Studies on Cultural Requirements of Bacteria: IX. Tissue Extractives in the Growth of the Diphtheria Bacillus.

It was shown in the sixth paper of this series (Mueller, 1935c) that the organic substances present in a tissue extract (liver) which are essential for the growth of a strain of diphtheria bacillus, could be very considerably purified. The method consisted in vacuum concentration of a hot aqueous extract, precipitation with alcohol up to 95 per cent concentration, and removal of the alcohol in vacuo. The solution was precipitated with neutral lead acetate, the precipitate discarded and excess lead removed from the filtrate with H2S. The resulting material was adsorbed twice with norit charcoal, and the latter boiled out three times with acid 50-per-cent ethyl alcohol. Evaporation in vacuo until the alcohol was removed completed the preparation. The resulting concentrate is a clear, light brown limpid solution, acid in reaction and with an unpleasant bitter taste. One cubic centimeter is equivalent to about 85 grams of liver, and contains 41.0 mgm. solids with 3.9 mgm. nitrogen. (The point to which evaporation may be carried is entirely a matter of convenience.) From 0.1 cc. to 0.2 cc. of this solution, added to 10 cc. of otherwise extractive-free media containing suitable amino acids, inorganic salts and a source of energy, i.e., glycerol or sodium lactate, induces maximum growth with practically all the strains of Coryne-