A simplified method for detecting Gram-positive and Gram-negative bacteria in dental histological samples: A preliminary and comparative study

Objective: Bacteria identification inside the dental tissue is a complex procedure requiring specific protocols. This study aimed to compare two classical Gram staining methods with a new staining method proposed by the authors to detect Gram-positive and Gram-negative bacteria in dental histological samples of human dentin. Methods: Ten human teeth, extracted because of various pathologies, were decalcified, dehydrated, and paraffin-embedded. Then, approximately 100 serial sections of 4 µm thickness were made per sample. The serial sections were placed on glass slides and were stained according to Brown-Brenn, Brown-Hopps, and a proposed modification of Brown-Brenn staining. Both ATCC strains, smeared on glass slides, were stained following each method's instructions used in histological samples. Results: From a qualitative evaluation, the Brown-Brenn method resulted in better staining of Gram-positive bacteria, while the authors' proposed staining technique was more oriented towards Gram-negative bacteria. On the other hand, the Brown-Hopps showed quite a balance in detecting Gram-positive and Gram-negative bacteria. Unlike the Brown-Brenn stain, the other two protocols showed better stainability of Gram-negative microorganisms in bacterial-smeared samples. Conclusion: All staining techniques evaluated in this article can identify bacteria, but the outcome can change according to the staining procedure used.