A preparation of pure protoplasts of Geotrichum candidum became osmotically stable and colonies developed when the protoplasts were embedded in stabilizing thin-layer-agar and incubated with stabilizing basal medium. When growing protoplasts were exposed to distilled water and then reincubated with basal medium, the process of regeneration of protoplasts could be quantitatively demonstrated by counting colonies. The process was divided into three phases, lag, logarithmic, and stationary. Furthermore, the state of regeneration of protoplasts at each phase could be seen in detail by microscopic studies of protoplasts under similar growth conditions. In the lag phase, which lasted for 2 hr after inoculation, protoplasts were completely destroyed when placed in distilled water. During the logarithmic phase, from 2 to 5 hr after inoculation, protoplasts rapidly became osmotically stable and about 18% of them were growing. In the stationary phase, most protoplasts developed germ tubes within 2 hr. These results suggested that there are two main phases, although individual cells passed through three different conditions, osmotically labile, osmotically stable, and growing. No apparent structure of cell wall material could be detected by electron microscopy on the surface of the membrane of these osmotically stable cells. This suspension was contaminated with a large amount of ghosts and partially digested cells (Fig.

Glycine, at a concentration of 5-10%, inhibited the growth of Streptomyces griseoflavus. The lysozyme sensitivities of the cell walls of the organisms cultured with and without glycine were the same. However, lysozyme caused 40% lysis of whole cells from cultures with glycine but had no effect on cells from cultures without glycine. The cell walls of organisms grown with and without glycine contained glucosamine, galactosamine, muramic acid, lysine, glutamic acid, glycine, alanine and DAP. Lysine and DAP were present in equimolar amounts. Very small amounts of asparagine, threonine and serine were found in both cell walls. Protoplasts were obtained in a high yield by treating cells from cultures containing glycine with 10 mg/ml of lysozyme in 0 .01 M phosphate buffer (pH 7.0) containing 0.1 MgSO4 and 1 M sucrose for 3 hr. The protoplasts were spherical and were surrounded by cytoplasmic membranes. The nucleoplasm appeared less dense with a clear network of nuclear fibers and mesosomes, abundant in whole cells, were scarcely seen. Rod-shaped forms without cell wall materials were sometimes observed under the electron microscope.