A Method for the Cultivation of L Forms in Liquid Media

Abstract

Of all the bacterial L forms reported, only the L1 of Streptobacillus moniliformis will ordinarily grow well when transferred to liquid medium (Dienes and Weinberger, Bacteriol. Revs., 15, 245,1951). Tulasne, however (Compt. rend. soc. biol., 144, 1200, 1950 has succeeded in culturing L forms of Proteus on the surface of serum broth as a thin film, and Dienes (J. Bacteriol., 66, 274, 1953) has successfully applied this method of liquid culture to his Proteus strains. The disadvantages of this method are twofold: Suspended growth is not obtained, and the presence of serum in the medium may interfere with metabolic and serological studies.Pure cultures of L forms of Proteus vuaris OXK, Proteus sp. strain 52, and Proteu mirabilis strain K were obtained in liquid medium of the following composition, hereafter referred to as cmino acid broth (CAA broth): K2HPO4, 16 g; KH12P04, 2 g; Na citrate-55H20, 1 g; MgS04-7H20, 0.2 g; nicotinamide, 35 mg; vitamin-free csmino acids, 10 g; 1,000 ml distilled water.Casamino acid agar (CAA agar) was prepared by mixing equal amounts of sterile melted 2.2 per cent agar with double strength liquid medium following sterilization. Penicillin G, 300 units/ml, was added after autoclaving and immediately before inoculation of liquid media; 200 units/ml of penicillin were added to the solid medium immediately before pouring. The penicillin level was maintained in broth cultures which had long incubation periods by further additions of penicillin twice weekly.Liquid medium dispensed in quantities of 40 to 45 ml in 125 ml Erlenmeyer flasks was inoculated with agar blocks on which L colonies had been growing for 3 to 14 days. After incubation of these cultures at 37 C for 24 hours, they were then agitated continuously on a mechanical shaker for several days. Turbidity developed in three days in the case of P. mirabilis and Proteus strain 52 while it required seven days for visible growth of Proteus OXK. Serial transfer of the suspension of L forms was readily accomplished with P. mirabilis and Protu sp. strain 52. However, it was not found possible to initiate growth of Proteus OXK on new medium without the agar block inoculum. Where growth has been initiated in this manner, transfer of the original agar block to the new broth results in a shortened lag and more luxuriant growth.The L growth obtained in liquid media was composed primarily of small granular forms; although a few large spherical elements were present, no bacilli were observed. These granular elements were viable and reverted to bacilli within 18 hours on penicillin-free media.This method makes possible the production of abundant suspended growth of L forms in liquid medium without the addition of serum and should greatly facilitate the chemical, serological, and metabolic studies of L forms. NOTES 251 on July 31, 2020 by guest

Induction and cultivation of a stable L‐form of Bacillus subtilis

“…Three months after L-form initiation it was possible to obtain L-form growth in liquid culture. This was achieved by the traditional method (Abrams 1955) whereby LPM agar blocks bearing surface growth were introduced into L-phase broth (LPB). This consisted of LPM supplemented with 510 units/ml lysozyme and without the agar.…”

Growth of Bacterial L Forms and Bacterial Protoplasts

“…Growth conditions. The strains 9 and L9 of P. vulgaris were grown in 3-L Erlenmeyer flasks containing 500 ml of the liquid, serumfree medium described by Abrams (1955) for the cultivation of L forms. No penicillin was, however, included in the medium.…”

Growth and adhesion ofEnterococcus faeciumL-forms

“…There were noticeable differences in the growth curves of L-forms inoculated by different methods (Table 1). For example, when inoculated via agar blocks [17], loops, or a transfer from a batch culture, the difference in the lag phase was approximately 20 h (Table 1). Also, the maximum culture absorbance reached by the different inoculation methods was much lower than when subcultured by serial dilution.…”