The Hemolytic Amboceptor 285 



sents itself is how successful titration of reagents that may all be 

 more or less variable can be effected. To achieve this it is necessary 

 to begin with those that can be assumed to be least variable and 

 work up to those that are most so. 



(1) The Sheep Corpuscles. As these come from a healthy animal, 

 are always treated in precisely the same manner and used under 

 standard conditions of freshness, they can be looked upon as an in- 

 variable factor, i cc. of the 5 per cent, suspension forms a good 

 working quantity and constitutes the unit. 



(2) The Normal Guinea-pig Serum Containing the Complement. 

 As this also comes from a normal animal, is always treated in pre- 

 cisely the same manner, and is also used under standard conditions 

 of freshness, etc., it may also be looked upon as a factor subject 

 to very slight variation. Of this serum, o.i cc. (i cc. of a i :io 

 dilution, made with physiological salt solution) forms the unit, or 

 working quantity. 



These two reagents, therefore, may be regarded as the standards 

 of measurement through which the titer of a third is made possible. 



(3) The hemolytic serum from the rabbit treated with the sheep 

 corpuscles. 



This is subject to very great variation, according to the treat- 

 ment of the rabbit, and apparently, also, according to the ability 

 of the individual rabbit to respond to the treatment by the forma- 

 tion of hemolytic amboceptors. It is, therefore, imperative to make 

 a careful titration of it. 



To do this we proceed as follows, the quantities recommended 

 being such as experience has proved most satisfactory: 



Into each of a series of common test-tubes or culture- tubes i cc. 

 of the 5 per cent, suspension of sheep corpuscles and i cc. of the 

 i : 10 dilution of the normal guinea-pig serum (complement) are 

 measured with graduated pipettes, and then to each of these tubes 

 the rabbit serum (amboceptor), diluted with physiological salt solu- 

 tion so as to make the correct measurement of the minute quantities 

 necessarily employed a matter of ease and convenience, is added in 

 diminishing quantities for the purpose of determining the least 

 quantity that will bring about complete hemolysis in two hours at 

 the temperature of 37C. The occurrence of the hemolysis is shown 

 by a very striking change in the appearance of the fluids. The 

 mixture is at first opaque and pale red, but after hemolysis, or solu- 

 tion of the red corpuscles, becomes a beautiful transparent Burgundy 

 wine red. 



The actual " set-up" or working scheme for determining the unit 

 or least hemolyzing addition of the amboceptor serum may be 

 represented as follows, the tubes being placed in a thermostat and 

 observed every fifteen minutes: 



