42 THE PRINCIPLES OF IMMUNOLOGY 



toxin to induce active antitoxin production by the patient's own 

 body. In the work with animals the injections are given at inter- 

 vals of a few days, sometimes interspersed with rest periods of 

 about a week, until testing of small amounts of serum shows that a 

 satisfactory result has been attained. 



Technic of Producing Diphtheria Toxin, and Antitoxin. Some details of 

 the technic of producing diphtheria antitoxin may well serve as an example of the 

 general phases of the method. It was early noted that different strains of the 

 diphtheria bacillus produced toxins of variable strength and also that the same 

 strain showed slight variation. Finally the strain isolated by Park and Williams, 

 now well known as Park No. 8, a strong toxin producer, was selected as a standard 

 and is so used throughout the world. In order to obtain the best aerobic conditions, 

 a wide-bottom flask is employed so as to expose a large surface of the medium, 

 "bob" veal broth being selected as most desirable. The culture is planted super- 

 ficially and allowed to grow for seven or eight days. It is wise to use a culture that 

 has been grown for several generations on the surface of broth tubes, the tubes 

 so inclined as to expose a large broth surface. This accustoms the organisms to 

 freely aerobic conditions. After the period of growth in the flask the organisms 

 are killed by formaldehyde or by phenol, or even by heat, and the broth filtered 

 either through paper or through a porcelain filter, preserved with toluol and per- 

 mitted to " ripen." This ripening is made desirable because of progressive deteriora- 

 tion of the toxin, a phenomenon which will be more profitably taken up in the 

 discussion of the toxin-antitoxin combination. 



The following table shows a scheme as actually practised for producing 

 diphtheria antitoxin. All the injections are subcutaneous. 



INJECTION SCHEME FOR PRODUCTION OF DIPHTHERIA ANTITOXIN 

 July 22 6,000 units antitoxin 



25 400 minimum lethal doses of toxin (see page 45) 

 27 800 minimum lethal doses of toxin 



29 1,200 minimum lethal doses of toxin 



Aug. i i, 600 minimum lethal doses of toxin 



3 2,000 minimum lethal doses of toxin 



5 2,500 minimum lethal doses of toxin 



8 3,000 minimum lethal doses of toxin 



10 3,600 minimum lethal doses of toxin 



12 4,400 minimum lethal doses of toxin 



15 5,200 minimum lethal doses of toxin 



17 6,200 minimum lethal doses of toxin 



19 7,200 minimum lethal doses of toxin 



22 8,500 minimum lethal doses of toxin 



24 10,000 minimum lethal doses of toxin 



26 13,000 minimum lethal doses of toxin 

 29 16,000 minimum lethal doses of toxin 

 31 20,000 minimum lethal doses of toxin 



Sept. 2 24,000 



5 28,000 



7 32,000 



9 36,000 



12 40,000 



Lengthen intervals by 24 hours if made 

 necessary by severe reaction. 



Trial bleeding separation of and testing of serum, September 21. After that 

 if further immunization is necessary the dose is raised 5000 M.L.D. each injection. 



The Nature of Antitoxins. The serum thus obtained contains 

 the antitoxin. The exact nature of the antitoxin is unknown, but 

 chemical examination and other studies have thrown a certain 

 amount of light upon its properties. If we can accept the division 

 of the serum protein into fibrinogen, euglobulin and pseudo-globulin 

 by precipitation with magnesium sulphate or ammonium sulphate, 

 the antitoxin is found in that water-soluble fraction known as 



