STANDARDIZATION OF ANTITOXIN 267 



and toxicity of the toxin used and upon the antitoxic strength of the serum. 

 It is very important to know the antitoxic strength of the serum used, and 

 rules have been devised by which this may be determined (vide infra). 



Antitoxic serum is not bactericidal and contains no immune body (sensibili- 

 satrice) ; it has some power of agglutinating the bacillus but only in a feeble 

 and inconstant manner. For instance, it may agglutinate in dilutions of 

 1 in 10 and 1 in 20 and the agglutination is sometimes visible to the naked 

 eye, flocculi falling to the bottom of the tube (Nicolas) ; on the other hand 

 agglutination is often absent. The serum also of patients suffering from diph- 

 theria sometimes shows some slight property of agglutination (Bruno). 



Bacilli recently isolated from the living subject are often unaffected or but slightly 

 affected by the specific agglutinin ; prolonged sojourn in artificial culture, however, 

 seems to develop or increase the power of being agglutinated. It is important in 

 testing the agglutinating properties to use a good emulsion of bacilli, and for this 

 purpose a culture in 2 per cent, glucose broth, which is generally cloudy, is suitable. 

 Martin recommends collecting the bacilli from flasks which have been used for toxin 

 preparation and heating them to 100 C. For use, shake up these bacilli well with 

 normal saline solution and let the emulsion stand : a large number of the bacilli 

 will deposit, but the supernatant liquid remains cloudy permanently and serves 

 very well for the agglutination reaction. 



Antitoxic serum is both prophylactic and therapeutic in the case of animals 

 inoculated with a living culture of the diphtheria bacillus : the therapeutic 

 properties are exhibited even if the serum be not administered until 12 or 18 

 hours after the virus. 



In the case of a guinea-pig which has been inoculated with a living culture on 

 the mucous membrane of the trachea or vulva, the inoculation of serum, even though 

 it be administered before the virus, will not prevent the formation of the charac- 

 teristic false membrane, but does entirely prevent symptoms of intoxication or of 

 disturbance of the general health of the animal : and further the false membrane 

 becomes detached on the second day and the infected surface commences to heal. 

 If instead of being inoculated before the living virus, the serum be administered 

 after the false membrane has formed, it leads to the disappearance of the oedema 

 and swelling in a few hours and after two days to the casting off of the false membrane. 



The false membranes formed in the trachea of the rabbit as the result of infecting 

 the mucous membrane with a mixture of streptococci and B. diphtheric? are not so 

 readily affected by antitoxic serum. In such a case 5, and even 10 c.c. of antitoxin 

 are insufficient to save the life of the animal : but Roux and Martin have been able 

 to effect a cure in parallel cases by repeating the inoculation of antitoxin several 

 times. 



Roux and Martin have tested the value in these cases of mixing antistreptococcal 

 and antidiphtheria serums, but with only moderately successful results (vide The 

 Streptococci). 



(c) The standardization of antitoxin. 



1. Behring estimated the antitoxic content of an antiserum in terms of 

 the amount of the serum necessary to immunize 1 gram weight of animal 

 against the minimal fatal dose of toxin inoculated 12 hours after the serum. 



Thus, for example, if 1 c.c. of a serum immunized 1 kg. weight of guinea-pig 

 against the inoculation 12 hours later of the smallest dose of toxin which would kill 

 a control animal of the same weight within a given time the serum was said to have 

 a strength of f^Q. This method of titration is not very exact but it has the 

 advantage of being simple. 



2. Behring then altered his test inoculation. Instead of toxin he used 

 living bacilli and measured the value of the serum against an infection and 

 not against an intoxication. The unit of serum, now, was the amount necessary 

 to immunize 5,000 grams of guinea-pig (or 10 guinea-pigs of 500 grams each) 

 against the inoculation 24 hours later of ten times the amount of a forty -eight- 



