THE DIPHTHERIA BACILLUS 427 
nitol are not fermented. Neither indol nor phenols are formed in 
sugar-free broths,' but small amounts of ammonia are produced 
in this medium, the amount increasing with the age of the culture. - 
Enzymes. — y.0 enzymes acting upon ])roteins, carbohydrates or fats 
have been detected in cultures of the organism. 
Toxin.— The most important and characteristic of the })roducts 
formed by the diphtheria bacillus is a potent, soluble (extracellular) 
toxin. The potency of the toxin Aaries somewhat with the culture 
used, some strains producing more than others. An occasional strain, 
typical in other respects, fails to form toxin. Prolonged cultivation 
of the organism in artificial media may lead to a diminution in toxin- 
producing capacity, but this is by no means a general rule. Williams'* 
isolated a diphtheria bacillus from a mild case of tonsillar diphtheria 
(No. 8) which has retained its toxin-producing power unimpaired up 
to the present time. This culture is widely used throughout the world 
in the commercial preparation of diphtheria antitoxin. 
Conditions Favoring the Production of Diphtheria Toxin. — 1. Com- 
position of the Medium. — Park and ^Yilliams^ found that 2 per cent 
of peptone added to meat infusion broth increased the yield of toxin 
very materially, and Theobald Smith^ made the very important 
observation that the presence of muscle-sugar (glucose), commonly 
found in small amounts in meat-juice, prevented the formation of 
diphtheria toxin; he demonstrated conclusively that small amounts 
of glucose (less than 0.2 per cent) delay the appearance of toxin; 
in sugar-free broth toxin production increases with the growth of the 
organisms. Diphtheria toxin is formed from the protein constituents 
of the medium; when utilizable carbohydrate (glucose) is present 
in the medium, the bacilli ferment it instead of attacking the protein.*"' 
It is customary to add 0.1 per cent of glucose to broth for the pro- 
duction of diphtheria toxin; the initial development of the bacilli is 
greater, and this amount of glucose is rapidly used up, leaving greater 
numbers of organisms to form toxin from the protein constituents. 
The culture must be grown at 87° C. to insure a potent toxin. 
2. Oxygen.— Free oxygen is an essential factor in the production 
of toxin. It is customary to distribute the broth in shallow layers 
with a relatively large surface exposed to the air. 
3. Pellicle. — Cultin-es of diphtheria bacilli which grow habitually 
on the surface of fluid media must be used for the preparation of 
toxin. Diphtheria bacilli can be "trained" to develop on the surface 
by repeated transfers in broth.'' Surface development insures a 
maximal exposure of the bacilli to the air. 
1 .Jour. Exp. Med., 1897, 2, 54.3. 
- Kendall, Day and Walker: Jour. Am. Chcm. Soc, lfli:3, 35, 1210. 
3 Jour. Med. Res., 1902, 8. 8.3. 
* Jour. Exp. Med., 1896, 1, 164. 
5 Trans. Assn. Am. Phys., 1896: Jour. Exp. Med., 1N99, 4, .373. 
« Kendall: Boston Med. and Surg. Jour., 1913, 168, S25. 
' Theobald Smith: Jour. Exp. Med., 1899, 4, 373. 
