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

Abstract

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+/PO42- was indistinguishable from that of from thatof 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. A method for direct measurement of the microbial mass of cells growing on HT beads is also described. The method entails immobilizing a sample on a membrane filter (Millipore), staining it with amido black dye, and eluting the dye for spectrophotometic 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 μg of 'protein' corresponding to about 1.5 x 106 colony forming units, determined by viable count. For bacteria colonizing the beads, 1 μg of protein corresponded to about 2 x 107 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.