38 R. MAEKHAM 



ribs, may increase the yield from a given weight of starting material several- 

 fold, and is of advantage when the yields expected are small. In general, 

 however, this is a waste of time, and when working with viruses, such as the 

 tobacco mosaic viruses or turnip yellow mosaic viruses, it often pays to 

 throw away a large proportion of the virus in the plants and to gain m the 

 ease of handling of the larger volimies of material involved. 



B. The Handling of the Virus-Containing Sap 



In order to extract the virus from the "sap" of the plants, which amounts 

 to some 80 % of the weight of the fresh leaves, the leaves must be subjected 

 to gross damage. This is best accomplished by means of a domestic meat 

 grinder, preferably electrically operated, and with a worm which compresses 

 the material before it reaches the cutters. Such grinders are obtainable 

 commercially, but with difficulty. The use of the more recent blenders is 

 not recommended because they are not at all suited to dealing with plant 

 materials. 



When the plant cells are ruptured the virus becomes suspended in a 

 medium which is generally referred to as "sap." It is important to recognize 

 that this material is essentially a pathological fluid, in which the virus is 

 subjected to a change in its osmotic environment, and which contains a 

 miscellany of enzymes and their substrates, and intracellular materials 

 in various stages of disintegration. Many viruses tolerate such an environ- 

 ment reasonably well, and it is not usual to add materials to stabilize the 

 osmotic pressure of the sap, although this might be profitable in case of the 

 larger plant viruses. Much of the solids in the leaves may be eliminated before 

 the actual crushing of the cells by freezing the leaves to about — 10°C. for a 

 day or two before they are actually ground, and this may also assist in the 

 grinding, because many leaves are more easily disintegrated in the frozen 

 state. The leaf tissue is then allowed to thaw and the sap expressed through 

 muslin or some other coarse cloth. On a large scale the sap may be obtained 

 by means of a basket centrifuge or a large screw press. 



When the plant sap is exposed to air, the phenolic substances which it 

 contains tend to become oxidized to quinones, which combine with proteins 

 including the virus. This may be prevented by the addition of a reducing 

 agent, such as NaHSOg, during the grinding, some 0.1 % being sufficient, or 

 by adding a small quantity of ethylenediamine tetraacetate (Versene), or 

 cyanide, which inhibits the metal catalysis involved in these oxidations. 

 Bisulfite is preferable, because some procedures of purification take advantage 

 of the presence in the sap of divalent cations such as Ca2+, Avhich would be 

 removed by the ethylenediamine tetraacetate. Usually, if sufficiently young, 

 actively growing plants are used, trouble from quinone formation is not 

 serious, and the addition of substances to prevent it is not necessary. 



