

METHODS FOR CONDUCTING THE SYPHILIS REACTION 447 



first method. It is well to prepare this dilution in such a manner 

 that the hemolytic dose will not be more than 0.1 c.c. If the am- 

 boceptor is being constantly used, so that its titer is well known, it will 

 not be necessary to titrate, but 5 times this unit is used in sensitizing the 

 corpuscles. Otherwise small amounts of complement and corpuscle 

 suspension must be prepared, and the amboceptor titrated as follows : 

 Sufficient complement is prepared by diluting 0.5 c.c. of fresh 

 guinea-pig serum with 9.5 c.c. of salt solution (1 : 20), each cubic 

 centimeter of which contains 0.05 c.c. undiluted serum. It is well 

 at this time to prepare only sufficient 2.5 per cent, suspension of 

 sheep's cells for the titration, which is then conducted after the 

 technic given in the first method. For instance, if 0.1 c.c. of ambo- 

 ceptor dilution represents one hemolytic unit, 0.5 c.c. would be used 

 in sensitizing each dose of corpuscles. If this quantity of ambo- 

 ceptor dilution is added to a 2.5 per cent, suspension of cells, the 

 emulsion would be considerably reduced, which would not be advis- 

 able. It is well, therefore, to calculate the amount of amboceptor and 

 corpuscle suspension required for the work on hand-, measure out the 

 amboceptor dilution in a flask, and add the corpuscles and then 

 sufficient salt solution to bring the total volume to the required amount 

 to make a 2.5 per cent, suspension of cells. To continue the fore- 

 going example: five hemolytic doses of amboceptor and sufficient 

 corpuscular suspension are required for 100 tubes; this requires 50 c.c. 

 of amboceptor dilution, 2% c.c. of washed corpuscles, and sufficient 

 sterile normal salt solution to bring the total volume up to 100 c.c. 

 Permit the mixture to remain for at least half an hour at room tempera- 

 ture for the process of sensitization to take place before the complement 

 titration is performed. 



2. Complement. Fresh clear guinea-pig serum is diluted with 

 four parts of normal salt solution and titrated to determine the hemo- 

 lytic unit. 



Complement Titration. Into a series of eight test-tubes place 

 increasing amounts of the diluted complement serum (using the 

 special 0.2 c.c. pipets graduated in 0.01 c.c.), as follows: 0.01, 0.02, 

 0.03, 0.04, 0.05, 0.06, 0.08, 0.1 c.c.; add 1 c.c. of sensitized corpuscles 

 and sufficient salt solution to make the total volume about 3 c.c. 

 After incubating for one hour at 37 C., the smallest amount of comple- 

 ment giving complete hemolysis represents one hemolytic unit. 



3. Antigen. Browning and Mackenzie use a 0.75 per cent, dilu- 

 tion of lecithin and cholesterin in alcohol, prepared according to their 



