362 SPECIFIC MICRO-ORGANISMS 



for 30 minutes and then refrigerated. The separated serum is 

 then drawn off with a pipette and 2 c.c. of it are mixed with 18 c.c. 

 of cold 0.9 per cent salt solution. This 10 per cent solution of 

 guinea-pig's serum is kept in a cold place, preferably immersed in 

 ice water. It is prepared on the day it is to be used. The unit 

 of complement is contained in 0.2 c.c. of this solution. 



3. The hemolytic amboceptor is prepared by injecting 2 c.c. 

 of thoroughly washed (five times) sheep's corpuscles intravenously 

 into a large rabbit at intervals of three days, until four injections 

 have been given. Ten days after the last injection the animal is 

 allowed to fast for 12 hours and the blood is then aseptically 

 drawn from the carotid artery, allowed to clot and the serum 

 separated by standing at 37 C. for two to five hours. The clear 

 serum is transferred to small glass ampoules in amounts of 0.5 to 

 i.o c.c. and hermetically sealed. These are then heated at 56 C. 

 for 30 minutes and stored in the refrigerator. The hemolytic 

 power of this serum is ascertained by titration. The unit is that 

 amount which, when mixed with 0.2 c.c. (i unit) of corpuscles 

 and 0.2 c.c. (i unit) of complement and sufficient salt solution 

 (0.9 per cent) to make a total volume of i c.c., will cause complete 

 laking of the red blood cells in exactly i hour after being placed 

 in the incubator (air) at 37 C. The unit of amboceptor is ordi- 

 narily contained in o.i c.c. of a dilution of i part of serum in 

 1000 to 2000 parts of salt solution. After the strength is ascer- 

 tained the diluted amboceptor is made up so that o.i c.c. contains 

 i unit. 



The amboceptor is quite permanent under ordinary refrigera- 

 tor conditions, but when diluted it may deteriorate after a few 

 days. The relation of complement, red blood cells and ambo- 

 ceptor is tested always immediately before undertaking a comple- 

 ment-fixation test. If the mixture of one unit of each of these in 

 a total volume of i c.c. produces complete hemolysis at the end 

 of an hour, the hemolytic system is considered satisfactory. If 

 there is only a slight discrepancy this may be corrected by em- 

 ploying a little more or a little less (within limits of 20 per cent) 



