LYSINS 165 
It must be emphasized that precision of measurement is an absolute 
requirement for success. The activating power of complement for hem- 
olysin does not follow the law of multiple proportions— it is rather an 
inverse ratio, as Xoguchi' has pointed out. Relatively less comple- 
ment is required to induce complete hemolysis in a system containing 
4 units than is required for a system containing but a single hemolytic 
unit. 
The serum to be examined for specific antibodies by the method 
of complement-fixation must be sterile and free from hemoglobin. 
The products of bacterial growths in serum may be anticomplementary 
and the presence of hemoglobin in serum also tends to inhibit hemo- 
lysis. Blood, therefore, should be withdrawn with aseptic precautions 
from the median basilic vein of the patient into sterile test-tubes, and 
either centrifugalized at once and the serum removed from the clot, 
or placed in an inclined position in a cool place until the serum has 
separated. The serum must be clear'^ and free from erythrocytes 
or hemoglobin.^ It is inactivated at 54° to 55° C. for one-half hour in 
a water-bath."* 
The Technic of the Test. — It is essential that the hemolytic system 
—erythrocytes, hemolysin, complement — be standardized daily. Vary- 
ing amounts of hemolysin are added to constant amounts of erythrocyte 
suspension and complement, as outlined above. A known positive 
syphilitic serum and a known negative syphilitic serum, together with 
suitable controls, must be examined along with the unknowTi serum 
to be tested. Stillians^ has prepared a "positive serum control," 
consisting of a mixture of strongly positive sera diluted with an equal 
volume of glycerin, which keeps well, and can be used not only for the 
actual test, but also as a means for the comparison for antigens at 
various times. Weakly positive sera are unsuited for this purpose. 
The following reagents are required : 
1. Sterile physiological salt solution. 
2. Fresh guinea-pig serum (complement)— add 0.1 cc. to each tube. 
3. Five per cent suspension of washed sheep erythrocytes in normal 
salt solution— use 1 cc. to each tube. 
4. Hemolysin (amboceptor)— use twice the hemolytic unit (the 
unit must be determined daily). 
5. Known syphilitic serum— inactivate and use 0.2 cc.** 
6. Known normal (non-syphilitic) serum, inactivated— use 0.2 cc.^ 
1 Serum Diagnosis of Syphilis 
2 Blood is best obtained early in the morning, before the patient has eaten; blood 
obtained at the height of digestion frequently contains fats which make the serum 
turbid. 
' Small amounts of blood, yielding a few drops of serum, may be obtained from the 
finger-tip or the lobe of the ear. Massage must not be practised, for there is danger of 
damaging erythrocytes with the liberation of hemoglobin. 
^ Noguchi (Serum Diagnosis of Syphilis) states that inactivation at 54° C. should be 
practised — the higher temperature weakens the reactive substance somewhat. 
6 Am. Jour. Syphilis, 1917, 1, No. 4. 
* This must be shown not to be anticomplementary. 
