212 BACTERIA PATHOGENIC TO MAN 



hours of toxin formation, a double process going on one of deteri- 

 oration in the toxin already accumulated, which tends to increase 

 the neutralizing value of the fatal dose ; the other of new toxin forma- 

 tion, which probably tends to diminish the neutralizing value. The 

 chemical changes produced by the growth of the bacilli in the bouillon 

 tend to aid one or the other of these processes, and so to make, from 

 hour to hour, slight changes in the value of the fatal dose. Later, with 

 the period of cessation of toxin production, the gradual deterioration of 

 the toxicity alone continues, and the fatal dose gradually and steadily 

 increases in its neutralizing value. 



With greater information Ehrlich has had to modify greatly the 

 details of his explanation of the reason of the variation in the ratio 

 between toxicity and neutralizing value of toxin. He now accepts the 

 fact that diphtheria culture fluid contains at least two toxins. 



Partial Saturation Method of Study. Much additional information 

 concerning the nature of toxin has been gained by experimenting with 

 mixtures of toxin and antitoxin, in which the two are present in varying 

 proportions. This is the "partial saturation" method of Ehrlich. 

 Through a number of experiments Ehrlich obtained information which 

 permitted him to estimate that 200 "binding units" are represented 

 in the amount of diphtheria toxin (hypothetically pure) which is exactly 

 neutralized by one antitoxin unit. If the entire amount of antitoxin 

 i. e., 200/200, is added to the amount of toxin in question, complete 

 neutralization of the latter, of course, occurs. In case the toxin is entirely 

 pure, 199/200 of the antitoxin unit would destroy all but 1/200 of the 

 initial toxicity; and 150/200, or 100/200, or 75/200, etc., of the antitoxin 

 when added would permit corresponding degrees of toxicity to be 

 demonstrated through animal inoculations.- It was found, however, 

 that neutralization according to this simple scale did not take place. 

 The results were complicated, and Ehrlich has found it convenient to 

 express them graphically in the form of the "toxin spectrum." For 

 example, let 199/200 of the antitoxin unit be added to the proper amount 

 of the toxin, 198/200 to another similar amount, 197/200 to another, etc., 

 down to 150/200. In the last mixture, 50 out of the 200 binding units 

 which the toxin possesses are free, and these 50, rather than some other 

 50, are free because they have less affinity for the antitoxin than the 150 

 units which were bound. It has been found that those units which 

 first become free are much less toxic than a corresponding amount of 

 the original toxin. It was thought that they might have lost their toxo- 

 phore groups i. e., that they were toxoids; and because of their weak 

 affinity for antitoxin they were called epitoxoids. It was found, how- 

 ever, that they possessed a rather constant though low degree of toxicity 

 and that the toxic action was characteristic. Injection was followed 

 by some local oedema, then by a long incubation period, and finally by 

 cachexia and paralysis. On account of this characteristic toxic action 

 and the long incubation period, Ehrlich has concluded that the so-called 

 epitoxoid is in reality a separate toxin secreted by the diphtheria 

 bacillus. 



