Growth of streptococcal protoplasts and L-colonies on membrane filters

Membrane filters (Millipore Corp.; pore sizes 1.2 to 0.22 mum) were placed on the surface of L-phase growth medium solidified with agar. The filter and the surrounding medium were inoculated with either protoplasts or stable broth-grown L-phase variants obtained from Streptococcus faecium strain F24. The L-phase inoculum gave rise to viable L-colonies on the filters and on the medium, whereas protoplasts gave colony formation only on the medium. However, when the Millipore filters were covered by a layer of solid L-phase medium, 75 mum or greater in depth, before inoculation with protoplasts, colony formation resulted but with atypical morphology. In contrast, inoculation of protoplasts on Nuclepore and Sartorius membrane filters did give rise to L-colonies on the surface and underneath the filters after 2 days of incubation at 37 C. Submicroscopic, viable L-phase elements produced during colony formation were capable of passing through membrane filters with pore channels as small as 0.22 mum; these elements required transfer from underneath the filters to fresh agar medium in order to develop into L-phase colonies. Membrane filters were also placed on the surface of L-phase growth medium solidified with gelatin. Inoculation of the filters and surrounding medium with a lysozyme-prepared protoplast suspension gave rise to streptococci on the surface of the filters and on the medium. However, inoculation with the stable broth-grown L-phase variants gave rise to atypical colonies on the medium and only small patches of abortive growth on the filters.