THE CYTOPLASM 211 



identify mitochondria. The isolated and washed preparations contained no elements 

 stainable with neutral red (e.g., secretory granules and lipid droplets) and, as far as 

 could be determined by examination in the electron microscope, contained very little 

 material of submicroscopic dimensions. It was therefore concluded that the prepara- 

 tions were essentially homogeneous suspensions of mitochondria. 



A number of subsequent observations have indicated that the "large-granule" 

 fraction isolated from liver homogenates prepared in isotonic (0.25 M) sucrose solu- 

 tion also consists of mitochondria, despite the fact that in this medium the particles 

 are uniformly spherical and are not readily stained by Janus Green B. Thus, after 

 appropriate fi.xation, the granules are intensely colored by mitochondrial stains. 

 Furthermore, when the suspension is diluted somewhat with water and examined in 

 the phase microscope, the swollen granules show the typical morphological charac- 

 teristics of mitochondria described by Zollinger*^ and Harmon," i.e., a delicate, 

 transparent sphere capped at one or both poles bj' crescent-shaped, dense material. 

 A number of biochemical studies^* ■'* have indicated that there are no appreciable 

 differences in the properties of liver mitochondria isolated in isotonic (0.25 M) or 

 hypertonic (0.88 M) sucrose solution. The latter medium, because of its density and 

 viscosity, requires relatively high centrifugal forces in the fractionation of liver 

 homogenates and on occasion makes it necessary to add such large amounts of sucrose 

 to reaction mixtures that the activity of some enzymes is inhibited (cf. footnote 8). 

 For these reasons, the medium of choice for the isolation of mitochondria would ap- 

 pear at present to be isotonic sucrose. It has been the authors' experience that the 

 addition of salts to the medium, even at low concentrations, causes sufficient aggre- 

 gation of both mitochondria and microsomes to interfere considerably with the sub- 

 sequent fractionation of liver homogenates. 



The components of the microsomal fraction have not is yet been identified with 

 certainty, mainly because it is only since the advent of electron microscopy that the 

 fine details of cytoplasmic structure have been disclosed. Recent observations of 

 Forter^^ have indicated, however, that isolated microsomes include the endoplasmic 

 reticulum of the cytoplasm. Since this structural network is probably identical to 

 the basophilic ground substance of cells," Claude's" initial suggestion that the 

 microsomes actually' represent the basophilic ground substance seems well founded. 

 Additional confirmation of this view is found in the work of Brenner.^' From time to 

 time, however, microsomes have been considered to be artifacts derived from other 

 cell structures, e.g., from mitochondria. i^'" That the latter concept is of doubtful 

 validity will be demonstrated later in a discussion of the biochemical properties of the 

 microsomal fraction. 



Finally, it should be pointed out that the conditions for the fractionation of liver 

 may not be applicable to other tissue. It may be necessary to use a different means 

 of homogenization, a different isolation medium, or a modified isolation procedure to 

 attain the desired separations. In using cell fractionation methods, it is essential that 

 careful microscopic examination of the fractions be made and supplemented with 

 suitable cytological methods for the identification of the isolated cell structures. Un- 

 less such observations are made, the nature of the fractions isolated must be consid- 

 ered as highly questionable. 



56 H. U. Zollinger, Experientia 6, 14 (1950). 



"J. W. Harmon, Exptl Cell Research 1, 394 (1950). 



58 W. C. Schneider and G. H. Hogeboom, J. Biol. Chem. 183, 123 (1950). 



" S. Brenner, S. African J. Med. Sci. 12, 53 (1947). 



