differential 

 centrifugation, as in the case of the nucleolar material described by Kra- 

 kauer. However, the size and morphology of the starfish egg nucleoli are 

 such that it is unhkely that a mistake could have been caused by carrying 

 out a direct isolation from the homogenate rather than from previously 

 isolated nuclei. 



188 G. E. Palade, J. Exptl. Med. 95, 285 (1952). 



18' E. Borysko and F. B. Bang, Bull. Johns Hopkins Hosp. 89, 468 (1951). 



19" W. Bernhard, F. Haguenan, and C. Oberling, Experientia 8, 58 (1952). 



1" W. H. Lewis, Cancer Research 3, 531 (1943). 



'92 R. Krakauer, /. Cellular Comp. Physiol. 39, Suppl. 2, 66 (1952). 



1" R. Krakauer, A. M. Graff, and S. Graff, Cancer Research 12, 276 (1952). 



