SPIROCILETJE 363 



amboceptor, that is, down to o.c8 c.c. or up to 0.12 c.c. as may be 

 necessary in place of the usual o.io c.c. If the discrepancy 4s 

 greater than this it is well to obtain a new sample of complement 

 or of sheep's cells or of both. The hemolytic system should be- 

 have much the same from day to day when the technic is 

 accurate. 



4. The patient's serum is obtained from 5 to 10 c.c. of blood 

 drawn from the elbow vein. The serum must be free from sus- 

 pended matter, centrifugalized if necessary. The serum is heated 

 at 54 to 56 C. for 30 minutes just before use. 



5. The antigen is a 3 per cent solution in methyl alcohol of the 

 acetone-insoluble lipoids extracted by alcohol and ether from the 

 heart muscle of beef. The strength of antigen to be used must be 

 ascertained by careful titration. A dilution of i c.c. of the antigen 

 in 9 c.c. of salt solution is first prepared. Then various quanti- 

 ties, o.i c.c., 0.2 c.c., 0.3 c.c., 0.4 c.c. and 0.5 c.c. of this suspension 

 are placed in separate tubes. To each tube is added i unit of 

 complement and sufficient salt solution to bring the total volume 

 to i c.c. The tubes are incubated i hour at 37 C. (air). Then 

 one unit of corpuscles (0.2 c.c.) and two units of hemolytic ambo- 

 ceptor (0.2 c.c.) are added and the tubes are again incubated an 

 hour. Of those tubes in which hemolysis is not complete, the one 

 containing the least antigen marks the concentration at which 

 the antigen is distinctly anti-complementary. The second test 

 of the ant'gen is now undertaken. Various amounts of a i to 100 

 dilution, o.oi c.c., 0.03 c.c., 0.05 c.c., o.i c.c. and 0.2 c.c., are meas- 

 ured into tubes. To each tube is then added i unit of comple- 

 ment, 0.02 c.c. of serum from an active untreated case of syphilis 

 and sufficient salt solution to make a total volume of 0.6 c.c. 

 The tubes are incubated an hour. Then i unit of corpuscles 

 (0.2 c.c.) and 2 units of hemolytic amboceptor (0.2 c.c.) are added 

 and the tubes are again incubated one hour. Of the tubes show- 

 ing no hemolysis (complete fixation), that one which contains the 

 least antigen marks the lowest effective concentration of the an- 

 tigen. This amount of antigen should be very much less than the 



