THE DIPHTHERIA BACILLUS 393 



observation that the presence of muscle-sugar (dextrose), commonly 

 found in small amounts in meat-juice, prevented the formation of 

 diphtheria toxin; he demonstrated conclusively that small amounts 

 of dextrose (less than 0.2 per cent.) delay the appearance of toxin; 

 in sugar-free broth toxin production increases with the growth of the 

 organisms. Diphtheria toxin is formed from the protein constituents 

 of the medium; when utilizable carbohydrate (dextrose) is present 

 in the medium, the bacilli ferment it instead of attacking the protein. 1 

 It is customary to add 0.1 per cent, of dextrose to broth for the pro- 

 duction of diphtheria toxin; the initial development of the bacilli is 

 greater, and this amount of dextrose is rapidly used up, leaving greater 

 numbers of organisms to form toxin from the protein constituents. 

 The culture must be grown at 37 C. to insure a potent toxin. 



2. Oxygen. Free oxygen is an essential factor in the production 

 of toxin. It is customary to distribute the broth in shallow layers 

 with a relatively large surface exposed to the air. 



3. Pellicle. Cultures of diphtheria bacilli which grow habitually 

 on the surface of fluid media must be used for the preparation of 

 toxin. Diphtheria bacilli can be "trained" to develop on the surface 

 by repeated transfers in broth. 2 Surface development insures a 

 maximal exposure of the bacilli to the air. 



4. Incubation. It requires from seven to ten days' incubation at 

 37 C. for the maximum accumulation of toxin. Deterioration of the 

 toxin after this time sets in, and the formation of new toxin fails to 

 keep pace with the recession in potency of the toxin already formed. 3 



Storage of Toxin. At the end of the period of incubation carbolic 

 acid or other preservative is added to the broth to kill the bacilli; 

 they rapidly settle out, leaving a clear supernatant fluid free from 

 bacteria, containing the toxin, which is either decanted off from the 

 bacilli or filtered through unglazed porcelain to remove the bacteria. 

 It is then stored in amber bottles which are completely filled and 

 kept in cold storage. Under these conditions the toxin deteriorates 

 comparatively slowly. 



Testing Toxin. Toxin produced by the diphtheria bacillus kills 

 the ordinary laboratory animals, guinea-pigs, rabbits, dogs, and birds; 

 but it is practically without effect upon rats and mice, unless the 

 toxin is injected directly into the nervous system. The general method 



1 Kendall, Boston Med. and Surg. Jour., 1913, clxviii, 825. 



2 Theobald Smith, Jour. Exp. Med., 1899, iv, 392. 



3 Ibid. 



