Immuno-electron microscopic identification of a dental plaque microorganism: Streptococcus mutans

The aim of the present investigation was to test a procedure useful for the electron-microscopic in situ identification of the presumptive cariogenic microorganism Streptococcus mutans (serotype d) growing in the human dental plaque. For this purpose, different parameters of an indirect immunohistochemical method were tested in three sets of experiments. In experiment set I, all serological and histochemical procedures were performed en bloc on specimens fixed only with glutaraldehyde before embedding in Vestopal W. Different marking substances, such as ferritin, alkaline phosphatase and horseradish peroxidase (HRP) were tested. The en bloc method using HRP-labelled antibodies was found useful for staining of in vitro-grown bacteria but failed when applied to dental plaque. In experiment set II, ultrathin sections of glutaraldehyde-fixed and glycol methacrylate-embedded in vitro-grown bacteria were section-stained. The bacteria became outlined with a highly electron-dense HRP reaction product which accumulated on top of the cell envelope. The same type of HRP reaction product was found on some bacteria in the 2-day-old dental plaque after section-staining. This system was further tested in a series of controls also using consecutive serial sections. In exp. set III, a number of different stationary and transient oral bacteria were immunohistochemically section-stained. A cross-reaction with bacteria belonging to S.salivarius was discovered and removed by absorbing the anti-S.mutans serotype d serum with S. salivarius (NCTC 8618). The present work described the distribution of horseradish peroxidase reaction product obtained after immunohistochemical staining of consecutive ultra-thin sections of 5-day-old experimental dental plaques using differently absorbed anti-S.mutans sobrinus, anti-S.mutans mutans, and normal rabbit γ-globulins. It was found that an in situ identification of S.mutans subspecies sobrinus could be obtained using thoroughly absorbed anti-S. mutans sobrinus γ-globulins and consecutive sections. The anti-S.mutans mutans γ-globulins were after tests on pure cultures of several oral bacteria found to cross-react with S.milleri. Furthermore, crude and absorbed normal rabbit γ-globulins were observed to contain antibodies that cross-reacted with several plaque microorganisms.