16i ANTIGENS AND THE TECH NIC OF SERUM REACTIONS 
sodium citrate, or in sterile centrifuge tubes. If the former is used, 
9 volumes of blood are allowed to flow into the flask and immediately 
mixed intimately with the citrate solution, which prevents clotting. 
This method is applicable if the blood cannot be centrifuged imme- 
diately. If centrifuge tubes are used, an amoimt of blood not more 
than one-third the capacity of the tube (about 5 cc.) is collected 
and twice the volume of sterile salt solution is added to it. The 
corpuscles are sedimented, the supernatant solution is pipetted off, 
fresh salt solution is poured in, and the corpuscles resuspended by 
careful stirring with a clean glass rod. This process is repeated three 
times, each time discarding the washings. The last time the volume 
occupied by the erythrocytes is read off on the graduations of the 
tube and they are suspended in a volume of salt solution twenty 
times that occupied by the erythrocytes. This makes a 5 per cent 
suspension. Erythrocytes are obtained by centrifugalization from 
the citrated blood in precisely the same manner. This suspension 
of red blood cells, kept in a cool, dark place, may be used for two days, 
but not longer. Beyond that time the cells deteriorate and hemolyze 
with abnormal readiness, thus vitiating the value of the test. 
(c) Standardization of Hemolytic System. — It is very important to 
know with exactness the amount of hemolytic serum (inactivated, 
of course) which will effect complete hemolysis of 1 cc. of a 5 per cent 
suspension of sheep erythrocytes in the presence of a constant amount 
of complement. The determination of this factor gives the hemolytic 
titer of the hemolytic serum. It is readily determined by adding to a 
series of tubes, 0.1 cc. of fresh guinea-pig serum (complement), 1 cc. 
of erythrocyte suspension, and varying amounts of the inactivated 
hemolytic serum. The smallest amount of hemolysin which will 
effect hemolysis under the conditions stated is the hemolytic titer or 
unit. Thus, the following tubes incubated at 37° C. for one hour 
showed : 
3 . 
4 . 
5 . 
6 . 
7 . 
8 . 
9 . 
10 . 
11' 
122 
Complement, 
cc. 
. 0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
0.1 
1 
0.1 
. 
5 per cent 
suspension 
sheep 
erythrocytes, 
cc. 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
1 
Inactivated 
hemolytic 
serum, 
cc. 
0.10 
0.075 
0.050 
0.025 
0.010 
. 0075 
. 0050 
0.0025 
0.0010 
00075 
. 00000 
. 00000 
Complete hemolysis. 
Partial hemolysis. 
No hemolysis. 
0.005 of this serum is 1 unit; the hemolytic titer is 0.005 cc, in other words. It is 
customary to use 2 units in the actual test, consequently 0.01 would be the amount 
used. 
1 Complement control. 
'^ Erythrocyte control. 
