STANHOPE BAYNE- JONES 761 



(1924) define as the amount of toxin equivalent to i unit of antitoxin as determined 

 by flocculation, is 0.131 cc, and i cc. of the toxin is equivalent to 7.65 units of anti- 

 toxin. 



The titration of antitoxin is made in a similar manner, by noting the initial floccu- 

 lation occurring when it is mixed with a toxin whose Lj value has been established. 

 Methods of rough orientation measurements followed by closer titrations and for- 

 mulas for calculating values from the results of flocculation tests are given in papers 

 by Ramon (1922-23), Glenny and Okell (1924), Scholz (1924), and H. Schmidt 



(1925)- 



In order to obtain dependable results, at least 1-2 cc, of toxin should be placed in 

 each tube, the tubes should be clear and all of the same diameter, and they should be 

 immersed to the same depth in the water bath. 



A minimal flocculating value of an antitoxin may be established by mixing an 

 unknown serum with a standardized antitoxin. This method described by Renaux 

 (1924), for the titration of slowly flocculating or non-flocculating antitoxins has been 

 applied with useful results by myself, Ramon (1926), and others to the titration of 

 purified antitoxic sera, composed largely of the pseudoglobulins which Ramon (1922) 

 found to be devoid of flocculating power. The flocculation value thus obtained in- 

 dicates the total neutralizing properties of the mixed antitoxins. As the same results 

 are obtained whether the fractions are mixed at one time or at intervals, no definite 

 evidence of a Danysz phenomenon has been conclusively educed. Theoretically, it 

 is not to be expected that the Danysz phenomenon which is dependent upon a toxicity 

 factor would be demonstrable by the flocculation method which is an indication of the 

 total combining capacities of the components of the mixtures. But Dr. Elliott Robin- 

 son writes me it may be possible to demonstrate by flocculation that a smaUer amount 

 of toxin wfll flocculate a given amount of antitoxin if the toxin is added in several 

 small fractions than if it is added all at once. The experimental difficulties of demon- 

 strating this will be very great, 



CONDITIONS AFFECTING THE FLOCCULATION REACTION 



Temperature. — Flocculation may occur after several days at 0° C. Increase in 

 temperature up to 55° C. increases the speed of flocculation. Above 55° C. the re- 

 sults become irregular, and temperatures above 60° C. often completely inhibit the 

 reaction. Both the toxin and antitoxin can be rendered unsuitable for flocculation by 

 heating them separately at 60° C. for one hour. The most suitable temperature is 

 45°-So° C, although especially close watch of the tubes must be maintained to ob- 

 serve the occurrence of initial flocculation. A procedure which permits the observa- 

 tion of a number of titrations in a short time is based upon taking advantage of the 

 slow union of the components at o°-5° C. and their rapid flocculation when placed at 

 45°-So° C, The mixtures may be prepared during an afternoon, placed in the icebox 

 over night, and then in the water bath at 45°-5o° C, the next morning. 



Reaction. — The H-ion concentration of the toxic broth after filtration seemed to 

 have no appreciable effect upon the occurrence or rate of flocculation. The reactions 

 of the toxins have varied from pH 6,4-8.4. Attempts to study the effect of changing 

 the reaction were made by adding 11/ 1 HCl to toxins in order to bring the pH to 4, 5, 



