CONTRIBUTIONS TO THE THEORY OF LYSIN ACTION. 7 



as time and temperature conditions were concerned, but the result 

 was always the same; the red blood-cells did not combine with a trace 

 of addiment. This is in direct contrast to their behavior toward the 

 immune body. 



Having now determined the behavior of the blood-cells to immune 

 body and addiment separately, it remained to see what the affinities 

 of the blood-cells were when both of these bodies were present at the 

 same time. The solution of this problem offers many technical 

 difficulties. Practically it will be best to make the mixtures so that 

 there will be just the proper amount of the two ingredients to effect 

 complete solution of the blood-cells. We found that if we mixed 

 1.0 to 1.3 cc. of our inactivated goat serum with 0.5 cc. normal goat 

 serum, this would just suffice to dissolve 5 cc. of a 5% mixture (in 

 saline) of sheep blood-cells. If this mixture is placed in the ther- 

 mostat, complete solution will ensue; but because an excess of the 

 solvent substances has been avoided, the process does not take place 

 rapidly. Usually it is completed at the end of 14- to 2 hours. 



If the mixture is kept at 0-3 C., no solution occurs, and if it is 

 then centrifuged and examined according to the methods just studied, 

 the red blood-cells will be found to have loaded themselves with 

 immune body, leaving the addiment in the fluid. The experiment 

 shows that under the conditions mentioned, addiment and immune 

 body exist in the fluid entirely independent of one another. 



It still remained to determine the combining affinities at higher 

 temperatures. A preliminary trial showed that if we used the pro- 

 portions above mentioned and kept such mixtures in an Ostwald 

 water-bath at 40 C. for six, ten, thirteen, and eighteen minutes 

 respectively and then centrifuged, only in the first two tubes did 

 the fluid remain colorless, while in the other tubes it was distinctly 

 red. In the experiments at this temperature we therefore adopted 

 a time limit of ten minutes. A tube of the above-mentioned mixture 

 was allowed to remain in the water-bath at 40 C. for ten minutes 

 and then centrifuged. The results were as follows: 



The sediment mixed with salt solution shows haemolysis of a 

 moderate degree. (This occurs even if the sediment is mixed with 

 ice-cold salt solution, centrifuged, and then again mixed with salt 

 solution. By this manipulation the last trace of fluid originally 

 adhering to the cells is removed.) Solution becomes complete when 

 new addiment in the form of normal serum is added to the 

 mixture. The centrifuged fluid does not, by itself, dissolve blood 



