302 H. K. SCHACHMAN AND R. C. WILLIAMS 



particles are, at best, present as only mmor constituents of the suspension 

 and the problem is to determine their sedimenting characteristics in the 

 presence of large amomits of particulate impurities. 



The methods of identifying virus particles with the aid of the ultracentri- 

 fuge have been developed and refined particularly by Lauffer and his col- 

 leagues (Epstein and Lauffer, 1952), and the work done by them on Southern 

 bean mosaic virus (SBMV) can be used as an example to illustrate the 

 present status of the techniques. SBMV can be purified sufficiently to form 

 a homogeneous suspension. Upon sedimentation its particles form a con- 

 centration gradient, or boundary, and the sedimentation coefficient of this 

 boundary has been determined to be 115 S under standard conditions. This 

 gives a measure of the ratio of the molecular weight of the particle to its 

 shape factor. To determine something about its effective density it is neces- 

 sary to sediment it in solvents of different densities, such as sucrose solutions. 

 To see if the particle class so characterized by these two types of experi- 

 ments carries infectivity, it is necessary in prmciple to ascertain that the 

 infectious entity sediments in the same manner. 



To reduce the hazards of convective disturbances it is desirable to use a 

 partition cell, although this may not eliminate them entirely. The SBMV sus- 

 pension is first sedimented, while observing the position of its boundary by . 

 optical means, to a degree that it is known that the boundary has passed 

 completely through the partition. The contents above the cell are assayed 

 for a "base infectivity." This is usually very low. A series of centrifugations 

 and assays is performed, varying the time and, consequently, the degree to 

 wliich the boundary approaches and passes through the partition. In this way 

 it can be told with some precision whether or not the disappearance of infec- 

 tivity from the upper portion of the cell coincides with the disappearance of 

 the boundary from this portion. A refinement of this technique that will effec- 

 tively cancel out the effects of residual convection is possible, at least in 

 principle. This is to use indicator particles in concentrations such that their 

 boundaries are discernible. The particles should have free sedimentation 

 coefficients that are known, at least relative to that of SBMV, and two lots 

 of them should be available; one with a sedimentation coefficient greater 

 than the virus under study and one with a smaller coefficient. (In the case 

 of SBMV two such types were not available, but two were used: TMV with 

 s = 185S, and bushy stunt virus [BSV] with s = 132S.) The procedure is then 

 to set upper and lower limits upon the sedimentation coefficient of the 

 infective entity by use of the two types of indicator particles. 



By sedimenting the virus-containing suspension, either with or without 

 the refinement of indicator particles, in solvents of differing sucrose concen- 

 trations additional information is found about the coincidence of sedimenta- 

 tion behavior between the characteristic particles and the infective one. 



