ISOLATION AND PURIFICATION | 
with the enzymes from snail gut. The next step is to clarify the cruq 
extracted sap; this can be done by heating to §5°C for a few minutes or, 
alternatively, by adding 2-4 per cent disodium phosphate. The sap 
is then centrifuged for about 15 minutes at 3000 r.p.m. to remove 
the bulky coagulum which is discarded. To the clear supernatant 
fluid is now added half its volume of a saturated solution of ammonium 
sulphate to give one-third saturation. The virus is thrown out of 
solution and comes down as a white flocculent precipitate. This 
precipitate is spun down on the centrifuge and re-suspended in a 
volume of water equal to about one-fifth to one-tenth of the original 
volume of sap; this is centrifuged again to remove any insoluble 
matter. The virus is then re-precipitated either by acidifying the 
solution with a 10 per cent solution of acetic acid or by one-third 
saturation, again with ammonium sulphate. The precipitated virus is 
centrifuged off and re-suspended in a small volume of water and 
dialysed against running water. When free from ammonium sulphate 
the solution should be centrifuged again to get rid of insoluble matter. 
The purified virus solution should be opalescent and almost colourless. 
If sufficiently concentrated such a solution will, on standing, separate 
into two layers; the lower layer is the more concentrated and is 
spontaneously birefringent. The upper layer is not birefringent when 
stationary but if gently shaken it exhibits anisotropy of flow. 
If acid or ammonium sulphate is added to the solution, the virus 
comes out of solution as a precipitate with a characteristic satin-like 
sheen. Under the microscope this precipitate is seen to be composed 
of needle-shaped paracrystals. These needles were first isolated by 
Stanley in 1935; they are not, however, true three-dimensional 
crystals. 
The virus of turnip yellow mosaic is more easily purified than that of 
tobacco mosaic or probably any other plant virus and forms true 
three-dimensional crystals. The leaves are harvested from infected 
plants of either turnips or Chinese cabbage and frozen overnight. 
Curiously enough much more virus is obtainable from old, rather 
hard, turnip plants growing in the field, than from young, sappy, 
Chinese cabbage grown in the glasshouse. This is the reverse of what 
happens with tobacco plants infected with the tobacco mosaic virus. 
The leaves are minced and thawed as before and the sap is expressed 
through muslin; to each litre of sap is added exactly 300 ml of 90 
per cent alcohol. This clarifies the sap and the precipitate is removed 
by centrifuging, the virus remaining in the supernatant fluid. It is 
