Isolation of L-forms from the spleens of Brucella suis-infected, penicillin-treated mice.

Previous attempts to obtain in vitro wall-deficient stable L-forms of various strains of Brucella have failed because the obtained spheroplasts revert quickly to bacterial form. Here, we report the isolation of L-forms from mice infected with a B. suis strain type 1 and treated with penicillin. In defined experimental conditions, L-type microcolonies associated with tissue debris were observed in primary spleen cultures, even on antibiotic free media. After several transfers on penicillin-containing medium. typical, tissue-free L colonies were obtained. At first, when cultivated on antibiotic-free medium, these colonies reverted to the bacterial form (identified as B suis, biotype 1). Later, after approximately fifteen transfers on penicillin-supplemented medium, they no longer reverted even after several subcultures on antibiotic-free medium. The L-forms' ultrastructural features included many giant empty bodies, considerable variation in size, shape and density of the wall-deficient cells, and many multilayered membranes. The stabilized L-forms were propagated in vitro and inoculated into mice, and then recovered from their spleens as tissue associated L-microcolonies. An occasional in vivo revertant was identified as B. suis, biotype 1. These data provide one possible explanation for earlier failures to detect the presence of atypical bacteria in clinical or experimental Brucella infections.

In vitro spheroplast and L-form induction within the pathogenic nocardiae.

Six strains of Nocardia asteroides, two strains of N. caviae, and two strains of N. braziliensis were grown in medium supplementted with glycine, lysozyme, D-cycloserine, glycine plus lysozyme, and glycine plus D-cycloserine. It was shown that three strains of N. asteroides, and two strains of N. caviae, readily formed spheroplasts and/or protoplasts when grown in the presence of glycine plus either lysozyme or D-cycloserine. This process was studied by both phase contrast microscopy and electron microscopy. The induced cultures were then plated on hypertonic medium for the isolation of L-forms. It was shown that the organisms differed greatly in their ability to produce spheroplasts and subsequently grew as L-forms or transitional-phase variants.