760 



TITRATION OF TOXINS AND ANTITOXINS 



of a certain time one of the mixtures began to flocculate. The first tube showing def- 

 inite flocculi was called the precipite indicateur. The toxin-antitoxin mixture in this 

 tube was neutral, while the mixtures as well as the precipitates on either side of the 

 one containing the initial flocculation were either toxic or antitoxic according to 

 whether they contained more or less of the antitoxic serum than the amount in the 

 first tube which flocculated. 



TECHNIQUE 



The technique of the procedure, which requires slight modification to suit different 

 conditions, can be described adequately by a summary of the method used by me in 

 1924 for the titration of diphtheria toxin and antitoxin. Into each of a series of test 

 tubes, 2 cc. of a standardized, rapidly flocculating toxin are placed, and to each tube 

 is added a small amount of antitoxic serum by means of 0.2-cc. pipettes graduated in 



TABLE I 



Standardization of Toxin 



C, cloudy; P, granular precipitate; F, flocculation; I, indicating tube. 

 Diphtheria toxin: Albany 308 B. 



Diphtheria antitoxic serum: Albany 164, 430 units per cubic centimeter. 

 Temperature of water bath: 55° C. 



o.oi cc, or by a micro-syringe like that devised by Trevan (1922), capable of deliver- 

 ing o.ooi cc. with an error of i per cent. The mixtures are then placed in a water bath 

 at 45°-5o° C. and observed at frequent intervals for the detection of initial flocculation 

 in the indicating tube. 



As long as the Ehrlich unit is used for the evaluation of antitoxin, it must be taken 

 as the point of reference for titrations of toxins as well as antitoxins. Hence, in Ra- 

 mon's method, the toxin to be employed subsequently for determining the values of 

 antitoxins must be standardized in terms of a known antitoxin, prepared in the usual 

 way. Therefore, the first step to be taken is to determine the amount of toxin which 

 will cause initial flocculation with i unit of antitoxin. An example of such a standard- 

 izing titration is given in Table I. In this titration (Table I) the initial flocculation 

 occurred in the fifth tube. This tube, then, contained the indicating mixture composed 

 of 2 cc. of toxin and 0.035 cc. of the antitoxic serum. As the serum contained 430 

 units of antitoxin per cubic centimeter, i unit was represented by 0.0023 cc. of the 

 serum. Therefore, the amount of serum in the indicating tube 0.035 cc. was equal to 

 15.3 units of antitoxin. From these figures, the L/ amount, which Glenny antl Okell 



