Positive stain for light and electron microscopic demonstration of spirochetes in subgingival plaque and crevicular fluid samples from periodontal diseased sites

Abstract

In a recent study of 400 subgingival plaque samples from over 110 adult periodontitis patients, spirochetes were the overwhelming microbial type, averaging about 45% of the microbial count. This finding supports earlier arguments that spirochetes are pathognomonic in periodontal disease. Other studies had shown clear-cut differences in the microbial composition of healthy and diseased subgingival sites — the proportion of spirochetes being significantly higher in the latter. Another study indicated that periodontal deterioration at these sites could be predicted better by increased proportions of motile rods and spirochetes than by clinical measurements. However, spirochetes of all sizes and species do not show the same degree of association with periodontal breakdown. Moreover, spirochetes are usually difficult to culture and stain; they are generally monitored by darkfield or phase contrast microscopy.The PATS reaction, a modified periodic acid-Schiff(PAS) reaction which deposits silver for light and electron microscopy appears to stain Gram(-) bacteria positively as well as neutrophils and activated macrophages. When studying the stained Gram(-) bacteria on coverslip smears of subgingival plaque or crevicular fluid samples of patients by light microscopy, varying numbers of intensely stained spirochetes of different sizes were observed (Figs. 1,2). More spirochetes were usually seen in samples from diseased sites. After drying replicate PATS-stained coverslips with hexamethyldisilazane they were sputter coated with gold, and. then examined by the SEI and BEI modes of scanning electron microscopy (Figs. 3-6). A permanent record of the proportions of large, medium and small spirochetes at each site could thus be obtained. Generally, greater numbers of gram negative bacteria including some spirochetes were stained in samples from diseased sites. At some sites, however, spirochetes were the predominant microbes in both crevicular fluid and subgingival plaque (Fig. 1).