210 GEORGE H. HOGEBOOM AND WALTER C. SCHNEIDER 



between the pestle and test tube. Apparently, the major factor in cell disruption with 

 this homogenizer is the rapidity with which the tissue suspension can be forced be- 

 tween the wall of the tube and the rotating pestle rather than the amount of clearance 

 that exists between the pestle and the tube wall. In the case of liver, homogenization 

 for 2 minutes is sufficient to break over 95% of the cells. ^^ Other investigations have 

 indicated that longer periods of homogenization might lead to disruption of cellular 

 particulates as well.*^ Since all-glass homogenizers were used in the latter experi- 

 ments, it is possible, however, that fragments of glass may have contributed to the 

 disruption of the particles. The use of homogenizers is discussed also in Chapter 18. 



d. The Effect of Various Media on Cytoplasmic Particles and the Cytological 

 Identification of the Components of Cell Fractions 



The medium in which the cells are disrupted is of great importance in that it in- 

 fluences not only the morphological and cytological properties of cell structures, but 

 also their physical and biochemical properties. In 1947, a reinvestigation of Claude's 

 original method" '^^ for the fractionation of cytoplasmic particulates was undertaken 

 by the present authors'''*' with the aim of improving the yields and of identifying 

 more positively the cellular elements present in the fractions. The experiments were 

 mainly concerned with the "large-granule" fraction, which was thought by Claude 

 to consist of a mixture of mitochondria and secretory granules. 



When released into isotonic solutions, the large granules appeared as refractive 

 spherical bodies, 0.5 to 2 /x in diameter. When exposed to h.ypotonic salt solutions or 

 to water, they became greatly swollen, and as Claude had shown previously," •*'' lost 

 appreciable amounts of soluble material. It was also noted that marked aggregation 

 of the granules occurred in the solutions of neutral electrolytes that had been em- 

 ployed previously as media, e.g., isotonic NaCl, KCl, and phosphate buffer. * The 

 clumps of granules were of such a size that they sedimented together with nuclei and 

 intact liver cells. It was therefore impossible to remove nuclei, intact liver cells, and 

 connective tissue from the broken cell suspensions by centrifugation without sedi- 

 menting 40 to 80% of the large granules. When released into isotonic (0.25 M) sucrose, 

 however, the granules showed no tendency to aggregate.'' By means of a method de- 

 scribed below, it was possible with this medium to separate efficiently the nuclei and 

 microsomes from large granules and to obtain the latter in yields of 80% or greater. 



The problem of identifying the structures present in the isolated large-granule 

 fraction was simplified to a considerable degree by the finding that most of the gran- 

 ules were rod-like in shape when released into hypertonic solutions of sucrose.'' When 

 a sucrose concentration of 0.88 M was employed, the granules retained their mor- 

 phological characteristics after having been separated from other cell constituents 

 and washed several times in the centrifuge. It was evident that they were similar in 

 size and shape to the mitochondria of the liver cell. In addition, they could be stained 

 supravitally with Janus Green B, and, after fixation with osmium tetroxide, they 

 showed the other staining characteristics that for many years had been used to 



62 T. A. F. Quinlan-Watson and D. W. Dewey, Australian J. Set. Research Bl, 139 



(1948). 

 " A. Claude, J. Exptl. Med. 84, 51 (1946). 

 " A. Claude, J. Exptl. Med. 84, 61 (1946). 

 " G. H. Hogeboom, W. C. Schneider, and G. E. Palade, Proc. Soc. Exptl. Biol. Med. 



65, 320 (1947). 



