THE CYTOPLASM 213 



of the nuclear fraction is transferred to Lusteroid centrifuge tubes and centrifuged 

 for 10 minutes at 9200 r.p.m. (5000 g) in the International rotor No. 295. The sediment 

 is resuspended in 8.0 ml. of 0.25 M sucrose and recentrifuged 10 minutes at 20,800 

 r.p.m. in the same rotor. At this point the sediment is seen to consist of two definite 

 layers, a lower tan layer and an upper, poorly packed layer of a lighter color. If the 

 livers have not been perfused, the color of the upper layer is a striking pink and easily 

 differentiated from the tan color of the lower layer. The opalescent supernatant fluid, 

 together with the poorly sedimented material, is removed and combined with the 

 supernatant from the first centrifugation. The sediment is resusp>ended and recentri- 

 fuged for 10 minutes at 20,800 r.p.m. A small amount of poorly sedimented material 

 is again obtained and removed along with the clear supernatant fluid. The sediment 

 is made up to a definite volume with 0.25 M sucrose and rehomogenized briefly. As 

 indicated by the cytological observations described earlier, this suspension consists 

 of mitochondria essentially uncontaminated by other cellular elements. The yield of 

 mitochondria, as shown by direct count, is approximately 80%. 



The layer of poorly sedimented material ("fluffy layer") appearing in the prepara- 

 tion of the mitochondria (see above) requires additional comment. Since microscopic 

 examinations in the authors' laboratory have indicated that this material is largley 

 submicroscopic in character, it has been routinely separated from nitochondria and, 

 by subsequent centrifugation, included in the microsomal fraction. Muntwyler et 

 al.^^ independently reached the same conclusions on the basis of the staining charac- 

 teristics of the fluffy layer. Furthermore, Potter et al.^^ have found that, if Janus 

 Green B is added to a tissue suspension and the mixture centrifuged and incubated 

 at 38°, a sharp color boundary occurs between the firmly packed mitochondria, which 

 stain red, and the partially sedimented fluffy layer, which stains blue. The reduction 

 of Janus Green B to yield the red dye, diethylsafranine, is considered to be a specific 

 means of identification of mitochondria. Smellie et al.^^ have found in electron micro- 

 scopic observations both of mitochondria freed of the fluffy layer and of microsomes 

 containing the fluffy layer, that the morphology of the two fractions is distinctly 

 different. On the other hand. Laird et al.^^ have claimed that the fluffy layer consists 

 of small mitochondria that can be isolated in purified form. These mitochondria were 

 reported to have a succinoxidase activity equivalent to that of the main mass of 

 mitochondria on the one hand, and to contain a high concentration of PNA, similar 

 to that of microsomes, on the other. Novikoff et al.,^^ using the procedure communi- 

 cated to them by Laird for the isolation of the small "mitochondria," reported that 

 the preparation was, in fact, a mixture of mitochondria and microsomes. Further- 

 more, Kuff and Schneider in unpublished experiments in the writers' laboratory were 

 unable to obtain any fraction from the fluffy layer with a succinic dehydrogenase 

 activity approaching that of mitochondria, although the fractions were rich in PNA. 

 The correctness of the original conclusion that the fluffy layer is microsomal in its 

 properties, rather than mitochondrial, seems inescapable. 



(4) Isolation of Microsomes. The supernatants and washings remaining after the 



" E. Muntwyler, S. Seifter, and D. M. Harkness, /. Biol. Chem. 184, 181 (1950). 

 63 V. R. Potter, R. O. Recknagel, and R. B. Hurlbert, Federation Proc. 10, 646 (1951). 

 " R. M. S. Smellie, W. M. Mclndoe, R. Logan, J. N. Davidson, and I. M. Dawson, 



Biochem.J. 54, 280 (1953). 

 6* A. K. Laird, O. Nygaard, and H. Ris, Cancer Research 12, 276 (1952). 

 66 A. B. Novikoff, E. Podber, J. Ryan, and E. Noe, J. Histochem. and Cytochem. 1, 



27 (1953). 



