LYSINS 163 
As a general rule, the hemolytic titer is higher than the anticom- 
plementary titer. The test is readily made, if desired, by using the 
same amounts of antigen mixed with 1 cc. of red blood cell suspension 
and sufficient salt solution to bring the volume to 4 cc. 
It is customary to use one-fourth the anticomplementary titer as 
the standard amount of antigen to be used in the actual test. In 
the example cited, 1 cc. of the antigen was found to be anticomple- 
mentary, consequently 0.25 to 0.3 cc. would be the proper amount of 
antigen to employ in the test. 
Complemoit. — Fresh guinea-pig serum is the usual source of com- 
plement for fixation reactions. The animal should be healthy and 
not previously injected with protein of any nature. The serum of 
pregnant pigs is not trustworthy. Blood may be obtained directly 
from the heart of the living animal by aspiration through a hypo- 
dermic needle, from a severed carotid artery, or, more expeditiously 
by cutting the throat of the animal, avoiding the esophagus, and 
collecting the blood in sterile Petri dishes. The freshly drawn blood 
is allowed to stand for a few hours at a low temperature and the serum 
is pipetted off. In practice, serum from three or four guinea-pigs is 
"pooled ;" experience has shown that more constant results are obtained 
from pooled serum. Complement must be kept cold (below 16° C.) 
and in the dark. It must be used fresh, for it deteriorates rapidly. 
In a frozen condition, however, it will remain active for two or three 
weeks. It may be kept with little deterioration also if it be made 
hypertonic with sodium chloride, to a concentration of 20 to 25 per cent. 
Both the "activating" and combining properties of normal fresh 
guinea-pig serum are sufficiently constant for the reaction of comple- 
ment-fixation. 
Hemolytic System.— (a) Hemolytic Servm (Hemolysin) .—Hemolytic 
serum is obtained from rabbits which have been injected with 2 cc, 
4 cc, and finally 8 cc. of a 50 per cent solution of washed sheep red 
blood cells^ at intervals of four or six days. The injections may be 
made intraperitoneally or intravenously, the former being preferable. 
The seriun should be tested for potency. When the desired titer is 
reached the injections are discontinued. Not less than nine days after 
the last injection the animal is bled to death from the carotid artery 
under anesthesia, the blood being received in sterile test-tubes, which 
are placed in an inclined position in the ice-box. The serimi is removed, 
centrifugalized if not wholly free from blood corpuscles, and placed in 
small amber bottles with aseptic precautions. These are heated to 
56° C. for one-half hour to effect inactivation (to destroy complement). 
(6) Red Blood Cf-Z/^. — Erythrocytes of the sheep are used. The 
blood of a sheep is collected either in small sterile flasks containing 
1 volume of a mixture of 0.85 per cent salt solution and 0.5 per cent 
' Fresh red blood cells of the sheep are freed from serum by repeated washings with 
physiological salt solution — usually five washings suffice. The corpuscles arc then 
suspended in a volume of salt solution twice that of the corpuscles themselves- 
