194 BACTERIA PATHOGENIC TO MAN 



The Production of Toxin in Culture Media. The artificial production 

 of toxin in cultures of the diphtheria bacillus has been found to depend 

 upon definite conditions, which are of practical importance in obtaining 

 toxin for the inoculation of horses, and also of theoretical interest in 

 explaining why cases of apparently equal local severity have such 

 different degrees of toxic absorption. The researches of Roux and 

 Yersin laid the foundation of our knowledge. Their investigations 

 have been continued by Theobald Smith, Spronck, ourselves, and 

 others. After an extensive series of investigations we (Park and Wil- 

 liams) came to the following conclusions: Toxin is produced by fully 

 virulent diphtheria bacilli at all times during their life when the condi- 

 tions are favorable. Under less favorable conditions some bacilli are 

 able to produce toxin while others are not; or it may be that some 

 conditions favor some bacilli while they are deleterious to others. Diph- 

 theria bacilli may find conditions suitable for luxuriant growth, but 

 unsuitable for the production of toxin. The requisite conditions for 

 good development of toxin, as judged by the behavior of a number of 

 cultures, are a temperature from about 35 to 36 C., a suitable culture 

 medium, such as a 2 per cent, peptone nutrient bouillon of an alkalinity 

 which should be about 8 c.c. of normal soda solution per litre above 

 the neutral point to litmus, and prepared from a suitable peptone (Witte) 

 and meat. The culture fluid should be* in comparatively thin layers 

 and in large-necked Erlenmeyer flasks, so as to allow of a free access 

 of air. The greatest accumulation of toxin in bouillon is after a dura- 

 tion of growth of the culture of from five to ten days, according to the 

 peculiarities of the culture employed. At a too early period toxin has 

 not sufficiently accumulated; at a too late period it has begun to degen- 

 erate. In our experience the amount of muscle-sugar present in the 

 meat makes no appreciable difference in the toxin produced when a 

 vigorously growing bacillus is used, so long as the bouillon has been 

 made sufficiently alkaline to prevent the acid produced by the fer- 

 mentation of the sugar from producing in the bouillon an acidity suffi- 

 cient to inhibit the growth of the bacilli. If the sugar does interfere 

 this can be prevented by its previous destruction through the fermenta- 

 tion caused by the growth of the colon bacilli. After the fermentation 

 0.1 per cent, of glucose should be added. Besides the sugar and allied 

 bodies in the meat there are other substances whose nature is unknown, 

 which hinder or aid a full growth of the bacilli or production of toxin. 

 This is true of bouillon made directly from fresh meat, fermented meat, 

 or meat extracts. With the meat as we obtain it in New York, we get 

 better results with unfermented meat than with fermented. In Boston, 

 with the same bacillus, Smith gets more toxin from the fermented bouil- 

 lon. Instead of colon bacilli, yeast may be added to the soaking meat, 

 which is allowed to stand at about 25 C. 



Under the best conditions we can devise, toxin begins to be produced 

 by bacilli from some cultures when freshly sown in bouillon some time 

 during the first twenty-four hours; from other cultures, for reasons not 

 well understood, not for from two to four days. In neutral bouillon 



