Synthetic Medium for Calcification of Bacterionema matruchotii

Abstract

A synthetic medium was developed for further study of intracellular calcification by Bacterionema matruchotii. The medium contained metastable calcium phosphate, six other salts, three purines, two pyrimidines, nine vitamins, pimelic and thioctic acids, vitamin-free casein hydrolysate, and glucose. The vehicle was 0.1 M N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid butler at pH 7.4. This work was undertaken to develop a chemically defined medium in which Bacterionema matruchotii would calcify. The investigation is part of a continuing program designed to provide a microbiologic model for studying vertebrate calcification. Several workersl-3 have established that under appropriate conditions B matruchotii forms intracellular apatite. The subject was reviewed by Ennever and Creamer.4 Ennever, Vogel, and Takazoe5 have demonstrated that B matruchotii calcification is initiated by a cell component that is soluble in chloroform-methanol. Subsequently, these researchers6 found that a phospholipid-peptide complex contains the factor responsible for calcification. The role of specific metabolites in synthesis of the complex is uncertain. Such information may be obtained by the use of a defined environment in which metabolic parameters can be controlled and measured. The medium described herein provides such an environment. Materials and MethodsThe synthetic medium (Table) was prepared as follows: 0.1 M (22.93 gm) of N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) was dissolved in 500 ml of deionized water. Purines and pyrimidines were dissolved in 100 ml of 0.1 M KOH and added to the TES. The volume was increased to 850 ml and adjusted to pH 7.4 with NaOH. The vitamins were triturated with glucose and added to the buffer. The remaining ingredients were added and the volume was brought to 1 liter. The medium was filter-sterilized* and dispensed in 250 ml volumes. The final pH was 7.4Each of three strains of B matruchotii: Richardson 13, ATCC 14265, and ATCC 14266, was inoculated into separate volumes of the medium. Inoculums were obtained from brain-heart infusion agar slants,t or cells grown in Takazoe's synthetic medium.7 Cultures were incubated aerobically at 37 C for seven days. Cells were harvested by centrifugation at 10,000 X g for five minutes at 5 C, then water-washed and stored at -14 C.Most of each yield was air-dried and ashed4 at 55 C for two to four hours to determine whether calcification had occurred. The residues were examined by X-ray diffraction using a powder camera film technique with nickel-filtered copper radiation generated at 30 kv 20 ma anode current for five hours. The films were compared visually to known standards. To confirm that the calcifications were intracellular, the remaining portion of each yield was fixed with 3% gluteraldehyde in pH 7.2 cacodylate buffer, followed by 1% osmium tetroxide. Cells were dehydrated in ethanols, embedded in epoxy resin, § and sectioned at 600 A with a diamond knife in * Millipore filter type HA, Millipore Corp., Bedford, Mass.

Basic Biological Sciences Pre-apatitic Mineral Deposition in Bacterionema matruchotii

“…Ultrastructure of calcified cells (containing apatite) was identical to that already reported. 1 Calcium, magnesium, and phosphate concentrations determined for calcifying and noncalcifying B matruchotii are shown in Table 2 as Ca/Pi, Ca/Mg, and Ca + Mg/Pi molar ratios. Ca concentration was slightly higher in calcifying cells as early as day two.…”

Calcification of Bacillary and Streptococcal Variants of Bacterionema matruchotii

“…The procedures and instrumentation have been described previously. 5 Results Both morphologic variants calcified during cultivation in the synthetic medium. The ashed residues gave X-ray diffraction patterns for apatite (Fig 1).…”

Calcification of a Cariogenic Streptococcus and of Corynebacterium (Bacterionema) matruchotii

“…Calcification media.-The cultivation/calcification broth (E-fluid) was formulated as described by Ennever et al(1971). It supports the growth of C. matruchotii and 'many types of microorganisms' (Ennever et al, 1972).…”