Morphological and histochemical study of supragingival human calculus and dental plaque using ruthenium hexammine trichloride and acridine orange.

Ruthenium hexammine trichloride (RHT) and acridine orange were used to preserve and visualize anionic groups in human plaque and dental calculus. RHT-reacting material was present on the membrane of micro-organisms and in intermicrobial spaces of the calcifying areas, and seems to correspond to, and derive from, acidic glyco- and phospholipids of the plasma membrane of the micro-organisms. However, the presence of acidic salivary peptidoglycans cannot be ruled out. Two types of calcification were found: extramicrobial and intramicrobial. The former consisted of calcified deposits irregularly scattered in the intermicrobial matrix. They were in close relationship with RHT-reacting material, or were placed inside vesicular structures delimited by a membrane. Intramicrobial calcification consisted of small aggregates of needle-shaped crystals and/or of granular deposits; in both cases, they either masked the whole cytoplasm of the micro-organisms, or were located only over the plasma membrane. These results suggest that mineral deposition occurs in connection with acidic components of intermicrobial matrix, microbial plasma membranes, and cytoplasms. The addition of RHT and acridine orange to fixing and decalcifying solutions yields satisfactory preservation of dental calculus and plaque, and apparently reduces loss of their anionic organic components and increases their electron density. However, these substances are not sufficient to preserve all ultrastructural details in decalcified areas, probably because the inorganic substance prevents reaction of acridine orange and RHT with the organic components of the calcified matrix.