324 STUDIES IN IMMUNITY. 



EXPERIMENT 2 // the comparison is exact it must be true that dissociation of 

 barium sulphate by serum is due to an adhesion of the colloidal substances of the 

 serum similar to that which takes place between the corpuscles and sulphate in their 

 agglutination. Repeated washings in salt solution of these masses formed by the 

 adhesion of corpuscles and suspension lead to no change in the clumps. Shall we 

 find in a similar way that barium sulphate that has been dissociated by serum will 

 remain dissociated after washing, in other words, that the adhesion of the suspension 

 to the dissociating colloids of the serum will persist? Twelve drops of our emulsion 

 of barium sulphate are mixed with a large dose of horse serum, that is, with 3 c.c. of 

 fresh serum diluted 1-4, and after 5 minutes the mixture is centnfugalized. The 

 sediment is subsequently washed in salt solution until the wash water contains no 

 trace of free serum, as evidenced by the fact that on boiling it does not become white and 

 that it fails to dissociate fresh sulphate. We then suspend this sulphate treated by 

 serum in salt solution and find that it remains in the form of an emulsion. This 

 emulsion differs absolutely from fresh barium sulphate in salt solution; it is very 

 colloidal in appearance and resembles milk; it finally clarifies, but without any evidence 

 of an agglutination of particles, since a slight jar suffices to restore it to the form of 

 an emulsion. 



This experiment is the complement of the preceding; serum 

 owes its dissociating property to colloidal substances that adhere 

 to the barium sulphate, just as red blood cells adhere to it. 



EXPERIMENT 3. We have endeavored to demonstrate more directly the union 

 that occurs between the colloids of serum and the dissociated suspensions. As we 

 shall see, we have succeeded in liberating or separating these colloids from the suspen- 

 sion to which they were attached. In order to do this it is necessary to eliminate the 

 original suspension by dissolving it. For this purpose we have employed tricalcium 

 phosphate as an emulsion in distilled water instead of barium sulphate, which is 

 difficult to dissolve. This tricalcium phosphate is similar to barium sulphate in that 

 it sediments rather rapidly in salt solution and distilled water, but remains for a long 

 time as an emulsion in the presence of horse serum. 



Three drops of our emulsion, for example, remain dissociated for a long time in 

 1 c.c. of serum diluted 1-10. We saturate a rather large dose of tricalcium phosphate, 

 for example, 20 drops of our emulsion, with 4 c.c. of serum diluted 1-4 in salt solution, 

 and after 15 minutes' contact centrifugalize the precipitate and wash repeatedly with salt 

 solution until the wash water contains no free serum. TJie phosphate is then suspended 

 in as much salt solution as the amount of serum employed in the first place and gives 

 a homogeneous emulsion impregnated with serum as in the case of barium sulphate. 



We place 1 c.c. of this emulsion in tube 1, and an equal amount of fresh phosphate 

 in tube 2, a corresponding amount of fresh phosphate plus 1 c.c. of salt solution and 

 a very small amount (0.025 c.c.) of serum in 1 c.c. of salt solution in tube 3. We 

 then add to each tube one drop of acetic acid. There is no change in tube 3; a certain 

 amount of the ptiosphate in tubes 1 and 2 is dissolved and the rest remains suspended. 

 Fifteen minutes later these two tubes are centrifugalized and the supernatant fluid 

 decanted. These fluids differ in this respect: The one is obtained by dissolving the 

 fresh phosphate (tube 2), and the other by dissolving phosphate that has previously 

 been impregnated with the dissociating substance of serum (tube 1). // we have 

 succeeded in liberating the dissociating substances in tube 1 by dissolving the phosphate 



