Comparison of fluorescence microscopy and culture assays to quantitate adhesion of Porphyromonas gingivalis to mono- and multi-layered pocket epithelium cultures

Background: The present study compared 2 different methods (direct versus indirect evaluation) for the quantification of the adhesion of Porphyromonas gingivalis strains to in vitro cultured monolayers of pocket epithelium. Methods: The indirect culture viability assay (calculation of colony forming units) was compared to a direct microscopic evaluation using a novel fluorescent stain. The fluorescent kit was found to stain both bacteria and epithelial cells and enabled a differentiation between dead and living cells. Results: Comparing the visual to the culture data, a high and significant correlation was found (Pearson's correlation = 0.75; P < 0.001). The adhesion capacity was in general higher for dead epithelial cells than for living cells (P < 0.01). Although comparable numbers of bacteria of 2 P. gingivalis strains (Pg 4 and Pg 5) were applied, Pg 4 showed a significantly lower adhesion capacity. This intra-strain variability was observed by the culture assay (2.3 x 10(6) versus 7.8 x 10(6) +/- 2.7 x 10(6); P < 0.01) and by the direct microscopy (P < 0.01) for both live and dead epithelial cells. A second goal was to see whether there was a difference in the amount of bacterial adherence to mono- and multi-layers of in vitro cultured epithelium. No significant differences were found for the 5 examined P. gingivalis strains. However, interstrain differences in adhesion capacity were evident for both tissues. Conclusions: This study highlights the reproducibility of a direct microscopic evaluation of bacterial adhesion to in vitro cultured epithelial cells, and suggests both intrastrain (P. gingivalis) and inter-cell (live versus dead) variation in adhesion capacity, Studies are needed to determine the extent to which P. gingivalis strain variation is reflected in variation of other strains in humans.