Atypical colony-like structures developing in control media and in clinical L-form cultures containing serum

Abstract

Solid and liquid media designed to support growth of cell wall deficient variants were evaluated for the presence of endogenous variants. The media compared in this study consisted of brain heart infusion base containing 10% sucrose, 3% NaCl + 5% sucrose, 3% NaCl, or 5% NaCl + 20% sucrose, and supplemented with filtrates of 10–20% horse serum - Sources A, B, C, suppliers of slaughterhouse (Type I) serum; and S, a supplier of standing herd (Type II) serum - and yeast extract. Three types of yeast supplements used in this study were aqueous extracts of: (1) fresh, or (2) active dry yeast; and (3) 0.5% commercial dehydrated yeast extract. Liquid control media prepared from one supplier gave rise to a pleomorphic Gram positive rod on extended incubation ( > 10 days) in spite of rigorous controls for asepsis during preparation and use. The second and more serious problem concerned repeated encounters with ‘pseudocolony’-like structures in work with clinical material. Correlation with severity of disease, different rates of appearance, and promotion of development by high osmolarity, led to a comparison of this phenomenon in control Type I- vs. Type II-BYE medium. Type I gave rise to numerous ‘pseudocolony‘- like bodies within 3 weeks, while Type II media containing standing herd serum developed < 20 per plate. Media variations known to promote growth of L-phase variants and mycoplasmas were noted to affect time of appearance and numbers of atypical colonies in control, uninoculated Type II-BYE. These included serum level, quality of yeast extract, pH, osmolar supplements, and the O 2 tension. The development of these structures in only certain Type II-BYE cultures of clinical fluids does not support the view that disturbance of an agar surface, or the presence of cells (or nuclei) promotes ‘growth’. No atypical colony-like structures or amber bodies developed in plates incubated for 7 weeks in the presence of vapors of phenol + formaldehyde, nor during an additional 10-day interval in a normal gaseous environment. It is suggested that all horse sera tested in this study contained unclassified atypical L-forms with unusual resistance to heat and chemicals.