Spheroplast formation of Mycobacterium smegmatis and morphological aspects of their reversion to the bacillary form.

Cell wall-deficient forms (spheroplasts) of Mycobacterium smegmatis strain P53 were prepared by combined treatment with glycine, lysozyme, and lytic enzyme no. 2 as the spheroplasting agents. Quantitative mass conversion to spherical forms was effected by pretreatment of the intact cells with 1.2% glycine in nutrient broth, followed by transfer to spheroplasting medium containing the above agents. Two apparent modes of reversion to the bacillary form were observed under electron microscopy. The first one was initiated by budding from the spheroplasts. The buds gradually elongated to become the mycelial form, which showed branching, septation, and fragmentation. The second resulted from the intracellular formation of tiny cells, possibly the elementary bodies, and their release from the spheroplasts.