Analytical electron microscopy of chlorhexidine‐induced tooth stain in humans: direct evidence for metal‐induced stain

Using analytical electron microscopy, we directly tested the hypothesis that transition metals and sulfur are the cause of chlorhexidine-induced tooth stain. Plaque scraped from the teeth of water-treated individuals or chlorhexidinetreated “non-stainers” was non-or lightly-stained and contained low concentrations of sulfur and transition metals. Tooth scrapings from heavily-staining chlorhexidine-treated individuals consisted of distinct unstained and stained regions. The stained regions were organic but were in close proximity to mineralized areas. Enhanced levels of sulfur and transition metals, particularly iron, were found in stained regions, whereas unstained regions contained low sulfur and metal levels similar to the water-treated or non-staining individuals. Excluding decreased mineralization, the major elemental change in heavily-stained plaque was an increase in sulfur, and to a lesser extent, iron and other transition metal. Sulphur and iron levels were directly correlated. Following chlorhexidine treatment with iron supplementation, staining was enhanced, the Fe/S ratio increased, and sulfur and iron remained correlated. These data support the hypothesis that transition metals and sulfur are the cause of chlorhexidine-induced tooth stain. The data are consistent with chlorhexidine treatment altering the incorporation into plaque of a natural sulfur-containing organic component of saliva or bacteria. This natural component appears to readily interact with transition metals, particularly iron, producing stained material.