P DIPHTHERIA TOXIN 257 



3. Toxin. 

 Hphtheria, as Roux an,d Yersin showed, is an intoxication with the highly 

 poisonous products of the diphtheria bacillus. These products, as the same 

 observers proved, are also present in broth cultures of the bacillus. 



In their first experiments Roux and Yersin were only able to manufacture 

 a very weak toxin of which 30 c.c. were required to kill a guinea-pig. Martin 

 now prepares a toxin which is fatal to adult guinea-pigs in doses of ^J^ c.c. 

 and even -^ c.c. [Toxins even more powerful than this have been prepared. ] 



(a) Preparation of diphtheria toxin. 



Conditions under which toxin is elaborated. Diphtheria toxin is obtained 

 by growing a toxigenic 1 bacillus in presence of air. 



A. Selection of the organism. The strain to be used for the preparation of 

 toxin should first of all be tested on animals. To be suitable for the purpose, 

 1 c.c. of a broth culture of the organism should when inoculated beneath the 

 skin of a guinea-pig weighing 300-400 grams prove fatal in 24-36 hours. 

 But as it has been found that bacilli isolated from very severe cases of diph- 

 theria are not always powerfully toxigenic [and on the other hand some 

 bacilli are toxigenic and non-virulent] the toxigenic capacity of the organism 

 should always be tested before embarking upon the manufacture of large 

 quantities of toxin. [At the present time a bacillus known as Park and 

 Williams' bacillus No. 8 is extensively used for the preparation of toxin. 

 Park and Williams recovered this organism from a mild tonsillar case of 

 diphtheria. ] 



To preserve the toxigenic properties of a diphtheria bacillus sow it on Martin's 

 broth, and after incubating for a week at 33 35 C. remove the tubes from the 

 incubator and keep them in the dark. Martin keeps 48-hour cultures on coagulated 

 serum at 10-12 C. Old cultures stored in this way on being revived by a couple 

 of sub-cultivations yield a bacillus which has a very considerable power of toxin 

 production. 



B. The choice of medium. The amount of toxin produced depends upon 

 the composition and especially upon the reaction of the medium. 



It has already been pointed out that when the diphtheria bacillus is culti- 

 vated upon an alkaline broth the medium is first turned acid but after a few 

 days again becomes alkaline ; and it is just when the acid reaction begins to 

 diminish that the toxin begins to be formed, after that the toxicity of the 

 broth increases pari passu with the alkalinity of the medium, and the more 

 rapidly the alkalinity increases the more rapidly does the amount of toxin 

 increase. If the formation of acid be prevented toxin will be formed both 

 more rapidly and also in larger quantity. As the result of experiment many 

 methods have been devised for diminishing or altogether preventing the 

 initial formation of acid. Roux, Yersin and Martin shortened the period 

 of acid reaction by growing in a current of air, and this method has been 

 used for a long time in the preparation of toxin on a large scale. Many 

 observers have aimed at excluding from the culture medium all those sub- 

 stances (e.g. glucose, laevulose, saccharose, galactose, glycerin) from which 

 the diphtheria bacillus forms acid. Nicolle, for instance, obtained a satis- 

 factory toxin by using fresh meat meat, that is, in which the glycogen had 

 not had time to be converted into glucose (glycogen not being convertible 

 into acid) ; Spronck on the contrary suggested the use of meat which was 



[ l The disease-producing power of a diphtheria bacillus may be regarded as made up 

 of two elements, toxicity and virulence. The former represents the rate of accumulation 

 by it of toxin in culture fluids, the latter the behaviour of the bacillus towards living tissue 

 (Theobald Smith).] 



R 



