Serial subculture of BCG on solid and liquid media

To follow an earlier study of the effects on BCG of deep subculture in liquid media, transfers on the surface of Löwenstein-Jensen and Sauton's media have been made. When a vaccine preparation that yields on culture 99+% of spreading-type colonies, with a small minority of non-spreading forms, is transferred on Löwenstein-Jensen medium, the minority population can be readily selected out. Growth of BCG on this medium is not homogeneous, and the selection of either the minority or majority populations appears fortuitous, whereas it was previously demonstrated that deep subculture in Dubos medium tends to favour selection of a minority yielding non-spreading colonies. When such a minority population has been favoured during transfer on Löwenstein-Jensen medium, subsequent subculture in Dubos medium usually accentuates the effect. Subculture as a surface pellicle on Sauton's medium has a powerful effect in reducing the relative size of a minority population yielding non-spreading colonies, and thereafter maintaining 99+% of spreading forms. The relationship between transfers on Löwenstein-Jensen and Sauton's media and those on potato-Sauton medium is briefly discussed.