TOXINS AND ANTITOXINS 45 



differences in bacterial strains and variations in the same strains 

 growing under even slightly different conditions. Ehrlich sought to 

 reduce the factor of error by determining the antitoxic " unit " as 

 the amount of antitoxic serum necessary to protect against ten times 

 the minimum lethal dose. Even this was unsatisfactory, and Behring 

 and Ehrlich in collaboration settled upon an arbitrary method of 

 determining a " normal " toxin and a " normal " therapeutic serum. 

 The "normal" toxin contained in i.o c.c. one hundred times the 

 minimum lethal dose for a guinea-pig of 250 grams. The " normal " 

 therapeutic serum was tested and diluted so that o.i c.c. contained 

 sufficient antitoxin to neutralize i.o c.c. of the "normal" toxin or, 

 in other words, i.o c.c. antitoxic serum, as a unit, was capable of 

 neutralizing 100 minimum lethal doses of toxin. The fundamental 

 error, however, had not been overcome by this method, and it was 

 found that no method which had as its basis a toxin, could be ap- 

 plied over a large area and the method was finally abandoned. 

 The toxin deteriorates rapidly on standing, and even though 

 after a time it becomes fairly stable, it still is insufficiently so 

 to justify its use for purposes of standardization. On the other 

 hand, the antitoxic serum resists drying for an indefinite period, and 

 if used as a standard can be shipped great distances. The standard 

 in this country has been established by the United States Public 

 Health Service. The unit of antitoxin as now used has no direct 

 relation to the unit of " normal " therapeutic serum as defined by 

 Behring and Ehrlich, but by interchange between nations it is 

 practically constant throughout the world. Hence, if a laboratory 

 wishes to prepare an antitoxin the standard unit of antitoxin can be 

 obtained from the Public Health Service. With the standard anti- 

 toxin on hand, the antitoxic content of a newly prepared antiserum 

 may be determined. This must be done through the medium of a 

 toxin whose strength is titrated against the standard antitoxin ; the 

 toxin is thereby standardized, so that the strength of the new anti- 

 toxic serum can be measured. The toxin must be one which has 

 been ripened, so that any deterioration during the few days' time 

 necessary for titration against the standard immune serum and then 

 against the new serum, is reduced to a negligible minimum. In 

 order to make the titration against the standard antitoxic unit some- 

 what easier it is well to know the minimum lethal dose of toxin. 

 There are then to be determined : 



1. The minimum lethal dose of toxin (M.L.D.). 



2. The L dose of antitoxin. 



3. The L + dose of antitoxin. 



The minimum lethal dose of toxin is determined by injecting subcuta- 

 neously, varying doses of toxin into a series of guinea-pigs 250 grams in 

 weight. Healthy pigs of this weight are usually young and less expensive 

 than fully-grown animals. The M.L.D. is the smallest dose that kills a 

 pig in from four to five days. Less than four days means too great strength, 



