516 HEMOLYTIC SENSITIZER AND RED BLOOD CELLS 



5 cc. of 10 per cent cell suspension. The tubes were stoppered with 

 paraffined corks, gently agitated, and kept in the water bath at 

 38°C. for 35 minutes, with gentle agitation every 5 minutes. The 

 tubes were then centrifugated and the supernatant fluid was drawn 

 off into two equal portions. To the first portion indicator was added 

 and the pH determined colorimetrically by comparison with a stand- 

 ard series. The second portion served as a color screen for the 

 standard tube. After the determination of the pH value, the first 

 tube was titrated with n/100 HCl or n/100 NaOH to a pH between 

 6.5 and 6.0 and the amount of acid or alkali so required added to the 

 second portion of test fluid. This portion was then diluted with 

 saline solution and its content of sensitizer determined by titration 

 in the usual way, with physiological saline solution as the medium. 

 0.04 cc. of guinea pig serum was used as complement. The cells 

 used were 0.5 cc. of a 3 per cent suspension of the same sheep cells 

 used in the first part of the experiment. The total volume of each 

 tube was 2.0 cc. In consequence of the adjustment of the reaction 

 of the test fluid and the buffer action of the complement added the 

 hydrogen ion concentration of the tubes in this titration was sensibly 

 constant. For each experiment the immune serum itself was titrated 

 directly, using the same saline solution, complement, and cells. The 

 hemolytic value was determined by interpolation, as, for instance, 

 where with a 1 : 10 dilution of the supernatant test fluid the readings 

 were 0.4 cc. complete, 0.35 cc. almost complete, 0.3 cc. d= ; the value 

 chosen was 0.375 cc. By this means and by the use of a 3 per cent 

 cell suspension the error is within 5 per cent of the true value for any 

 given reading. In the calculations the alteration in volume of the 

 test fluid by the two additions of acid or alkali was taken into 

 consideration. 



The efi"ect of electrolyte was determined by adding 1 or 2 cc. of 

 physiological saline solution together with 4 or 3 cc. of saccharose 

 solution to a series of tubes and then adding acid or alkali, sensitizer, 

 and cells as before. 



The dissociation of sensitizer from cells was investigated as follows: 

 5 cc. of concentrated cell sediment from saccharose were sensitized 

 with approximately 50 units of sensitizer per unit of cells in a volume 

 of 50 cc. Saccharose solution was used as the medium so that the 



