226 CLINICAL BACTERIOLOGY. 



most careful experimental observations the limit is deter- 

 mined at which just that excess of toxin becomes manifest 

 which causes death of the animal in the first four days. 

 The amount of toxin thus obtained represents the imme- 

 diate test-dose. By means of the same dose of serum, for 

 the more exact characterization of the toxin, the determi- 

 nation of a second limit is made, for the purpose of learning 

 the dose of toxin that is just neutralized by admixture with 

 the amount of serum named. 



IV. The determination of the strength of a diphtheria- 

 antitoxin is made by means of the test-dose of toxin (see 

 paragraph III) as follows: The test-dose of toxin in question 

 for instance, 0.355 cu - cm - f tested toxin at the time 

 present in the laboratory is mixed with 4 cu. cm. of anti- 

 toxin corresponding to the test-figures given. As the test- 

 dose of toxin is estimated for I cu. cm. of antitoxin of 

 normal strength, or for 4 cu. cm. of antitoxin ^ the normal 

 strength, an antitoxin of x-strength will have to be diluted 

 i^ x, and in testing an antitoxin 100 times the normal 

 strength, ^ 



V. The mixture obtained is injected unmodified sub- 

 cutaneously' into guinea-pigs weighing from 250 to 300 

 grams. If the animals die in the test-experiments made by 

 two observers in the laboratory within the first four days, 

 the antitoxin does not possess the required strength. 

 Should death occur within five or six days, the antitoxin 

 is close to the required strength, and in order to avoid the 

 early withdrawal to be anticipated an improvement of from 

 5 to 10 per cent, is recommended the manufacturers. In- 

 durations that occur in the animals experimented upon do 

 not, however, constitute sufficient ground for objection. 

 In the case of the dead animals an autopsy should be held, 

 and careful attention directed to complications with previ- 

 ously existing disease (tuberculosis, pseudo-tuberculosis, 

 and pneumonia) that may induce undue susceptibility on 

 the part of the test-animals. 



VI. Both liquid and solid toxins may be employed for 

 test-purposes, if the limits defined in paragraph III can be 

 accurately estimated, and the difference between them does 

 not exceed fifteen simple lethal doses. If liquid toxins 

 preserved in toluol are employed, this should be done 

 only if as a result of preliminary investigation the perma- 

 nency of the test-constants is demonstrated, if the test- 



