182 ANTIGENS AND THE TECHNIC OF SERUM REACTIONS 
The potency of any serum is determined according to the formula 
S = 4D, where S is the serum potency in terms of reacting units and 
D the highest dilution ratio giving a positive reaction. The preceding 
table (p. 181) illustrates some typical reactions. If a serum gives a 
positive reaction with a dilution ratio of 60, examine still higher dilu- 
tions with antigen suspension until a negative reaction is obtained. 
Qualitative Spinal Fluid Procedure.— In this procedure the greater 
part of the spinal fluid globulins is precipitated by means of ammonium 
sulphate and redissolved in an amount of normal saline equivalent to 
one-tenth of the original spinal fluid volume. The concentrated 
globulin solution thus obtained is then tested with antigen suspension. 
Preparation of Globulin Solution. — (a) Centrifuge spinal fluid to 
render it free from cells and foreign particles; (6) add 3 cc. of the clear 
fluid to a conical-shaped 10 or 15 cc. centrifuge tube; (c) to the same 
tube add 2 cc. of a saturated solution of ammonium sulphate (the 
ammonium sulphate must be of highest purity such as Merck's Reagent 
or Baker's Analyzed) ; (d) mix vigorously and place in 56° C. water 
bath for fifteen minutes; (e) centrifuge at high speed for fifteen minutes 
to throw down the precipitated globulins; (/) remove with capillary 
pipette the supernatant fluid as completely as possible; ig) add 0.3 cc. 
normal saline to the precipitate and redissolve it by gentle shaking. 
This saline should be added by lowering the pipette close to the bottom 
of the tube to avoid washing down ammonium sulphate. The use of 
a 0.2 cc. pipette is preferred for adding saline. With this pipette, the 
fluid is gently drawn up and expelled to assure the complete solution 
of the precipitate. This redissolved globulin solution is now ready to 
be tested with antigen suspension. 
Performance of Test.— Mix salt solution with antigen in the same 
manner as for the test with serum, according to the required antigen 
titer for spinal fluid. ^ The antigen suspension should stand ten minutes 
before its use in the test and should be used within thirty minutes. 
With a 0.2 cc. pipette graduated to 0.001 cc, measure 0.01 cc. of 
antigen suspension to the bottom of a test-tube. Measure 0.15 cc. of 
concentrated globulin solution into the antigen suspension tube, using 
a 0.2 cc. pipette. Shake tests vigorously for a few seconds to mix 
ingredients. 
Controls.— liichide positive and negative spinal fluid controls; also 
observe each concentrated globulin solution to establish that it is free 
from foreign particles. 
' The antigen titer for the qualitative spinal fluid procedure is not the same as for the 
serum procedures due to the fact that the dissolved globulin solution contains from 2 to 
3 per cent of ammonium sulphate. To counterbalance the presence of the ammonium 
sulphate which tends to reduce the solubility of the antigen suspension, the amount of 
saline in the antigen titer is somewhat increased. Usually an increase of 0.3 cc. over the 
serum titer will be ample. Thus, if the antigen titer for serum is 1 cc. antigen + 1.1 cc. 
saline, for spinal fluid the titer will be found to be 1 cc. antigen + 1.4 cc. saline. A 
trial with a known positive and negative spinal fluid will help establish the correctness 
of the titer. The negative spinal fluid test should be opalescent and clear. If cloudy, 
the saline in the titer is further increased, usually by 0.1 cc. 
