\ iii„-20 MANUAL OF METHODS FOR PURE CULTURE STUDY 



and different antitoxins. The tubes must be observed every 15 

 minutes at these temperatures in order to observe the tube in which 

 initial flocculation occurs. The reaction is probably due to a com- 

 bination of the antigenic portion of the toxin and the antitoxin. The 

 "floe" formed is composed of both toxin and antitoxin in dissociable 

 union — dissociated by heat or sodium iodide. 



There is no difficulty in obtaining flocculation with the first frac- 

 tion in the concentration of antitoxins but later fractions may not 

 flocculate. Concentrated toxoids likewise may not flocculate. The 

 method of titration has been applied mainly to diphtheria and tetanus 

 toxins, toxoids, and antitoxins. It has been applied to others and 

 may be applied still further. 



PROCEDURE 



To a series of ten test tubes (4" X 3^") add serially amounts of anti- 

 toxin, differing by 0.005 ml. (or 0.001 ml.) from tube to tube. This 

 may be done by means of a 0.2 ml. pipette graduated in 0.01 ml. (or 

 0.001 ml.) or, if greater accuracy is desired, by means of a Trevan 

 micro-syringe. Add to each tube from a 10 ml. pipette 2 ml. of the 

 toxin or toxoid to be titrated for its Lf value. The tubes are now 

 shaken, placed in a ivater bath at 40 to 50° C. and observed every 15 

 minutes for the first appearance of flocculation. 



TABLE 7 



TITRATION OF A DIPHTHERIA TOXIN WITH RESULTS IN A TYPICAL INSTANCE 



(Flocculation method) 



C = Cloudiness; P = Precipitate; F = Flocculation. 

 Temperature of the water bath 50°C. 



Depth of the tubes in water — water % distance to top of the liquid in the tubes. 

 Size of tubes 4"X3^" (inside dimensions). 



Antitoxin used in above titration contained 425 units per ml. (or 1 unit is contained 

 in 0.00235.) 



In Table 7 is given a protocol for the titration of an unknown 

 diphtheria toxin with the results obtained. For greater accuracy the 

 toxin would be retitrated using 0.050 to 0.60 ml. of antitoxin with 

 differences of 0.001 ml. between tubes. 



Calculation of the typical instance given in Table 7 is as follows: 



