394 PHARMACEUTICAL BACTERIOLOGY 



incubator at 37 C., for an hour and then examined to determine the smal- 

 lest quantity of serum that produced hemolysis. (The proper quantity 

 is usually i cc. of a i in 2000 dilution, in normal salt solution. The 

 quantity necessary for the reaction is two minimal units, thus i cc. of a 

 1000 dilution is used for the reaction). The dilution used should never be 

 lower than 1:1000. If it happens to be lower it will be necessary to give 

 the rabbit a few more injections of blood-corpuscles, before using its serum. 



5. Lamb's Blood Corpuscles. Five cc. of defibrinated lamb's blood are 

 collected and washed with normal salt solution in the same way as the 

 rabbit's blood. Then a 5 per cent, suspension in normal salt solution is 

 made. 



The antigen, the patient's serum and the hemolytic serum must be in- 

 activated (to destroy complement) before using, by heating them for three- 

 quarters of an hour at 56 C. The two sera should be inactivated as soon 

 as made. 



The antigen, antibody (patient's serum) complement, and hemolytic 

 serum should each be so diluted with normal salt solution that i cc. of the 

 dilution will contain the necessary quantities needed for the reaction. 



Technic for Performing the Reaction. Into a test-tube place 0.2 cc. 

 of the antigen, 0.2 cc. of the patient's serum (antibody), and o.i cc. 

 of the complement. Incubate at 37 C. for three-quarters of an hour and 

 then add i.o cc. of the solution of hemolytic serum, containing two mini- 

 mal doses and i.o cc. of the 5 per cent, suspension of lamb's blood- 

 corpuscles. Incubate the whole for two hours, place in the refrigerator 

 over night, and then note if hemolysis has occurred. If the antibody of 

 syphilis is present in the suspected blood serum, hemolysis will not occur 

 because the complement is "fixed" to the immune body by the aid of the 

 antigen and the reaction is positive. Should the suspected blood serum 

 not contain the specific antibody, hemolysis will occur because there is no 

 immune body to "fix" the complement, therefore causing the hemolytic 

 amboceptor (hemolytic serum), by the aid of the red corpuscles, to fix the 

 complement, producing hemolysis and the reaction is then negative. 



The substances employed are subject to many external influences, and 

 it is, therefore, necessary to control their action. The controls made are 

 necessary in order to demonstrate that none of the employed substances 

 alone "fix" the complement, and that the occurrence of either a positive or 

 a negative reaction, when testing a suspected serum, is due to and depen- 

 dent upon the fixation or non-fixation of the complement by means of the 

 immune body. 



The quantity of antigen used for the reaction may have to be either 

 increased or decreased. The controls will indicate when a change is 

 required and the proper quantity necessary is determined by the method 

 given under the preparation of the antigen. 



