Strain M, classified as a Staphylococcus aureus, behaves like the other rare encapsulated staphylococcal strains. It was clumping-factor negative, grew in diffuse-type colonies in serum-soft agar, and produced rapidly fatal disease in mice. Strain M was highly resistant to phagocytosis by human or mouse leukocytes and required both specific antibody and heat-labile serum factor(s) for efficient ingestion by human polymorphonuclear leukocytes. Electron micrographs confirmed the presence of a large capsule. Agglutination studies, active or passive mouse protection experiments, and opsonic studies revealed that strain M represents a new, immunologically distinct strain of encapsulated staphylococcus. Strain M differs from other known encapsulated staphylococci in several other respects: its cellular and colonial morphology is atypical; its LD50 in the mouse peritoneal model is 100 times less than that of other mouse lethal strains; it is poorly opsonized by normal human serum; and, finally, it possesses an unusually large capsule as seen in electron micrographs. MATERIALS AND METHODS Cultures. Strain M. This encapsulated strain was isolated from a human infection in 1962 and has been described by Scott (20). The organism (NCTC 10649) was obtained from the National Collection of Type J. Bacteriol, in press). Smith diffuse. This variant has been previously described and has been used extensively as the prototype of an encapsulated mouse-virulent staphylococcus in this laboratory (9). T mutant. This is a mutant of S. aureus H possessing an additional surface polysaccharide antigen (24). The organism was kindly supplied by J. T. Park, Tufts University, Boston, Mass. Wiley. This strain (Wound) was kindly supplied by B. B. Wiley. When grown under certain conditions this strain has been reported to undergo a specific capsular reaction (23). Giorgio. This strain of S. aureus has been previously described (14) and is a prototypic non-ecapsulated S. aureus. All cultures were maintained on brain heart infusion agar (Difco) slants at 4 C. Unless otherwise noted, 18-h subcultures in brain heart infusion broth were used in all experiments.

A solid medium was developed for callus cultures originating from explants of root, hypocotyl, and cotyledon of seedlings of bush bean, and a liquid medium was developed for the growth of cell suspension cultures derived from the callus cultures. Some unsatisfactory media are recorded. Concentrations of mineral salts for cell suspension cultures are lower than for callus cultures. Both coconut milk and other organic substances are required for maximum growth. With cell suspensions the effect of deproteinized coconut milk is the same as that of raw coconut milk but, with callus cultures, deproteinized coconut milk gives greater yield. There are no obvious differences in yield of callus derived from root, hypocotyl, or cotyledon. Few differences in yield were obtained between cell suspension cultures from root, hypocotyl, and cotyledon but those from root gave the highest yield in dry weight. However, in the same medium, cells from the three origins are very similar in form and appearance. Some effects of different media on cell form and clumping are described. The yield in suspension culture is very high. Increase in cell number, fresh weight, and dry weight is about 100-fold in 12 days involving about six to seven divisions per cell.

The chemical structure of the surface polysaccharide from Staphylococcus aureus M was investigated by a combination of methanolytic, hydrolytic, and chromatographic techniques. The repeating unit that was most consistent with the data was a hexasaccharide composed of N-acetyl-D-aminogalacturonic acid, N-acetyl-D-fucosamine, and taurine in molar ratios of 4:2:1. A disaccharide was isolated and characterized, by combined gas-liquid chromatography-mass spectrometry, as N-acetyl-D-aminogalacturonyl-(1-+ 3)-N-acetyl-D-fucosamine. Taurine is linked to a carboxyl group of N-acetyl-n-aminogalacturonic acid via an amide bond. We have previously reported the isolation and identification of the constituent components of the surface polysaccharide ofStaphylococcus aureus M (2). This polysaccharide is composed of D-aminogalacturonic acid, D-fucosamine, and taurine. It thus differs from the surface polysaccharides of the S. aureus Smith diffuse variant, which is composed of D-aminoglucuronic acid and L-alanine (1), and S. aureus mutant T, which is composed of D-aminomannuronic acid and D-fucosamine (11). This report is concerned with structural analysis of the strain M surface polysaccharide. (This work was presented in part at the 59th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N.J., 1975.) MATERIALS AND METHODS Unless otherwise indicated, the materials and methods for the isolation and identification of the components of the surface polysaccharide antigen (SPA) from S. aureus M are the same as previously described (2). Other materials used in this study were obtained from the following sources: AG50W-X8 resins, Bio-Rad Laboratories; thin-layer chromatographic (TLC) plates (no. 6064 cellulose, no. 6060 silica gel with fluorescent indicator), Eastman Kodak Co.; coiled glass columns (1/8 inch by 6 feet [0.32 by 183 cm]) filled with 3% OV-1 on Gas-Chrom Q (100 to 200 mesh) and coiled stainless-steel columns (1/8 inch by 6 feet [0.32 by 183 cm]) filled with GP 2% SP-1200/1% H3PO4 on Chromsorb WAW, Applied Science Laboratories; [14C]methanol (4 mCi/ mmol), New England Nuclear Corp.; carbodiimide [1ethyl-3-(3-dimethylaminopropyl)carbodiimide

The surface polysaccharide antigen of Staphylococcus aureus M was isolated, and the component parts were identified. The antigen is composed of taurine, d -aminogalacturonic acid, and d -fucosamine. The capsule is chemically distinct from all previously reported staphylococcal capsules. Taurine and aminogalacturonic acid have not been reported previously in staphylococci.

Changes in cell number, cell size, fresh weight, and dry weight were recorded for the growth cycle of a serially subcultured cell suspension culture derived from hypocotyl of bush bean (var. Contender). The various cell types and growth patterns from single cells were also recorded. The culture showed unique features including a relatively sharp separation of a phase of exponential cell division from a phase of cell expansion. Complete separation of cell clumps gave a free cell culture, of viable cells, by day 16. The growth cycle, cell types, and growth patterns are compared with those in other plant cell suspension cultures. The potential value of the culture for study of growth regulation is discussed.