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 mono-layers 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.
Department of Periodontology, Faculty of Medicine, Catholic University, Leuven, Belgium.
W
W
Papaioannou
D
D
van Steenberghe
J J
JJ
Cassiman
J
J
Van Eldere
M
M
Quirynen
D001422:Bacterial Adhesion; D001431:Bacteriological Techniques; D001681:Biological Assay; D018929:Cell Culture Techniques / Q000379:methods*; D015169:Colony Count, Microbial; D046508:Culture Techniques; D004847:Epithelial Cells / Q000382:microbiology; D005260:Female; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D010514:Periodontal Pocket / Q000382:microbiology*; D016966:Porphyromonas gingivalis / Q000502:physiology*; D015203:Reproducibility of Results; D013045:Species Specificity; D018709:Statistics, Nonparametric