CALVIN B. COULTER 773 



heated sample can be obtained by reading the abscissa at which the 

 percentage of hemolysis given by 1 cc. of the diluted and heated 

 specimen intersects the base-Hne curve. The various corrections 

 used by Brooks were not made. By the use of this principle as Brooks 

 has shown, one compares amounts which produce hke results, and 

 can thereby obtain the actual ratio between the unheated control 

 and the heated sample with respect to the complementary function 

 under consideration. For each complete experiment there was used 

 one stock emulsion of 5 per cent sheep cells sensitized with two 

 units of sensitizer. The total volume in each tube of the titration 

 and of the test was 5 cc. before the addition of acid or alkali. 



The H ion concentration was determined by diluting three 2.5 cc. 

 portions of each specimen of serum to 5 cc. with distilled water or 

 saline, according to the medium chosen, adding methyl red and brom- 

 thymol blue separately to two of the tubes and determining by com- 

 parison with a standard series the H ion concentration reached after 

 the addition of successive drops of n/40 HCl in equal number to each 

 tube. The third tube served as a color screen after each addition 

 of acid.^^ For each specimen of the isolated fractions of complement 

 a similar titration was carried out, using for the end-piece n/40 

 and for the mid-piece n/160 HCl and NaOH. 



The partition of the complement into the fractions was carried 

 out by bringing the fresh serum diluted 1 to 20 with cold distilled 

 water to a pH between 6.2 and 6.4, using for this purpose the nec- 

 essary amount of HCl as calculated from the titration described 

 above. This narrow range of reaction is that which was found 

 optimal for the precipitation of the euglobuhn under these conditions. 

 A titration was found necessary for each specimen of serum because 

 of differences in the amount of acid required to bring the pH to the 

 desired reaction, and the blood of a single animal for the same reason 

 was used for each experiment. The different methods which have 

 been used for the partition of complement obviously depend upon 

 the insolubility of the euglobulin within a narrow range of reaction 



^^ Electrometric control of the colorimetric determinations indicate that the 

 limit of error in the latter is pH 0.2. Between pH 5.8 and 6.5 the error is 

 consistent in sign and lies between -1-0.15 and -f-O.lO, the electrometric pH is 

 more acid. No corrections have been made on the curves given here. 



