L-forms of a strain of Pseudomonas aeruginosa were produced by serial subculture of the bacterial form on agar medium containing sucrose as an osmotic stabilizer and carbenicillin. L-forms eventually became stable, i.e., would not revert in the absence of antibiotic, and were adapted to grow well in broth with the osmotic stabilizer. Gross morphology and light microscopic colony morphology were typical of an L-form. L-form cells were approximately spherical and bounded in part by a plasma membrane; they lacked the triple-layer cell wall structure and coarse, electron-dense nucleoidal granules of the parent bacterial form. The L-form, but not the bacterial form, contained cores, organelles previously reported only in group D streptococci. Antibiotic disc-sensitivity studies showed the stable L-form to be as sensitive as, or more sensitive than, the bacterial form to most antibiotics. Exceptions were polymyxin B, colimycin sulfate, and gentamicin, which were more active against the bacterial form. The remainder of the aminoglycosides and cell wallactive antibiotics showed no inhibition of either form. The L-form was more susceptible to cidal activity of normal human serum than the parent form. The L-form exhibited fewer biochemical activities than the parent bacteria or bacterial forms derived by reversion at a time when the L-form was still unstable. L-form colonies appeared colorless, and chemical analysis demonstrated that, if the L-form produces pigment at all, which was not demonstrated, it could not have been more than 3.6%o of that produced by the bacterial form. The role of L-forms in infectious diseases in general and in urinary tract infections in particular has not been clarified. However, there is inferential evidence that these wall-deficient variants may be significant, not only as primary etiologic agents, but particularly may account for relapse after apparent cure (23). Postulated mechanism for production in vivo is based on the in vitro demonstration that wall-deficient variants can be induced by serum action on the parent form (1, 37). L-forms have been isolated from urine of patients with urinary tract infection (14) and inferential evidence has been obtained by treatment of experimental animals with antibiotics (15). Pseudomonas infections present an important problem, particularly in urinary tract infections and burn patients, because antibiotics in general use are not active against this organism. The semisynthetic penicillin, carbenicillin (disodium a-carboxybenzylpenicillin; BRL 2064), has been demonstrated to possess significant activity against strains of Pseudomonas aeruginosa, both in vitro (40) and in vivo (21, 39, 44). In view of this activity, it was felt that carbenicillin provided an opportunity to determine whether L-forms could be produced in vitro from P. aeruginosa and to study some biological properties of the L-forms. This study details initial production of the Lform of P. aeruginosa and the achievement of nonreversion in the absence of antibiotic. Comparisons of morphology,...

Spherules of C. immitis have been grown in vitro in modified Roessler's medium under COs tension and continuous cultures now maintained for over 18 months. Transformation of hyphae and development of the spherule form have been studied by thin section electron microscopy. Cells of organisms in the hyphal stage have thin (ca. 50 m#), apparently structureless walls and a cytoplasmic membrane. Many nuclei, elongated mitochrondria with both transverse and longitudinal cristae, and lipid particles are present. The hyphal wall thickens and the cell transforms into spherules. A large central accumulation of electrontransparent polysaccharide appears in the spherule. The peripheral cytoplasm contains nuclei, each enclosed in a double-layered membrane, mitochondria, and small dense particles. Prior to cleavage the polysaccharide droplets are lost, while mitochrondria become small and spherical. Endospores are formed and liberated when the spherule wall breaks. These begin to grow and repeat the cleavage cycle.

No abstract