An L-form isolated from Bacillus subtilis 168 was adapted to growth in a 340 mOsm minimal salts medium without the addition of osmotically protective solutes. This L-form had no chemically detectable peptidoglycan residues on its surface, but 0.8% of the dry weight of washed membranes was hexosamine. The osmotic stability and susceptibility to bacitracin and vancomycin of the L-form adapted to growth in 340 mOsm osmotically unprotected medium was twice that of the L-form grown in 2,680 mOsm medium supplemented with 1.2 M NaCl.

Several methods were used to monitor the growth of a stable L-form in batch culture. The end of the exponential growth phase was determined with greatest accuracy by the amounts of deoxyribonucleic acid per milliliter of culture. Optical density and viable count data were not as reliable because the L-forms began to lyse at the end of exponential growth. Lysis was detected visually, by phase-contrast observations of wet mounts, and by release of ultraviolet-absorbing material into culture supernatant fluids.