New Bacterial Staining and Decalcification Methods Applied to Bone-Tooth Tissues

Introduction. Decalcification of teeth and bone is a crucial phase during histological processing of these tissues. The effect of decalcifying substances on the tissue and its staining characteristics are significant considerations, which impact the selection of decalcifying agents. Gram and Brown-Breen stains are used to detect bacteria; however, it is not usual to perform bacterial staining on bone and teeth. Objective. The purpose of this study was to modify the Brown-Breen stain and develop a new method to decalcify bone and teeth tissues in a period of 5 days. Methods. The new staining technique was standardized with 90 samples; furthermore, these tissues were decalcified with the new method using: trichloroacetic acid (20%), neutralizing with soapy water (tris (hydroxymethyl) aminomethane) and peptides solution; and sample processing with: 50^o, 70^o, 80^o and 96^o ethyl alcohol (1h, each one), isopropyl alcohol, Xylem (2h), embedding in paraffin at 56^oC (1h). Results. Nighty six percent of the samples could be cut and stained without problems. Bacteria were detected in 94.5% of samples. About the decalcification time using the new method: we obtained decalcified incisors and canines teeth in 5 days; while for the premolars, molars teeth and bone, ten days were required. Discussion and conclusion. In general, decalcification methods have duration of three weeks or longer. With the decalcification method proposed, time was saved and this allowed the assessment of dental caries and osteomyelitis using the new stain.