76 TOXIN AND ANTITOXIN 



Guinea-pigs surviving a dose of toxin may after two to four weeks begin to show 

 paresis first of the hind, then of the fore extremities, and finally even of the muscles of 

 the back and respiration. The most severe types of such conditions, however, may 

 fully subside. They may be considered as analogous to the post-diphtheritic paral- 

 ysis taking place in man, which is usually of a benign nature. 



Besides guinea-pigs other animals suitable for diphtheria experimental work 

 are rabbits (especially by intravenous injection) and pigeons (by intramuscular 

 injection). 



The susceptibility of animals towards diphtheria toxin varies greatly, as is seen 

 from the following scale of Behring, the least susceptible animals being mentioned first: 

 mouse, rat, dog, guinea-pig, rabbit, sheep, cow, horse, goat. 



The strength of the diphtheria toxin is estimated as follows: 

 Estimation Guinea-pigs of equal size (250 gms.) receive subcutaneous 

 of Strength injections of decreasing amounts of toxin. With a strong 

 of Diphthe- toxin, centi- and milligrams or even smaller quantities are 

 na Toxin. o f su ffi c i en t potency to produce death. Doses such as 

 these are not injected unless diluted in normal salt solution. 

 For exact results one must not depend upon the findings from the 

 injection of a single animal with each dilution; several should be in- 

 oculated with the same dose and the effects, which should be the same in 

 all cases, noted. It is impossible to state beforehand how many dilutions 

 may be necessary. If the various actions dependent upon the successive 

 gradations of dosage are successfully represented, the experiment may be 

 taken as conclusive; that is to say, the smallest doses must leave the animal 

 entirely unaffected, the moderate produce slight local and general symp- 

 toms, and the larger ones cause death of the animals. If it should so 

 happen that they all die, a new set of experiments employing a lower 

 scale of dosage should be undertaken. 



Thus it is seen that the action of diphtheria toxin is subject to the quantity 

 of the toxin injected. If several different diphtheria toxins are tested at 

 the same time, it is at once evident what far reaching differences may 

 arise. While o.ooi c.cm. of one diphtheria toxin kills a guinea-pig in 

 twenty-four hours, a different diphtheria toxin will do the same 

 with a dose ten times as great, e.g., o.oi c.cm. The second toxin thus 

 contains only one-tenth as many of the active substances. In order to 

 obtain a uniform method for estimating the strength of a diphtheria toxin 

 and thus get comparative values, a standard unit has been adopted. And 

 this consists of the smallest amount of toxin that will kill a healthy 

 guinea-pig weighing about 250 gms. in four to five days. This is known 

 as the minimum lethal dose or dosis letalis minima. In addition to this 

 "direct toxic value/' it is frequently important, especially for the stand- 

 ardization of curative sera, to estimate the " indirect toxic value" 

 by which is meant the amount of antitoxin which a toxin can bind or 

 neutralize. 



