Some effects of nutritional components on the morphology of BCG colonies

Under appropriate conditions of growth colonies showing fine wrinkling (rugosity) of their surface and characteristic of certain BCG strains can be distinguished from colonies with a smoother non-rugose morphology that are characteristic of some other BCG strains. This study has been concerned with the influence of nutritional and other media constituents on the evolution of these colonies. In a preliminary investigation nutritional components such as enzymic digest of casein, asparagine and salts were systematically eliminated from an agar medium, separately and together, and in the presence or absence of glycerol. From this and supplementary experiments, it appeared that the nutritional element with most effect on the size and morphology of colonies is the carbohydrate. Small inocula of BCG will normally grow on simple agar media only in the presence of enrichments such as blood or albumin, or if charcoal has been added. However, in a second investigation it was found that individual colonies would develop from such inocula placed 2 cm or more away from a concentrated inoculum that had been seeded onto the medium approximately 7-14 days earlier. It appears that a diffusible agent is produced from multiplying bacilli within a colony which counteracts toxic factors in the medium and may also assist nutrition within the colony. To examine the effects of carbohydrate on the evolution of colonies, in a third investigation glycerol was added in increasing quantities to an agar medium enriched with increasing quantities of bovine albumin and/or 5% of blood, and suspensions of French (Pasteur) and British (Glaxo) BCG vaccines were seeded onto it. Individual colonies cultured from these two strains have a rugose and non-rugose morphology respectively, and to highlight the effects in question, the evolution over a prolonged period of concentrated 'drop-colonies' seeded onto the medium from each strain was studied. There was a very marked difference between the two strains in the evolution of such 'drop-colonies', and it appeared that the lateral spread of fine rugosity from those of the Pasteur strain represented an enhanced ability of small numbers of bacilli to take up the nutrient.