NATURE OF THE TOXIN. 367 



this means the period of acid reaction was shortened and the 

 toxin formation favoured. This expedient is now considered 

 unnecessary if an alkaline medium free from glucose is used, as 

 in this no acid reaction is developed : it is then sufficient to grow 

 the cultures in shallow flasks. The absence of glucose an all- 

 important point may be attained by the method described 

 above (p. 80), or by using for the preparation of the meat extract 

 flesh which is just commencing to putrefy (Spronck). L. Mar- 

 tin uses a medium composed of equal parts of freshly pre- 

 pared peptone (by digesting pigs' stomachs with HC1 at 35 

 C), and glucose-free veal bouillon. In this medium he has 

 obtained a toxin of which -g-J-g- c.c. is the fatal dose to a guinea- 

 pig of 500 grms. He finds that glucose, glycerin, saccharose, 

 and galactose lead to the production of an acid reaction, whilst 

 glycogen does not. The latter fact explains how some ob- 

 servers have found that bouillon prepared from qitite fresh flesh 

 is suitable for toxin formaticjn. There is in all cases a period at 

 which the toxicity reaches a maximum, usually in 2-3 weeks, 

 occurring earlier the more rabidly the toxin is formed ; later the 

 toxicity diminishes. Martin found that in his medium the maxi- 

 mum was reached on the 8th-ioth day. It may be added that 

 the power of toxin formation varies much in different races of 

 the diphtheria bacillus, and that many may require to be tested 

 ere one suitable is obtained. 



Properties and Nature of the Toxin. The toxic substance 

 in filtered cultures is a relatively unstable body. When kept in 

 sealed tubes in the absence of light, it may preserve its powers 

 little altered for several months, but, on the other hand, it grad- 

 ually loses them when exposed to the action of light and air. 

 Heating at 58 C. for two hours destroys the toxic properties in 

 great part, but not altogether. When, however, the toxin is 

 evaporated to dryness, it has much greater resistance to heat. 

 One striking fact, discovered by Roux and Yersin, is that after 

 an organic acid, such as'tartaric acid, is added to the toxin the 

 toxic property disappears, but that it can be in great part re- 

 stored by again making the fluid alkaline. 



The toxic body in filtered cultures can be precipitated by 

 alcohol, and is also carried down by calcium phosphate. It is, 

 however, soluble in water and dialyses somewhat slowly through 

 animal membranes. By repeated precipitation and again dis- 



