Continuous-flow zonal ultracentrifugation was introduced as a technique for rapidly concentrating, and largely purifying, milligram amounts of infectious virus from very large volumes of tissue culture fluid (1). The first continuous-flow rotor available commercially (B-VIII)
suffered from the disadvantage, that during unloading, small amounts of displacing solution leaked through the fluid line leading to the top of the rotor core, so causing considerable mixing of the gradient in the rotor (2). The B-IX rotor also gave unsatisfactory recovery of gradient and banded material for the same reason. Further development work was carried out leading to modifications to these rotors, and the introduction of the B-XVI rotor, which incorporates a centrifugally operated valve to eliminat'e leakage (2). In our experience this rotor works well (3).
A method for using unmodified B-VIII and B-IX rotors has been described by Chervenka and Cherry (4)) which involves the introduction of an air bubble into the rotor, before unloading, to prevent this leakage. Tinfortunately, this method is not wholly satisfactory since, as the authors themselves point out, the volume of the bubble is extremely critical. The procedure must be carried out carefully in order to produce conditions in which mixing is prevented. In addition, this method does not eliminate the possibility of mixing during the loading procedure, another fault which has been experienced with the B-VIII rotor. This communication describes another method by which unmodified B-VIII and B-IX rotors can be used without mixing their contents, during either loading or unloading.
It seemed possible that the B-VIII rotor could be made to function as a reorienting gradient rotor, particularly since this method is used routinely with the larger K-11 continuous-flow rotors (5). The empty rotor was, therefore, placed in the centrifuge, the upper bearing and the fluid lines were connected, and the rotor was loaded while stationary, RS follows:
The overlay solution (200 ml) was allowed to syphon in through the line leading to the center of the rotor core, followed by the gradient (600 ml of 10% w/w-15% w/ w sucrose linear with volume) which was pumped in slowly, from a gradient-forming engine, to the bottom of t'he rotor, followed by the cushion solution (approximately 175 ml of 40% w/ w sucrose) until the overlay solution began to emerge