title: Effect of histological decalcifying agents on number and stainability of gram-positive bacteria.
abstract: In tests of the effects of restorative materials on dental pulp, it is important that one evaluate bacterial contamination, and this is usually done histologically. Preceding the usual paraffin-embedding of hard-tissue specimens for microscopical investigations, decalcification is performed. To study the influence of decalcifying agents (nitric acid, formic acid, and ethylenediamine tetraacetic acid) on the number and Gram-stainability of bacteria, we used a model system consisting of suspensions of formaldehyde-fixed Streptococcus faecalis. The Gram-positive organisms were stored in distilled water, in 4% formaldehyde solution, or in the decalcifying agents for various experimental periods. Counts were made by means of a hemocytometer, and smears were stained with the Brown and Brenn staining method. After periods which are averages for the decalcification of teeth, severe reductions of both the number and the Gram-positive stainability were found. After one week in formic acid, only one out of 15 organisms stained blue. With nitric acid and EDTA, the reductions were fewer. Since only blue-staining bacteria can be detected clearly in tissue sections, the results of these experiments indicate that, with limited numbers of organisms, the risk exists for false-negative scores for decalcified hard-tissue sections.
mesh_terms: D004492:Edetic Acid; D013293:Enterococcus faecalis / Q000302:isolation & purification; D005404:Fixatives; D005557:Formaldehyde; D005561:Formates; D006094:Gram-Positive Bacteria / Q000302:isolation & purification*; D006652:Histological Techniques; D009566:Nitrates; D017942:Nitric Acid; D013194:Staining and Labeling
chemical_list: D005404:Fixatives; D005561:Formates; D009566:Nitrates; C030544:formic acid; D005557:Formaldehyde; D017942:Nitric Acid; D004492:Edetic Acid