80 ANTIBODIES, IMMUNITY, WASSERMANN SERUM TEST 



before all of the rabbit's serum is collected. If the preliminary trial 

 shows a strong hemolytic power, the rabbit is killed by cutting 

 both carotid arteries and allowing the blood to flow into a sterile 

 cylindrical vessel. The blood is allowed to coagulate, and is then 

 placed on ice for a number of hours, after which the serum is 

 removed from the coagulum. If the serum is not perfectly clear and 

 still contains some red blood corpuscles, it must be centrifuged. 

 The clear serum is finally pipetted off. This is next heated on a 

 water bath at 56 C. for thirty minutes. By so inactivating the 

 serum, we destroy, as explained before, the hemolytic complement, 

 so that the rabbit's serum will now alone not hemolyze sheep's cor- 

 puscles. 



3. The complement necessary to produce hemolysis is obtained 

 in the blood serum of the guinea-pig. An animal of this kind is 

 bled to death by severing the carotid arteries. The blood is col- 

 lected at once in some centrifuge tubes, and after coagulation is 

 centrifuged and the serum pipetted off from the clot. The comple- 

 ment in the guinea-pig's serum is very easily destroyed, by simply 

 allowing the serum to stand at room temperature. If the serum cannot 

 be used at once it must be placed in the refrigerator, and if it is to 

 be kept for more than twenty-four hours, it must be frozen hard in a 

 mixture of salt and ice and kept in a thermo bottle in a freezing mixture. 



4. The rabbit's inactivated serum is now tested in the following 

 manner : 



(a) Take a number of test tubes and place into each ten drops of 

 a 5 per cent, physiologic salt solution emulsion of washed sheep's 

 corpuscles. 



(6) Add to them one drop of the guinea-pig's serum and nine drops 

 of salt solution. 



(c) Now add to the different test-tubes varying amounts of the 

 inactivated rabbit's blood serum plus enough salt solution to make 

 ten drops, say as follows: 



To tube No. 1, add one drop. 



To tube No. 2, add one-half drop. 



To tube No. 3, add one-fourth drop. 



To tube No. 4, add one eighth drop. 



To tube No. 5, add one-twelfth drop. 



To tube No. 6, add one-sixteenth drop. 



These additions have to be made exactly by previously diluting some 

 of the inactivated rabbit's serum accurately; for instance, dilution 

 No. 6 would be made by adding to one drop of serum fifteen drops 

 of salt solution and taking of this dilution one drop plus nine drops 

 of salt solution to test-tube No. 6. 



(d) Finally, add to each tube twenty more drops of the salt solution, 

 so that each tube contains exactly fifty drops. Shake them well and 

 place them all for thirty minutes into the incubator. Examine after 

 they have been incubated one-half hour. 



