Morphologic Elements in the Halo of Subtilis Colonies. ∗

Abstract

The most interesting question in connection with the phenomena which are the subject of the previous note 1 is whether they are life phenomena produced by the growth of living organisms or whether they represent only a peculiar combination of diffusion phenomena and enzymatic action. The number of successive transfers in which the halo substance so far was grown is not sufficiently high to exclude the supposition that the mucoid substances are synthesized in the medium by an enzyme excreted from the bacteria. The observations which will be described in this note show that the halo consists of organized elements and is not an amorphous substance. These observations furnish definite evidence for the living nature of the halo.

Direct examination of the halo under the microscope and examination of preparations stained with crystal violet reveals no formed elements in the halo. If a small piece of agar containing the deep colonies of halo is cut out and stained with crystal violet the colonies remain unstained in the deeply stained agar. These colonies often contain stained granules and sometimes fine bacillary forms but these can not be distinguished from the impurities present in the agar. Examined with dark field illumination the halo consists of a great number of faintly visible granules. The elements which build up the halo can be seen best in preparations stained by flagellar staining methods. For a long time our attempts to stain the halo elements with these methods remained unsuccessful and only after much experimentation were successful preparations obtained.