Abstract
In this study we have attempted to develop methodology to enable identification of periodontitis-related organisms in situ using a post-embedding immunogold labelling technique. Pure cultures of Porphyromonas gingivalis W50 strain and specimens of plaque were fixed in 0.5 per cent glutaraldehyde in 0.1 M sodium cacodylate buffer and subsequently embedded in LR white acrylic resin. This procedure resulted in good ultrastructural preservation of the tissue and good retention of antigenicity. Fluorescence microscopy of a range of organisms and immunogold labelling of ultrathin sections were then employed using a high titre (1:5 × 105 by ELISA) murine polyclonal antiserum to P. gingivalis W50. Analysis of specificity by fluorescence microscopy using 46 different bacterial species demonstrated minimal cross-reactivity of the test antiserum, with only Bacteroides corporis showing an unexplained cross-reaction. At the ultrastructural level, consistent labelling of the pure culture specimens was achieved and labelling in plaque was confined to selected Gram-negative coccoid cells, or to associated areas of matrix and polymorphonuclear cytoplasm. These results indicate that post-embedding immunogold labelling may be an appropriate technique for the identification of bacterial strains within a mixed flora such as that of dental plaque.