DIPHTHERIA TOXIN AND ANTITOXIN 487 



cent of the total number) gave serum ranging from 200 to 1000 

 units, 20 per cent (15.6 per cent of the total number) yielded the 

 high potency products from 1000 to above 1500 antitoxin units, 

 and 44 per cent (34.3 of all of the treated horses) came within the 

 moderately high range from 500 to 1000 a.u. The foregoing 

 and the fact that it has been possible in routine operation to im- 

 munize horses to an antitoxin strength exceeding 1500 units per 

 cubic centimeter, can be taken as proof that the toxin used is 

 fully satisfactory to meet all requirements of diphtheria anti- 

 toxin-production . 



SUMMARY 



1. A resume of the more important literature on the production 

 of diphtheria toxin is given This shows wide divergence of pro- 

 cedure. The recent scarcity of Witte peptone and failure of 

 many substitute products to allow of appreciable toxin formation 

 has further complicated the methods employed. 



2. The essentials for the routine production of high potency 

 diphtheria toxin are discussed. It is shown that, other condi- 

 tions being the same, the toxicogenicity of the culture employed 

 may vary within wide limits, according to the source. Th^ neces- 

 sity of verifying toxin production with any new strain is made 

 apparent. 



3. Plain beef infusion bouillon, containing peptone and salt 

 is recommended for toxin production. Preliminary fermenta- 

 tion of the infusion with a culture of Bad. coli is shown to be un- 

 desirable. The use of veal infusion in place of beef is unnecessary. 

 Even traces of fat must be avoided in the infusion as it interferes 

 with maximum pellicle formation and thus diminishes toxin 

 elaboration. 



4. A content of 2 per cent peptone with 0.5 per cent of salt 

 in the bouillon has been found to be most satisfactory. 



5. Maximum strength of the final toxin has been obtained 

 when the reaction of the broth comes within the limits of pH + = 

 8.0 to pH+ = 8.2. A procedure is given for adjusting the hy- 

 drogen ion concentration to these values. 



6. Cultivation for toxin production is best made in large 

 flasks, previously inoculated with twenty-four hour cultures in 



