ISOLATION AND PURIFICATION 49 
For the initial concentration of the virus, the clarified lysate is 
passed through the so-ml concentration bowl (20) of the Sharples 
centrifuge at a rate of 21 per hour. The bowl speed should be 45,000 
r.p.m. (49,0009). 
After washing with saline and cooling overnight the suspension 
from the Sharples should be opalescent; it shows a slight yellow 
colour due to a small amount of mucoid material still remaining. 
This can be removed by spinning on an angle centrifuge at 2000g for 
10 minutes. The supernatant from this should show a bright blue, 
opalescent, Tyndall effect, and should be devoid of yellow colour. 
The next step is to put it in the chilled rotor of the ultracentrifuge 
and spin at 20,000g for 40 minutes. The ultracentrifuge concentrate, 
when re-suspended, should be strongly opalescent and blue with sharp 
high-lights when viewed by transmitted light. 
