Isolation of L-forms of Bacillus subtilis which grow in liquid medium

L-forms of Bacillus subtilis can be isolated by treatment of the parent strain sequentially with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme and selection of the surviving protoplasts on semisolid medium containing 2,000 units of penicillin per ml. Some of these clones can be adapted to grow in liquid cultures containing 1.2 m NaCl. This method will aid in the isolation of cell wall mutants which require hypertonic medium for growth.