JOURNAL OF BACTERIOLOGY, Dec. 1968, p. 2171-2174 Copyright (D 1968 American Society for Microbiology NOTES Gelatin-induced Reversion of Protoplasts of Bacillus subtilis to the Bacillary Form: Photomicrographic Study IRVING L. MILLER,' WILLIAM W1EBE,2 AND OTTO E. LANDMAN Department of Biology, Georgetowni University, Washingtoni, D. C. 20007 Received for publication 28 June 1968 When lysozyme protoplasts of Bacillus subtilis 168 indole- are washed free of lysozyme and plated on hypertonic soft agar medium, each pro- toplast forms an L colony. The L colonies con- sist of cell wall-free L bodies which, in turn, give rise to new L colonies. This heritably propa- gated wall-less condition can be terminated promptl"'6 by plating the cells in 25%, gelatin medium. In this&t;environment, each body reverts to thX ba l1ary state (1). An electron-microscopic study of this process and experiments on the physical and nutritional requirements of rever- sion are presented in an accompanying study (2). For the present phase-constrast study of rever- sion, protoplasts were prepared in the standard manner (2). They were then centrifuged at 8,000 rev/min, washed once in SL2 medium (1) and re- suspended to a density of approximately 4 X 107 / ml in the same medium. This was the inoculum suspension. The gelatin reversion medium used in these photomicrographic studies had the fol- lowing composition (per liter): 250 g of gelatin (Difco), 1 g of NH4NO3, 0.7 g of K2HPO,, 0.3 g of KH2PO4, 2 g of glucose, 1 g of acid-hydrolyzed casein (Nutritional Biochemicals Corp., Cleve- land, Ohio), 0.02 g of L-tryptophan, 0.005 M MgC92, and 0.5 M sodium succinate, pH 7.3. (The gelatin medium of Landman, Ryter, and Frehel gives similar reversion rates and similar morpho- logical changes. However, a marked temporary loss of contrast between 30 and 360 min after inoculation interfered with photomicrography.) The gelatin medium surface for phase-microscope observation of reversion was prepared as follows. Two pieces of glass tubing (0.5 mm diameter) were placed 18 mm apart on a thin glass micro- ' Present address: Red River Valley Potato Re- search Center, East Grand Forks, Minn. 56721. 2 Present address: Department of Microbiology, University of Georgia, Athens, Ga. 30601. scope slide and fastened into position with warm petrolatum. A cover slip (22 by 22 mm) was then placed on the glass rods. Warm gelatin reversion medium was next allowed to flow between slide and cover slip; then the slide with its film of me- dium was chilled quickly on a cold metal surface. The cover slip was now carefully removed, leav- ing a smooth gelatin surface (18 by 18 mm). This surface was inoculated with approximately 0.003 ml of inoculum suspension. A cover slip (22 by 40 mm) was placed directly on the surface; edges of the cover slip were then sealed with petrolatum to prevent desiccation, but an air space was left sur- rounding the 18 by 18 mm gelatin square. The slides were then incubated at room temperature. A Zeiss research microscope, fitted with a plana- chromat, oil immersion, 100 X phase lens, was used for observations. Photographs were taken with a Wild camera attachment and plates (6.35 by 8.89 cm) and Kodak commercial film (ASA 50). The plate magnification was X 1,000. After scanning the somewhat unevenly inoculated gela- tin surface, fields with three to six protoplasts were selected and then kept under observation for about 20 hr. Figures 1 to 3 give a representative picture of the observations. A large proportion of all proto- plasts survived protoplasting and washing and began to revert in a fairly synchronous manner (Fig. 1). Later, division of the reverted cells be- came asynchronous. The pictures also show that the reverting protoplasts early took on irregular shapes, then often grew or branched into fila- ments, and only much later gave rise to rod shapes (not shown). Plating experiments have shown that multiplication of colony-forming elements occurs only after reversion. This finding has recently been confirmed decisively by the observation that, in appropriately pretreated protoplasts, the wall- synthesizing step in reversion, which takes place 2171 Vol. 96, No. 6 Printed in U.S.A. Downloaded from https://journals.asm.org/journal/jb on 01 January 2022 by 112.133.81.138.