Contribution to Study of Diphtheria Toxin. 



25 



(as estimated by the poured plate method), and at the same time 

 the toxicity of the broth, free from organisms, was estimated. 



The growth curve had the characteristics of those reported 

 at various times in the literature for other organisms; i.e., a period 

 of logarithmic growth, and then a continued rise which in general 

 reached a maximum in 48 hours, a sudden falling off of the number 

 of viable organisms and then a more gradual decrease, with some- 

 times small increases in the number of viable organisms during the 

 period of decrease similar to the observations of Graham-Smith 1 

 for staphylococcus. It is suspected that these small secondary 

 increases may only be apparent, and are due to the method of 

 counting. The toxicity of the broth was very low at the end of 

 24 hours (approx. 1 M.L.D. per 0.5 c.c), with a small increase at 

 the end of 48 hours and then a rapid increase (if the increase and 

 decrease of the cells was characteristic), and after a maximum was 

 reached a falling off in toxicity which was sometimes very sudden. 

 This sudden falling off in toxicity has also been reported by 

 Bunker. 2 



The hydrogen-ion concentration of the broth during growth 

 showed an initial increase and then a gradual falling off ; this agrees 

 with the findings of Bunker 2 and Davis. 3 Bunker in his paper 

 gives the limits of maximum toxicity at P H 7.8 — 8.25; contrary 

 to this, good toxin has continued to be formed at P H 8.7 



There are several ways in which the above results may be 

 interpreted : 



1. Reproduction and toxin production do not go on at the 

 same time; a cell produces toxin when it is incapable of division; 

 apparently it is not accumulation of toxin which inhibits repro- 

 duction since toxin — 200 M.L.D's per c.c. — will support growth 

 when replanted with B. diphtheria. 



2. A non-toxic substance may be produced during the period 

 of cell division which is transformed into toxin; or this non -toxic 

 substance acts on some constituent of the broth and produces 

 toxin. Non-toxic germ-free broths (after organisms had grown 

 f or less than 24 hours) which were sterilized in various ways — 



j 1 Graham-Smith, Journal of Hygiene, 1920 (19). 

 ^ 2 Bunker, Journal of Bacteriology, 1919, iv. 

 3 Davis, Journal of Bacteriology. 1920, v. 



we 



