ISOLATION AND PURIFICATION 45 
and found to contain no activity. The sedimented protein was 
suspended in 5-4c.c. of buffer and on centrifuging at slow speed 
about 0-6 mg of insoluble protein without activity was removed. 
In method (b) to 320 c.c. of allantoic fluid similar to that used in 
method (a) and cooled to 4°C were added 14 c.c. of a 23 per cent 
suspension of adult chicken red cells. After standing for five hours at 
4°C, the preparation was centrifuged for 10 minutes at low speed to 
separate the red cells. The supernatant was removed and found to 
have about 30 per cent of the activity, so that about 70 per cent of the 
original activity was adsorbed on the red cells. Next the red cells 
were eluted with 32 c.c. of 0:1 M phosphate buffer at pH 7 for 24 hours 
at 37°C and then centrifuged at low speed; the red cells were then 
eluted a second time. 
In method (c) the remaining 320 c.c. portion of allantoic fluid was 
transferred to celluloid centrifuge tubes of 90c.c. capacity, frozen 
overnight in a CO, ice-box, and then thawed at room temperature 
for about 2$ hours. Care was taken that the temperature of the liquid 
did not rise above about 3°C. The fluid was centrifuged at 3000 r.p.m. 
in a cold room and the precipitate was collected and extracted with 
32.c.c. of oI M phosphate buffer at pH 7 for 30 minutes at about 
23°C. After centrifugation at low speed, the residual precipitate was 
again extracted as described above to yield a second extract. The first 
extract contained most of the virus. 
In method (d) eighty-nine infected chick embryos were taken 
directly from the incubator after 36 hours’ incubation, opened and 
the blood vessels torn. The bloody allantoic fluid was harvested 
within a few minutes and collected in iced centrifuge tubes. The 
570 c.c. of fluid obtained by this procedure were allowed to stand 
at 4°C for 5 hours and then centrifuged at low speed in the cold room. 
The embryonic red cells were eluted twice for 24 hours at 37°C with 
two $7 c.c. portions of buffer. 
The supernatant liquid from the original allantoic fluid, after 
adsorption with embryonic cells, was found to possess some activity. 
In order to recover this activity, the preparation was treated with 
25 c.c. of a 23 per cent suspension of adult chicken red cells for 5 hours 
at 4°C. The red cells were then removed by centrifugation at low 
speed and eluted with $7 c.c. of buffer for 24 hours at 37°C. 
Of these four methods of purifying the influenza virus, method (a) 
gave a final preparation with three times the activity of method (c) 
which gave the next highest infectivity. 
