44 AN INTRODUCTION TO THE STUDY OF VIRUSES 
important that the exact amount of 90 per cent alcohol should be 
added, as otherwise the virus is liable to be thrown down and will be 
lost with the precipitate. To the supernatant liquid is now added 
half a volume of saturated ammonium sulphate solution to bring it 
to one-third saturation. 
This precipitates the virus in the form of small octahedral crystals 
(lower photograph, Plate VIII). Further purification may be 
effected by re-crystallization from salt solution. A variety of salts are 
suitable for this purpose, but all deposit the virus in the form of 
octahedra. 
An alternative method is to re-crystallize the virus from.20 per cent 
ethyl alcohol at a pH of 3:8. This must be carried out in the cold at a 
temperature of 5°C or less. Under these circumstances the virus does 
not form octahedra but is deposited as fine needles which frequently 
form globular clusters (Markham and Smith, 1949). 
In the purification of animal viruses the worker is handicapped 
because from the nature of his material he is not always able to obtain 
large quantities of virus in the manner available to plant-virus workers. 
A good deal of work on the purification of influenza virus was carried 
out during the war chiefly with the aim of obtaining vaccines and 
some of these methods as carried out by Stanley (1944) are briefly 
described. 
The PR 8 strain of influenza virus was used in the work and the 
starting material was in the form of frozen and dried allantoic fluid 
from the hen’s egg in which it had been cultured. The virus was 
isolated originally from a ferret, passed many times in mice and tissue - 
culture and finally several times in chick embryos. The material was 
concentrated and purified by four general methods involving (a) dif- 
ferential centrifugation, (b) adsorption on, and elution from, adult 
chicken red cells, (c) elution of the precipitate formed on freezing and 
thawing of allantoic fluid, (d) adsorption on and elution from embryonic 
chick red cells. 
In method (a) 320 c.c. of infectious allantoic fluid was centrifuged 
in the ultracentrifuge at 24,000 r.p.m. for two hours. At the end of 
that time the pellets were found to contain 97 per cent of the virus 
activity. The pellets of sedimented protein were then suspended in 
35. c.c. of o-1 M phosphate buffer at pH 7 and centrifuged at a low 
speed of 3000 r.p.m. to remove the insoluble protein. The 35 c.c. of 
supernatant fluid were again centrifuged for two hours at 24,000 
r.p.m. The upper two-thirds of the supernatant fluid were removed 
