Use of Percoll Density Gradients for Studying the Attachment of Bacteria to Oral Epithelial Cells

Abstract

An assay for studying the attachment of bacteria to oral epithelial cells has been developed which utilizes Percoll density gradient centrifugation to separate bacteria and epithelial cells. 3H-thymidinelabeled bacteria were incubated with suspensions of buccal epithelial cells in microtitration plates for 2.5 hr at 35°C. The mixtures were then subjected to density gradient centrifugation in 50% Percoll. Epithelial cells with attached bacteria formed a band near the top of the tube, while unattached bacteria formed a band near the bottom. The epithelial cells were collected on membrane filters, and the number of attached bacteria was determined by scintillation counting. Binding of S. mitis C5 was found to increase with time, and equilibrium was attained within two hr. Saturation of available binding sites occurred when 107S. mitis cells were incubated with 1.5 × 104buccal epithelial cells. The numbers of streptococci which attached as determined with this assay were in good agreement with values obtained by direct microscopic counts. Adsorption of S. mitis C5 cells was adequately described by a Langmuir isotherm (correlation coefficient 0. 998). This permitted calculation of estimates of the number of binding sites and the affinity of the organism. The assay proved reliable even when as few as 1000 epithelial cells were used. Treating the epithelial cells with neuraminidase or trypsin significantly decreased the number of S. mitis C5 cells which attached. In contrast, binding of A. naeslundii 12104 to neuraminidase-treated cells was increased, and attachment of B. gingivalis 381 was also enhanced, especially to epithelial cells which had been treated with trypsin.