138 LABORATORY COURSE IN SERUM STUDY 



the presence of free, unneucralized toxin by its death, just as 

 phenolphthalein in. acid-alkali titrations indicates the presence of 

 free alkali by a pink color. Since guinea pigs of different weights 

 and ages have varying resistances against the poison, a standard 

 weight for such measurements, namely 250 grams, was established. 



The first step toward such a standardization, of course, con- 

 sisted in establishing standards of measurement for toxin. Beh- 

 ring, later together with Ehrlich, established as a toxin unit or 

 MLD (minimal lethal dose) the amount of a toxin solution which 

 would kill a guinea pig of 250 grams. Because of the importance of 

 the time element this was later modified to represent the amount 

 which would kill such a guinea pig in from four to five days. 



The antitoxin unit later established mainly by the efforts 

 of these two workers was designated as the amount of the anti- 

 toxin, i.e. of the serum of a toxin immune animal, which would 

 neutralize 100 such MLD's (minimal lethal doses) for guinea pigs 

 of the standard weight. It was soon found by these workers, as 

 well as by others, that it was not easy to determine the exact 

 point of neutralization, that is, while the guinea pig might be 

 preserved from death from 100 such minimal lethal doses, in one 

 case in another slight local or systemic symptoms might easily 

 escape the observation of the investigator, and it seemed safer to 

 eliminate the personal equation entirely. This was done by es- 

 tablishing as an antitoxin unit not the amount that would neutral- 

 ize the 100 fatal doses, but a partial neutralization measured in 

 such a way that the toxin quantity left over in the mixture would 

 still kill the guinea pig in four to five days, giving the same effect 

 as an unneutralized single toxin unit. 



Subsequent developments in the investigation of toxins, espe- 

 cially by Ehrlich, disclosed many difficulties in the path of such 

 a relatively simple method of titration. It is a comparatively 

 easy matter to determine the minimal lethal dose of any poison 

 that one may have produced. However, this quantity or 

 MLD will not be identical in one and the same toxin filtrate 

 if this is measured at intervals of a few weeks or months, since 

 the true toxin is gradually converted into a non-poisonous prod- 



