SERUM AGAR 



312 



SHADOW-CASTING 



that of corresponding spherical parti- 

 cles; this difference is attributable to a 

 viscosity factor and to Brownian bom- 

 bardment (the larger surface area of 

 the non-spherical particles results in 

 an increased Brownian bombardment 

 and a greater tendency to keep the 

 particles in suspension). If the density 

 of the particle is equal to that of the 

 media no sedimentation will take place 

 regardless of the centrifugal force 

 applied. If the particle is less dense 

 than the suspending media (as lipid 

 particles) it will be subjected to a flota- 

 tion during centrifugation. 



The centrifugal force is defined as 

 the force in dynes acting on a mass of 

 one gram. Since it is customary to 

 express the centrifugal force in terms of 

 the earth's gravitational force, and 

 since the gravitational force acting 

 upon a mass of one gram is 980 dynes, 

 the centrifugal force is given by the 

 equation 



47r^rn^ 

 Centrifugal force = „„„ where n is 



yoU 



equal to the number of revolutions per 

 second and r is equal to the radius of 

 centrifuge. Thus the centrifugal force 

 is proportional to the square of the 

 number of revolutions and directly 

 proportional to the radius of the centri- 

 fuge. [Thus for an angle centrifuge 

 with a radius of 8 cm., the centrifugal 

 force is 12,400 X g. at a speed of 12,000 

 RPM. For the International centri- 

 fuge with a radius of 20 cm. the cen- 

 trifugal force is 1,400 X g. at a speed of 

 2500 RPM]. 



In carrying out the fractionation of 

 the cytoplasmic constituents, a short 

 preliminary centrifugation at low speed 

 removes the intact cell nuclei and larger 

 cell fragments. The mitochondria are 

 subsequently separated from the su- 

 pernatant by centrifuging at about 

 1,000-1,500 X gravity for 10-30 minutes. 

 Less contaminated preparations can be 

 obtained by successively centrifuging 

 the supernatant for several 10-minute 

 periods. The purity of the prepara- 

 tions is checked by microscopic ob- 

 servation of the separated fractions. 

 After the mitochondria have been re- 

 moved, the supernatant is recentrifuged 

 at about 10,000 X g. for 30-60 minutes. 

 This removes the submicroscopic par- 

 ticulate components of the cell (micro- 

 somes, or ultramicrosomes). There are 

 at least two types of submicroscopic 

 particulate components, a lipo protein 

 nucleic acid complex and a hydrated 

 glycogen-particle (Lazarow, A., Biol. 

 Symposium, 1943, 10, 9, edited by N. L. 

 Hoerr). The supernatant of this high 



speed centrifugation contains the solu- 

 ble proteins, metabolites, electrolytes, 

 etc. 



When sucrose is used as a suspending 

 media (Hogeboom, Schneider, and 

 Palade, J. Biol. Chem., 1946, 177, 610) 

 the morphology of the mitochondria is 

 better preserved (in salt solution the 

 mitochondria tend to swell and they 

 lose their rod -like shape). By using 

 various buffered citric acid solutions 

 it is possible to make a clean separa- 

 tion of the nuclei (Dounce, A. L., 

 Ann. N. Y. Acad. Sci., 1950, 50, 982- 

 999). 



In the past decade considerable 

 progress has been made with regard to 

 the localization of enzymes with the 

 component parts of the cell. In de- 

 termining the localization of enzymes 

 (etc.) it is necessary to consider both 

 the concentration [i.e., the amount of 

 enzyme per unit of weight or per unit 

 of protein nitrogen] and the relative 

 fraction [i.e., what fraction of the total 

 enzyme of the cell is present in a given 

 fraction]. The cytochrome-cyto- 



chrome oxidase system as well as many 

 of the components of the Kreb's tri- 

 carboxylic acid cycle are concentrated 

 within the mitochondria (Schneider, 

 W. C, and Hogeboom, G. H., Cancer 

 Res., 1951,11, 1-22). The submicro- 

 scopic particles (ultramicrosomes) are 

 thought to be concerned with protein 

 synthesis. 



Serum Agar, see Bacteria, Media. 



Setoglaucine O (CI, 658), a basic dye less 

 light fast than Malachite green (CI, 

 657), a constituent of some bacterio- 

 logical media (Emig, p. 47). 



Shadow-Casting — Written by W. T. Demp- 

 ster, Dept. of Anatomy, University of 

 Michigan, Ann Arbor, Mich., and R. 

 C. Williams, Dept. of Biochemistry, 

 University of California, Berkeley, 

 Calif. June 9, 1950— This is a tech- 

 nique for revealing the surface form 

 and texture of microscopical material 

 in either light or electron microscopy. 

 It is an outgrowth of R. C. Williams' 

 experience with vacuum deposited 

 metal films on astronomical mirrors 

 and of studies on the physics of metallic 

 films. Metal evaporated from a hot 

 filament in a high vacuum is of atomic 

 dimensions, and upon condensation on 

 a perfectly smooth surface it forms a 

 continuous film of uniform thickness. 

 The atomic particles of metal travel 

 in straight line paths from the filament. 

 When these are directed to an obliquely 

 placed surface, obstructions, however 

 small, cast a "shadow" that is metal- 

 free. The technique of condensing 

 metal films obliquely upon specimens 



