366 DIPHTHERIA. 



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 toxine 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 and to start the growth on the 

 surface so that a thick pellicle forms. (The latter can be 

 readily effected by having small fragments of cork floating 

 on the surface.) The absence of glucose an all important 

 point may be attained by the method described above 

 (p. 87), 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 

 H.C1 at 35 C.), and glucose-free veal bouillon. In this 

 medium he has obtained a toxine of which -i^ c.c. is the 

 fatal dose to a guinea-pig of 500 grms. He finds that 

 glucose, glycerine, 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 

 toxine formation. There is in all cases a period at which 

 the toxicity reaches a maximum, beyond this it begins to 

 fall. This varies in different cases, occurring earlier the 

 more rapid the toxine is formed. Martin found that in his 

 medium the maximum was reached on the 8th-ioth day. 

 It may be added that the power of toxine 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 Toxine. The toxic sub- 

 stance in filtered cultures is a relatively unstable body. 

 When kept in sealed tubes in the absence of light, it may 



