Mycoplasma growth factors in bovine serum fraction

Abstract

Mycoplasma growth factors in bovine serum fraction were separated by Sephadex G150 column chromatography and density ultracentrifugation. The major 3 parts of 50% (wt/vol) sucrose, divided into 1.2-ml portions, and stored at -60°C. M. pneumoniae 65-2161, originally isolated from a 2-year-old child with pneumonia and supplied to us by Jose Canchola from Children's Hospital, Washington, D.C., was similarly identified, cloned, subcultured on BSF-containing media, and frozen after passage 19. M. arthritidis strain PG27 was subcultured six times in BSF-containing broth medium and after passage 7 was pooled and frozen as described above for M. pneumoniae. Culture media. Two broth media, both similar to the formulation of Chanock et al. (2), were used throughout this study. The first, designated SSR2, was developed for growing M. pneumoniae on glass and 818 on August 5, 2020 by guest http://jb.asm.org/ Downloaded from Jr. (ed.), Serum proteins and the dysproteinemias. J. Lippincott Co., Philadelphia. 39. Zlatkis, A., and B. Zak. 1969. Study of a new cholesterol reagent. Anal.

Cholesterol as a limiting factor in the growth of Mycoplasma pneumoniae

“…Growth-promoting activity for M. pneumoniae strain 65-2161 was markedly different for components from each BSF lot (Table 1). With BSF-A, as reported previously (22), component 3 yielded about three times more GAM protein than did component 4. However, with BSF-B, component 4 was as active as component 3; with either component, growth equalled that of intact BSF-B.…”

“…Four different lots of serum fraction (Difco) were assigned letter designations BSF-A through BSF-D. Most of BSF-A (Lot 591725) had previously been consumed in a nutritional study on BSF (22). We had a limited sample stored and used it for lipoprotein studies only.…”

“…In other studies, Washburn and co-workers (22) used a sequential ultracentrifugation procedure for a density separation of lipoproteins from BSF. In contrast to previous work, the greatest activity of all the components was found in the material designed to contain LDL, even though no intact lipoprotein could be detected by electrophoresis.…”

“…In our study, we further fractionated three lots of BSF by ultracentrifugation, and we used M. pneumoniae to ascertain the growth-promoting ability of some of the components. Our growth conditions, in which mycoplasmas attach to glass, simplify the quantitation of cultures grown in broth (3,22). In addition, we used agar medium to confirm the results and to determine the morphological features of the colonies.…”

Conditions for growing Mycoplasma canadense and Mycoplasma verecundum in a serum-free medium

“…This results from the partial or total inability to synthesize the long-chain fatty acids and cholesterol required for membrane synthesis (13). These nutrients are usually supplied in the culture medium by the addition of horse serum, which presents various disadvantages, such as the dissimilar qualities of different horse serum preparations (8) and the presence of contaminants (17). In addition to all these disadvantages, serum supplies phospholipids, which are directly incorporated into the cell membrane.…”