Based on electron microscopic observations, the reproductive cycle of a relatively stable L-phase variant of Streptococcus faecalis was proposed. The small, dense, nonvesiculated L-form is envisioned as the central (core) element. It was seen to divide and bud rapidly. In addition, the dense forms appeared to be capable of growth and development within vesicles of mature mother forms. When these forms were released from the vesicles into the surrounding fluid medium, further growth occurred, resulting in the development of immature and ultimately mature mother forms. Under conditions unfavorable for L-form growth, these dense forms developed first into transitional forms and then into the bacterial form. These dense forms might therefore be considered as undifferentiated "stem cells" with the capacity to develop along several different routes, depending upon the stimulus received. A number of recent investigations have been concerned with ultrastructure, mode of reproduction, and heterogeneity of individual organisms within L-form colonies (1, 9, 18, 19, 23, 24). The result has been assignment of functional roles to elementary corpuscles, granules, and large bodies (1, 7, 8), and the recognition that L-forms derived from widely divergent classes of bacteria are strikingly similar from a morphological point of view (3). Although various modes of replication, in addition to reversion, have been observed by light and electron microscopy (1, 8, 13, 15, 22), no one to date has attempted to correlate all of the various modes of reproduction into a meaningful cycle based on ultrastructural characteristics. An understanding of the reproductive cycle of the L-form is essential in order to assess its role in persisting and relapsing bacterial infections, and to gain insight into clinical management of patients suspected of harboring L-forms. It was therefore the purpose of this study to attempt to organize into logical sequence observations made on behavior, morphology, and ultrastructure of a relatively stable L-phase variant (17) of Streptococcus faecalis, using gross observations, phase contrast, and electron microscopy.

A series of experiments was undertaken in which human embryonic kidney (HEK) fibroblasts were infected with either relatively stable or stable L-forms of Streptococcus faecalis. The infected cells were maintained by splitting over long periods (1 to 2 months) while samples were withdrawn for culture and electron microscopic studies. Relatively stable S. faecalis L-forms could be cultured from infected cells only during the first week after infection, although L-form-like material was frequently detected at later periods by electron microscopy. HEK cells continued to proliferate and showed no gross histopathology until reversion occurred. At reversion, electron microscopy revealed the presence of intracellular L-forms, transitional variants, and intra-and extracellular bacterial forms. Unlike relatively stable L-forms, stable L-forms were culturable throughout the experimental period. These experiments demonstrate the phenomena of persistence and reversion in vitro. Electron microscopy indicated that the L-forms entered the HEK cells, where they underwent morphological changes. Observations suggested that these altered L-forms (elementary bodies) persisted in the cell until reversion to the parent bacterial form was triggered. Infected cells, when treated with L-form antiserum and stained with fluorescein-conjugated goat anti-rabbit serum, were consistently positive regardless of cultural findings. bacterium (20).