Model system for studying colonization and growth of bacteria on a hydroxyapatite surface

A model system for the study of bacterial colonization and growth on a hydroxyapatite (HT) surface is described. Hydroxyapatite was crystallized over the surface of porous glass beads. Chemical analysis of the product showed that the ratio of Ca2+/P042- was indistinguishable from that of commercial HT powder. X-ray diffraction analysis supported the conclusion that the product was HT. A system employing [14C]polyethylene glycol, which selectively adsorbs to the glass surface of the beads, was developed to determine the amount of glass surface covered by HT. Over 90% of the glass surface could be covered by our method. The product, HT beads, consisted of approximately 20% (dry weight) HT. The HT beads possess several properties which make them potentially useful for studying microbial adherence, growth, and interactions. These include: (i) chemical similarity to the tooth surface, (ii) large surface area, and (iii) high density. We also describe a method for direct measurement of the microbial mass of cells growing on beads. The method entails immobilizing a sample on a membrane filter (Millipore), staining it with amido black dye, and eluting the dye for spectrophotometric measurement. Streptococcus mutans served as the test organism. For free-growing bacteria the values measured with the filter assay were directly proportional to cell number, with a value of 1 mug of "protein" corresponding to about 1.5 X 10(6) colony-forming units, determined by viable count. For bacteria colonizing the beads, 1 mug of protein corresponded to about 2 X 10(7) colony-forming units on the beads during logarithmic growth. As the culture approached stationary phase, the efficiency of the assay decreased. These data indicate that multiple random samples, taken at a given time, are representative of the entire culture.