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.
It has previously been shown experimentally using S. faecalis that the relative number of blue-staining Gram-positive bacteria was reduced by demineralizing agents as well as by disinfection (Wijnbergen & van Mullem 1987, van Mullem & Wijnbergen 1989). In this study the cumulative effect of disinfection, experimental period, fixation and demineralization was investigated. The use of additives to the fixative and the demineralizing agents to limit the loss of blue-staining bacteria was also studied. The numbers of bacteria were determined using a haemocytometer, and the percentage of blue-staining organisms were ascertained from smears. When compared with the start of the experiment, the sequence of disinfection with chlorhexidine, storage in PBS, fixation with neutral formaldehyde and demineralization with formic acid or EDTA appeared to reduce the relative numbers of Gram-positive staining bacteria. For storage periods of 0 and 4 days the reduction factors were 100 and 600, respectively, using formic acid. These factors were 50 and 95, respectively, using EDTA. Addition of cetyl pyridinium chloride to the fixative and the demineralizing agents, and addition of neutral formaldehyde to the demineralizing agents lowered these reduction factors to 80 and 200, respectively, for formic acid, and 40 and 85, respectively, for EDTA. If the results are extrapolated to animal experiments where disinfection, experimental period, fixation and demineralization form part of the experimental framework, even the lowest reduction in the number of blue-staining bacteria could lead to false interpretation of tissue sections.
Eronat N, van Mullem PJ, Wijnbergen M. An enzyme histochemical study of the infiuence of formocresol and calcium hydroxide on the dental pulp. Endod Dent Traumatol 1988; 4: 37-43.Abstract -Sections of goat incisor pulps were studied after a 5min application of formocresol (FC) or after calcium hydroxide or saline/ZnOE treatment followed by freeze-sectioning. After 3-7 days the incisal part of the pulp was necrotic, as shown by absence of lactate dehydrogenase (LDH) activity. The necrotic zone was much larger after FC than after saline/ZnOE or calcium hydroxide. The LDH negative area after application of FC appeared to consist of a layer showing nuclei that were wellpreserved by the formaldehyde from the FC, and an area of tissue breakdown, which showed no or a reduced structure according to hematoxylin atid eosin. Well-preserved nuclei were not found in the 2 other groups. After 14 days all necrotic tissue, except that subjacent to calcium hydroxide, had been replaced by vital (LDH-positive) tissue. After saline/ZnOE or calcium hydroxide treatment, hard-tissue formation was found in all teeth, and after FC treatment in the majority of teeth.
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