BACILLUS DIPHTHEEI^ 521 



most favorable constitution of this medium for the production of toxin. 

 All agree, however, in recognizing the importance of pepton, without 

 which, according to Madsen,' no satisfactory toxin has yet been pro- 

 duced. This is added in proportions of from one to two per cent. The 

 presence of sugars in the medium is not desirable in that it leads to acid 

 production; L. Martin^ removes the sugars from the meat by fermen- 

 tation with yeast. Smith ^ accomplishes the same purpose with B. 

 coli. According to Park and Williams,* however, this is superfluous, 

 the quantity of sugar present in ordinary butcher's meat not being 

 sufficient to exert unfavorable influence. 



Experience has shown that a primary alkaUne reaction offers the 

 most favorable conditions for toxin production. In all cultures of B. 

 diphtherias in non-sugar free broth, there is, at first, a production of 

 acid and, while this continues, there is, as Spronk ' has shown, little 

 or no evidence of toxin elaboration. It is only after this initial acid- 

 ity has given way to alkalinity that cultures become decidedly toxic. 

 Park and Williams," in an inquiry into the question of reaction, came 

 to the conclusion that the best results are obtained with a broth to 

 which, after neutralization to litmus, ^ NaOH is added in an amount 

 of 7 c.c. to the liter. In such a medium the largest yield of toxin is 

 obtained after about five to eight days' growth at a temperature of 

 37.5° C. If left at this temperature for a longer period a diminution 

 in the strength of the toxin takes place. 



Free access of oxygen to the culture medium during the growth 

 of the organisms has been found to be of great importance. Roux 

 obtained this by passing a stream of oxygen through the bouillon. The 

 supply is quite sufficient for practical purposes, however, if the medium 

 is distributed in thin layers in large-necked Erlenmeyer flasks. 



Chemical Nature and Physical Properties of Diphtheria 

 Toxin. — ^The chemical composition of diphtheria toxin is not known. 

 Brieger and Frankel,' by repeated precipitation with alcohol, succeeded 

 in extracting from toxic bouiUon a white, water-soluble powder which 

 possessed most of the poisonous properties of the broth itself. This, in 



1 Madsen, Kraus und Levaditi, " Handbuch d. Technic," etc., 1907. 



2 L. Martin, Ann. de I'inst. Pasteur, 1897. 



3 Th. Smith, Jour. Exp. Med., iv, 1899. 



' Park and Williams, Joum. Exp. Med., 1897. 



^Sprtmk, Ann. de I'inst. Pasteur, 1895. 



' Park and Williams, Jour. Exp. Med., 1897. 



' Brieger und Frankel, Berl. klin. Woch., xi-xii, 1889. 



