238 MEDICAL BACTERIOLOGY 



The presence of complement in the guinea-pig serum, shown by the occur- 

 rence of complete hemolysis in the tubes containing a unit or more of amboceptor; 

 that a sufficient number of tubes were run to determine the unit of amboceptor, 

 because in addition to the tubes showing complete hemolysis there were others, 

 containing smaller amounts of rabbit serum, in which hemolysis was incomplete, 

 slight, or did not occur. 



When a sensitized rabbit is bled from 10 to 20 cc. of serum is obtained, an 

 amount sufficient for many hundreds of complement fixation tests. As the 

 amboceptor content of this serum will remain practically constant, once the 

 unit of a particular rabbit's serum has been determined, that quantity is taken 

 as the measure with which to determine the unit of complement prior to the 

 performance of each subsequent complement fixation test. 



COMPLEMENT 



Fresh unheated guinea-pig serum is ordinarily used to supply the comple- 

 ment for complement fixation tests. Guinea-pig serum is selected because it 

 contains complement capable of uniting with amboceptors of either rabbit or 

 human serum; because the average normal guinea-pig serum will not hemolize 

 human or sheep red blood cells; guinea-pigs are comparatively easy to obtain, 

 keep, handle and bleed. 



The method of bleeding guinea-pigs is the same as described on page 231 

 for bleeding sensitized rabbits. After some practice one can save time and 

 labor by withdrawing the blood from the pig's heart with a syringe. There 

 are other advantages in so obtaining the blood: (i) When not more than 2 cc. 

 of serum is required the withdrawal of the amount of blood necessary to yield 

 the desired serum (about 5 cc.) can usually be accomplished without permanent 

 injury to the pig. (2) For reasons to be explained later, it is better to use a 

 mixture of the serum from several pigs rather than the serum of a single pig. 

 (3) When not more than 5 cc. of blood is withdrawn from a pig, if an equal 

 amount of normal salt solution is injected into the peritoneal cavity, the pig 

 usually survives and several weeks later may be bled again. Some pigs survive 

 as many as 10 bleedings. 



When there is no particular hurry it is best to bleed pigs into Petri dishes, 

 place the dishes in a refrigerator over night and collect the serum and make the 

 complement fixation tests the following morning. In this way the maximum 

 amount of serum is obtained with the least labor. To obtain the serum rapicUy, 

 bleed the pigs into centrifuge tubes, stir the blood with a glass rod and centri- 

 fugalize it at high speed for 5 or 10 minutes, the clear serum will rise to the 

 top and can be at once pipetted off and used. 



While the amount of complement per cc. in the serum of a guinea-pig is 

 nearly constant, individual exceptions to the rule are met. The variations will 

 be slightest when serum from several pigs is mixed. Although the serum of 

 most guinea-pigs will not hemolize sheep or human red blood cells, occasionally 

 a serum is obtained that does. For these reasons it is advisable to standardize 

 complement before using it. The method of standardization is as follows: 



