362 DIPHTHERIA 



on a mouse, whilst of this toxin even ^ c.c. produced extensive 

 necrosis of the skin of the guinea-pig. 



Preparation of the Toxin. The obtaining of a very active 

 toxin in large quantities is an essential in the preparation of anti- 

 diphtheritic serum. Certain conditions favour the development 

 of a high degree of toxicity, viz., a free supply of oxygen, the 

 presence of a large proportion of peptone or albumin in the 

 medium, and the absence of substances which produce an acid 

 reaction. In the earlier work a current of sterile air was made 

 to pass over the surface of the medium, as it was found that by 

 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. 75), or by using for the preparation of the meat extract 

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

 uses a medium composed of equal parts of freshly prepared 

 peptone (by digesting pigs' stomachs with HC1 at 35 C.), and 

 glucose-free veal bouillon. By this medium he has obtained a 

 toxin of which y ^ 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 observers have found 

 that bouillon prepared from quite fresh flesh is suitable for toxin 

 formation. There is in all cases a period at which the toxicity 

 reaches a maximum, usually in 2-3 weeks, but earlier if the toxin 

 is rapidly formed ; later the toxicity diminishes. Martin found 

 that in his medium the maximum was reached on the 8th-10th 

 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 

 gradually loses them when exposed to the action of light and 

 air. As will be shown later (p. 472), the toxin probably does not 

 become destroyed, but its toxophorous group suffers a sort of 

 deterioration so that a toxoid is formed which has still the 

 power of combining with antitoxins. 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 



