CHAPTER V 
ISOLATION AND PURIFICATION OF SOME 
REPRESENTATIVE VIRUSES 
FoR some years previous to 1935 several attempts to isolate the virus 
of tobacco mosaic from the plant sap had failed and the first to succeed 
in this task was Stanley (1935) who used salt-precipitation methods. 
Since that time much progress has been made in the isolation of 
viruses, not only of plants but of animals as well, and some of the 
methods used are briefly described in this chapter. It is proposed to 
discuss the isolation and crystallization of two representative plant 
viruses and the purification of three animal viruses, including one 
insect virus, and one bacterial virus. 
Plant viruses which are suitable for purification must be fairly 
stable, that is they must resist denaturation by low temperatures and 
by oxidation when expressed from the plant with the sap. They must 
also be present in high concentration in the host plant; this is very 
important because it is of little use attempting to isolate a weakly 
infectious and unstable virus. 
The two main methods employed for the purification of plant 
viruses are the chemical-precipitation method and isolation by high- 
speed centrifugation. The latter is not so drastic as chemical precipi- 
tation and can be used with viruses which are less stable and concen- | 
trated. Sometimes it is preferable to use a combination of the two 
methods. 
The following is a brief outline of the technique for isolating the 
virus of tobacco mosaic by chemical methods. The leaves are harvested 
from young, systemically infected, tobacco plants; young plants 
are preferable to old since the latter contain much tarry material which 
it is difficult to eliminate. The leaves are frozen overnight, minced 
in an ordinary meat mincer and then thawed. The freezing and 
subsequent thawing seem to make the sap easier to handle; the leaf 
pulp and sap are then pressed by hand through muslin, in order to 
remove large particles. More sap may be extracted from the “cake 
by means of a press, either a hand or hydraulic press according to the 
quantities used. It has recently been shown (Bawden and Pirie, 1946) 
that more virus can be obtained from the dried pulp by digesting it 
Ag 
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