ISOLATION AND PURIFICATION 47 
In one such experiment 50:4 mg of virus were obtained, a yield of 
0°33 mg of virus from I g of warts. 
Satisfactory yields of purified rabbit papilloma virus can thus be 
obtained by preliminary sedimentation with the Sharples super- 
centrifuge from large volumes of extracts of virus warts, followed by 
repeated high and low speed spinning in the vacuum-type air-driven 
ultracentrifuge. 
The next example of virus purification is given by one of the virus 
diseases of insects, known as polyhedral diseases, and here the tech- 
nique of isolation is rather different. In this type of disease, most of 
the virus is contained inside the polyhedral bodies and the method of 
isolation is based mainly on the work of Bergold (1946) and refers to 
a polyhedral disease of the caterpillars of the gipsy moth (Lymantria 
dispar). The dead and semi-liquefied bodies of the larvae are collected 
and put together into a glass vessel, preferably tall and narrow, with 
water or saline, and allowed to stand for some weeks. Part of the 
object of this is to allow bacterial decomposition of the tissues. During 
this time, the polyhedra sediment to the bottom, after which the fluid 
and decomposing bodies can be decanted off. The polyhedra should 
then be washed thoroughly with water and drawn through a piece 
of fine muslin with a suction pump. If properly clean, the deposit 
of polyhedra should be white. The next step is to dissolve the poly- 
hedra to free the virus and this must be done at an alkaline pH of 9 
or 10. About 35 mg of dry polyhedra are put in a clean dry flask 
and 7 c.c. of alkali are added, using 0-008 M Na,COs3 and o-os5 NaCl. 
This is left to stand, with the vessel closed, for two or three hours. 
The solution should become fairly, but not too, clear. The next step 
is to spin out the undissolved material at 6000 r.p.m. for minutes and 
decant the supernatant fluid which contains the polyhedra protein 
plus virus. The fluid must then be spun in a centrifuge at 10,000 
r.p.m. for one hour, which gives a small bluish-white precipitate. 
The supernatant should be decanted very carefully and 7 .c.c. of 
glass-distilled water added to the precipitate; it can then be left for 
one hour but the precipitate should dissolve in 5-10 minutes 
giving an opalescent fluid with little or no insoluble matter. 
This should be spun again at 10,000 r.p.m. for one hour (70009), 
after which the supernatant should be quite clear, with all the 
virus thrown down. The virus is then dissolved in water once 
more and the final solution should be opalescent. It is important 
to keep such a virus solution at pH 7 or above and it must not be 
