Effect of histological decalcifying agents on number and stainability of gram-positive bacteria

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.