The fluoride resistance and smooth surface adherence characteristics of Streptococcus mutans were examined using tooth model and radioactive cell assays. Resistance to 600 ppmF by S. mutans isolated from the plaque of radiation-induced xerostomia patients receiving daily topical applications of a caries preventive 1% NaF gel was transient. Resistance induced in vitro in two strains of S. mutans by exposure to gradually increasing levels of NaF was apparently permanent. Smooth surface adherence by both fluoride-sensitive and -resistant strains of S. mutans 6715 in a tooth model system was slightly diminished by 1% NaF gel. Fluoride-resistant strains retained 89 to 93% of their adherence capability in 600 ppmF, as determined by the cell radiolabeling assay.

Twelve consecutive wk of daily five-minute topical applications of 1% NaF gel by non-cancer control subjects did not significantly affect plaque concentrations of Streptococcus mutans or Lactobacillus spp. Plaque F- levels increased 150% (P less than .001), while production of acetate and lactate decreased 40% (P less than .007) and 66% (P less than .001), respectively. Long-term (12 wk to more than five yr) fluoride gel use by post-irradiation xerostomic cancer patients was associated with increases in plaque F- and decreases in acidogenesis similar to those observed in the control subjects. Plaque concentrations of cariogenic organisms increased during the first yr of radiation-induced xerostomia and fluoride gel use, before starting to decline. Although sustained fluoride treatment increased (P less than .001) the ratio of fluoride-resistant to fluoride-sensitive strains, the number of patients harboring detectable S. mutans was diminished (P less than .001).

Escherichia coli K-12, grown in a synthetic medium containing metastable calcium phosphate, formed intracellular biological apatite crystals.

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.

Nine strains of cariogenic Streptococcus mutans and two strains of Streptococcus sanguis were tested for their ability to form hydroxyapatite. The cells were examined by X-ray diffraction and electron microscopy for apatite crystals after growth in a synthetic calcification medium. Each of the test isolates, except for one strain of S. sanguis , produced intracellular mineral. Two strains of S. mutans formed both intra- and extracellular crystals. There was no apparent relationship between calcifiability and serotype.