DETECTION OF MUTANS STREPTOCOCCI IN SECONDARY CARIOUS LESIONS USING IMMUNOFLUORESCENT TECHNIQUES AND CONFOCAL LASER-SCANNING MICROSCOPY

Secondary caries is one of the major reasons causing restoration failure; however, little is known of its microbial etiology, mainly because of the difficulties in eliminating bacterial contamination during collection and sample preparation. Therefore, the purpose of this study was to investigate the feasibility of immunofluorescent techniques and confocal laser scanning microscopy for identification and quantification of bacteria in secondary carious lesions. Thirty-six extracted human teeth, clinically diagnosed as having secondary caries, were used in the study. The teeth were sectioned in half across the secondary carious lesion. One half of each tooth was processed using the Brown and Hopps histologic staining method for bacterial detection. Sections (100 mu m thick) were obtained from the other half of each tooth for immunofluorescence labeling to detect and identify mutans streptococci in the subsurface of the lesion using confocal imaging techniques. Mutans streptococci were detected in 88.9% of the samples analyzed with the immunofluorescent technique. The Brown and Hopps histopathologic examination demonstrated evidence of bacteria in 94.4% of the samples. In addition, quantification of bacteria was conducted by digitalization of confocal images. The results indicated that the immunofluorescence confocal laser scanning microscopy technique was sensitive and specific for detection and quantification of mutans streptococci in secondary carious lesions.