1. The growth of diphtheria bacilli in suger-free pure casein digest broth may be accelerated by the addition of dilute nitric, acetic and lactic acids, and delayed by certain monobasic fatty acids.2. The limiting acid zone for toxic and non-toxic varieties is unaffected by initial dilute acidifications by several acids, but incresaed by the addition of citric acid.3. The time periods and intensity of the reaction changes produced by diphtheria bacilli growing in a sugar-free casein digest medium are altered by the addition of certain acids. Toxic strains produce earlier and more progressive alkalinity when dilute acetic or nitric acid is included in the medium: and later and lessened alkalinity in the presence of butyric, formic, and propionic acids. Certain other typical acids do not exert any obvious action. Non-toxic strains are less affected by the selected acids, although butyric acid generally retards reaction changes.4. The time required for virulence testing is decreased, and the potency of the bacillus under examination is more evident when 1/200 η acetic or nitric acid is added to the culture medium. Acidified casein digest plus peptone broth yields practically the same results as acidified fermented veal peptone infusion. The acidification does not affect the metabolism of non-toxic strains.5. The disadvantages of unfermented meat infusions and the ascribed harmful effects of the fermentation products in fermented meat broths, may perhaps be avoided by the substitution of casein digest peptone medium. The advantages of the starting action of the glucose may be retained by adding acetic or nitric acid to the casein digest.6. Individual strains of diphtheria bacilli differ considerably in their capacity of altering the reaction of culture media, and in their toxic potency. It does not seem permissible to apply to newly isolated organisms all the published results obtained from cultures that have been repeatedly subcultured and established as toxin producers in nutrient bouillon. As a presumptive test in the virulence testing of bacilli just transferred from human tissues to artificial culture media the tendency of non-toxic strains to produce early formation of alkali in adequately buffered and adjusted unfermented veal broth may be thought worthy of extended trial.
