Driving the Mineral out Faster: Simple Modifications of the Decalcification Technique

Abstract

Introduction: Quicker decalcification is essential for faster diagnosis of hard tissue pathology. Heat and mechanical agitation are known to hasten decalcification.

Aim: To compare the rate of decalcification, cellular and staining characteristics of decalcified specimens of bone and teeth by using the conventional method (10% formal formic acid), heating to 45(o)C and by physical agitation with magnetic stirrer.

Materials and methods: Weight-matched samples of caprine-origin bone (n=15) and teeth (n=15) were decalcified using three methods namely: a) Gooding and Stewart's fluid; b) Gooding and Stewart's fluid heated to 45(o)C for 6 hours daily; and c) Gooding and Stewart's fluid agitated using a magnetic stirrer for 6 hours daily. Non-lesional skin tissue samples were placed along with each specimen. End point of decalcification (chemical test) was noted; 4 micron sections were taken and stained with H&E.

Statistical analysis: Differences in rate of decalcification and staining characteristics were assessed by Kruskal Wallis test and chi-square test respectively.

Results: Hard tissues decalcified faster with stirring and heating methods. The amount of osteocyte retraction noted in bone was significantly reduced in the stirring method. In tooth specimens, modified techniques resulted in poorer nuclear-cytoplasmic contrast of pulp cells. Heating affected the odontoblast layer. Soft tissues exhibited higher eosinophilia in stirring and conventional methods, whereas nuclear-cytoplasmic contrast and chromatin staining was poorest in heating and conventional methods.

Conclusion: Physical agitation of decalcifying fluid may be recommended while maintaining satisfactory quality of tissue morphology and staining.

Keywords: Bone; Histology; Tooth.

[Table/Fig-1]:

Sample distribution

[Table/Fig-5]:

Left: Bone specimen decalcified by the conventional method exhibiting intact osteocytes within lacunae. Right: Bone specimen decalcified by the heat method exhibiting osteocyte retraction. (H&E, 100X) Inset shows magnified image (400X)

[Table/Fig-6]:

Tooth decalcified by the conventional method: (a) exhibiting pulp cells which demonstrate good nuclear contrast. Nuclear contrast of pulp cells was diminished in the (b) stirring and (c) heating methods. (H&E, 400X)