428 THE DIPHTHERIA BACILLUS GROUP 
4. Incubation. — It requires from seven to ten days' incubation at 
37° C. for the maximum accumulation of toxin. Deterioration of the 
toxin after this time sets in, and the formation of new toxin fails to 
keep pace with the recession in potency of the toxin ah-eafly formed.^ 
Storage of Toxin. —At the end of the period of incubation carboHc 
acid or other preservative is added to the broth to kill the bacilli; 
they rapidly settle out, leaving a clear supernatant fluid free from 
bacteria, containing the toxin, which is either decanted off from the 
bacilli or filtered through unglazed porcelain to remove the bacteria. 
It is then stored in amber bottles which are completely filled and 
kept in cold storage. Under these conditions the toxin deteriorates 
comparatively slowly. 
Testing Toxin.— Toxin produced by the diphtheria bacillus kills 
the ordinary laboratory animals, guinea-pigs, rabbits, dogs, and birds; 
but it is practically without effect upon rats and mice, unless the 
toxin is injected directly into the nervous system. The general method 
of testing the potency of the toxin is to inject successively smaller 
graduated doses of it subcutaneously into guinea-pigs of 250 gm. 
weight and observe the results. The smallest amount of a toxin 
which will kill a guinea-pig weighing 250 gm. in four days is designated 
the minimal lethal dose (M. L. D.). The minimal lethal dose varies 
considerably with different strains of bacilli; in general it varies from 
0.25 cc. to 0.001 cc. The injection of a M. L. D. of toxin leads to an 
edematous swelling at the site of inoculation and the animal soon 
exhibits generalized symptoms as well; the temperature rises, the 
respirations are hurried, and death ensues from the results of the 
toxemia. The more acute the death, the less striking the symptoms 
and lesions. Guinea-pigs which have died on the fourth day exliibit 
a marked congestion of the abdominal and thoracic viscera and of 
the colon. A hemorrhagic infiltration and enlargement of the supra- 
renals is almost pathognomonic. Frequently the stomach wall is 
markedly injected with blood and small ulcerations are demonstrable 
in the mucosa.- The lesions present the same general appearance 
when both toxin and bacilli are injected, but a false membrane, 
composed of bacteria and a fibrino-purulent exudate, forms at the 
site of inoculation. The bacilli do not spread to other parts of the body, 
however, but remain strictly localized. The changes in the visceral 
organs are attributable to the absorption of the toxin. A sub-lethal 
dose of toxin or an attenuated culture of diphtheria bacilli does not 
cause death; an ulcer forms at the site of inoculation which eventually 
sloughs away and is completely replaced by scar tissue. 
Constitution of Diphtheria Toxin.— The composition of diphtheria 
toxin is unknown, although many investigations have been made upon 
it. Attempts to demonstrate that the toxin is non-protein in nature 
by growing the organisms in protein-free media have not been con- 
1 Theobald Smith: Jour. Exp. Med., 1899, 4, 373. 
2 Rosenau and Anderson: Jour. Inf. Dis., 1907, 4, 1. 
