Wijnbergen M, Van Mullem P J
J Dent Res. 1987 May;66(5):1029-31. doi: 10.1177/00220345870660050701.
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.
在研究修复材料对牙髓的影响的测试中，评估细菌污染很重要，而这通常通过组织学方法进行。在对硬组织标本进行常规石蜡包埋以进行显微镜检查之前，需进行脱钙处理。为了研究脱钙剂（硝酸、甲酸和乙二胺四乙酸）对细菌数量和革兰氏染色性的影响，我们使用了一个由甲醛固定的粪肠球菌悬浮液组成的模型系统。革兰氏阳性菌分别保存在蒸馏水中、4%甲醛溶液中或脱钙剂中，保存不同的实验时间。通过血细胞计数器进行计数，涂片用布朗和布伦染色法染色。经过牙齿脱钙的平均时间后，发现细菌数量和革兰氏阳性染色性均大幅减少。在甲酸中保存一周后，15个细菌中只有1个染成蓝色。在硝酸和乙二胺四乙酸中，细菌数量减少得较少。由于在组织切片中只能清楚地检测到染成蓝色的细菌，这些实验结果表明，在细菌数量有限的情况下，脱钙硬组织切片存在假阴性评分的风险。
