A dual-mode chromogenic and fluorescent in situ hybridization approach for bacterial 16S rRNA detection in oral formalin-fixed paraffin-embedded samples

The combined use of fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) targeting bacterial 16S rRNA might facilitate the discrimination of bacterial signals from background noise, supporting the interpretation of bacterial spatial localization in tissues. In this study, we applied a fluorescein isothiocyanate-labeled universal 16S rRNA probe to both FISH and CISH to enable direct comparison of these methods using oral formalin-fixed paraffin-embedded (FFPE) samples. FISH and CISH were performed on saliva-derived and tongue FFPE samples. Because formalin fixation can reduce hybridization efficiency, the effect of the duration of fixation on 16S rRNA detection was evaluated using FFPE samples prepared from cultured bacteria. Both FISH and CISH generated clear positive signals with comparable staining patterns in saliva-derived FFPE samples, accompanied by a strong positive correlation between their signal area ratios. 16S rRNA detectability was maintained after formalin fixation for up to 72 h, and both methods visualized the bacterial localization in human tongue FFPE samples. Thus, this approach permits comparison of FISH and CISH staining patterns and enables complementary chromogenic evaluation of bacterial localization findings obtained by FISH, as CISH is not affected by tissue autofluorescence. The preservation of 16S rRNA detectability after formalin fixation for up to 72 h suggests that this approach may be applicable to clinical FFPE samples fixed for similar durations. In addition, inexpensive oligonucleotide probes and the ability of CISH to produce permanent slides using routine pathology equipment enhance its practical applicability. This method might represent a useful platform for evaluating the spatial localization of bacteria in oral FFPE samples.