Conditions of the Halo Formation Around Subtilis Colonies and Growth of Halo Transplants

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An interesting phenomenon was described which certain B. subtilis strains show when grown on nutrient agar containing a small amount of saccharose. The amorphous, stringy material which is present in abundance in the cultures as an intercellular substance spreads out from the colonies and forms a wide halo around them. In the halo bacteria are not visible and transplants of it do not grow on the usual media. Transplanted on agar plates containing saccharose the halo substance grows in tiny transparent colonies. The observations described in the following note furnish convincing evidence that these growth phenomena are caused by the growth of living elements entirely different in morphology from the usual forms of bacteria. The observations here described concern the conditions under which the halo develops and which determine the abundance of growth in the transplants. The formation of the halo depends on the addition of saccharose to the nutrient agar. Usually as small an amount as 0.02% is sufficient and about 0.1% gives the best results. It is probable that the saccharose itself is effective and not an impurity in the saccharose preparation; because, by fractionation of saccharose with alcohol, no fractions were obtained which had increased or decreased potency. Other sugars have no effect on the halo formation. The formation of the halo depends largely upon the condition of the surface of the plate. The production of the halo can be greatly improved by rubbing the surface of the agar plate lightly with a glass rod. The different batches of agar vary very slightly and the addition of different animal proteins, fresh plant tissue, cystein, etc., produced no effect. When the pH is varied between 6 and 8.5 there are no marked changes.