188 MECHANISM OF COMPLEMENT ACTION 



at all times except for one period of about ^ hour while the acid was 

 acting; during this period a temperature of 10°C. was maintained. 

 Three solutions were prepared for admixture with the complement: 

 0.04 N HCl, 0.04 N NaOH (adjusted by comparison with HCl), and 

 a 0.02 N NaCl solution made by mixing equal volumes of these two 

 solutions. All these solutions were cooled to the temperature of the 

 diluted complement before mixing. 



The general plan of the experiments involved the addition of the 

 desired amount of acid, and after a certain length of time its neutrah- 

 zation by the equivalent amount of alkali. In each case enough 0.02 

 N NaCl was added to make the final volume and concentration the 

 same in all the tubes. The effect of the change in NaCl concentra- 

 tion (from 0.85 to 0.73 per cent) was negligible in view of the subse- 

 quent dilution with much larger amounts of balanced solution. The 

 dilution of the serum was taken into account in the calculations. 



Sets of clean sterile test-tubes were set in an ice water bath, each 

 set consisting of a tube in which was placed 10 cc. of 5 per cent com- 

 plement, and tubes for the acid, alkali, and NaCl solutions. At the 

 beginning of the experiment the samples of complement were in turn 

 poured into the tubes containing acid and quickly mixed by pouring 

 back and forth twice. After the desired interval these mixed samples 

 were poured in like manner into the tubes containing alkali, and then 

 into the final tube containing NaCl to equalize the volumes. The 

 same operations were performed even when no acid or alkali was to 

 be added, to obviate any possible effect due to the slight foaming 

 involved in mixing the samples even though this caused no demon- 

 strable inactivation. 



After this treatment the complement was further diluted to the 

 desired extent and titrated as described in a previous paper,^" by a 

 method in which the probable error of determining the relative effi- 

 ciency of samples of complement lies between 1 and 2 per cent. 



The action of the acid apparently takes place very quickly; cer- 

 tainly in less than 15 minutes at 0°C. A preliminary experiment 

 will serve to demonstrate this point. The efficiencies of two samples 

 of complement left in the acid for 15 and for 66 minutes respectively 



I'' Brooks, S. C, /. Med. Research, 1920, xli, 399. 



