DIPHTHERIA BACILLUS. 345 



temperatures only alter it very gradually. Kept from 

 light and air and in cold storage it keeps almost un- 

 altered for years. 



The Production of Toxin in Culture Media. The arti- 

 ficial production of toxin in cultures of the diphtheria 

 bacillus has been found to depend upon definite condi- 

 tions, which are of practical importance in obtaining 

 toxin for the inoculation of horses, and also of theoret- 

 ical interest in explaining why cases of apparently equal 

 local severity have such different degrees of toxic ab- 

 sorption. 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 Williams) came to the following conclu- 

 sions : Toxin is produced by fully virulent diphtheria 

 bacilli at all times during their life when the conditions 

 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. Diphtheria ba- 

 cilli may find conditions suitable for luxuriant growth, 

 but unsuitable for the production of toxin. The re- 

 quisite conditions for a good development of toxin, as 

 judged by the behavior of a number of cultures, are a 

 temperature from about 35 to 37.5 C., a suitable cul- 

 ture 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 

 and meat. The culture fluid should be in compara- 

 tively thin layers and in large-necked Erlenmeyer 

 flasks, so as to allow of a free access of air. The 



