Effectiveness Of Various Decalcification Agents On Quality Beef Rib Bone Tissue (Bos Taurus): A Comparative Study Of Formic Acid, Nitric Acid, And Their Combination With EDTA

Alifah Putri Safina; Charissa Virginia Serhalawan; Christian Pairan; Jessica Rose Whaite; Fransisca Probo Setyoningrum; Diana Intan Gabriella Lusiasa

doi:10.63922/ijevss.v5i03.5429

Alifah Putri Safina Universitas Pelita Harapan
Charissa Virginia Serhalawan Universitas Pelita Harapan
Christian Pairan Universitas Pelita Harapan
Jessica Rose Whaite Universitas Pelita Harapan
Fransisca Probo Setyoningrum Universitas Pelita Harapan
Diana Intan Gabriella Lusiasa Universitas Pelita Harapan

DOI: https://doi.org/10.63922/ijevss.v5i03.5429

Keywords: Decalcification, beef rib, formic acid, nitric acid, EDTA, histotechnology

Abstract

Decalcification is the process of removing calcium salts and other minerals from hard tissue such as bone to produce histological specimens that can be sectioned and observed effectively. Selecting the right decalcifying agent is crucial because it must soften the tissue without damaging its morphology. This study aimed to compare the effectiveness of various decalcifying agents on the quality of beef rib bone tissue (Taurus boss) using formic acid, nitric acid, and a combination with ethylenediaminetetraacetic acid (EDTA). The study was conducted in a comparative experimental manner using six treatment groups, namely 5% formic acid, concentrated formic acid, 15% nitric acid, 15% nitric acid + 10% EDTA, concentrated nitric acid + 10% EDTA, and concentrated nitric acid. The effectiveness of decalcification was evaluated based on the level of bone hardness using the needle method, changes in tissue morphology, and the duration of treatment. The results showed that 5% formic acid and concentrated formic acid were not able to soften bones optimally. 15% nitric acid produced soft bones after five days of immersion, while the combination of 15% nitric acid and 10% EDTA did not show optimal results. The combination of concentrated nitric acid and 10% EDTA produced slightly soft bones with minimal tissue damage compared to concentrated nitric acid without EDTA. In contrast, the use of concentrated nitric acid without EDTA caused tissue shrinkage, residue formation, and morphological damage. Based on the research results, the effectiveness of decalcification was influenced by the type and concentration of decalcification agent, with 15% nitric acid and a combination of 10% concentrated nitric acid–EDTA showing better results than other treatments.

References

[1] W. Widiawati, A. Durachim, W. Wiryanti, and M. F. Solihat, “Pengaruh Waktu Dekalsifikasi Jaringan Tulang Menggunakan Larutan Asam Nitrat 5% terhadap Tingkat Kelunakan dan Kualitas Preparat,” Jimmi J. Ilm. Mhs. Multidisiplin, vol. 2, no. 1, pp. 78–92, Feb. 2025, doi: 10.71153/jimmi.v2i1.222.
[2] S. K. Suvarna, C. Layton, and J. D. Bancroft, Bancroft’s Theory And Practice Of Histological Techniques, 7th Editio. Oxford: Elsevier Limited, 2013.
[3] D. Sivakumar, N. Ghani, M. Das Murtey, and R. Ramli, “Decalcification Agents for Histological Analysis of Human Dental Tissues: A Review,” Asian J. Med. Biomed., vol. 9, no. 1, pp. 132–146, May 2025, doi: 10.37231/ajmb.2025.9.1.788.
[4] J. Morcilla, L. Quinto, I. Al Bozom, A. Adham, and K. Murshed, “Investigation of an appropriate decalcifying agent for bone biopsy specimens submitted for histomorphologic analyses and immunohistochemical studies,” Int. J. Heal. Bus. Anal., vol. 1, no. 2, pp. 1–14, Jan. 2026, doi: 10.65166/y1s38453.
[5] C. Broomfield, N. Meis, J. Johnson, D. Regan, K. McGilvray, and C. Puttlitz, “Optimization of ovine bone decalcification for increased cellular detail: a parametric study,” J. Histotechnol., vol. 45, no. 1, pp. 29–35, Jan. 2022, doi: 10.1080/01478885.2021.1951053.
[6] S. Pang, F. Y. Su, A. Green, J. Salim, J. McKittrick, and I. Jasiuk, “Comparison of different protocols for demineralization of cortical bone,” Sci. Rep., vol. 11, no. 1, p. 7012, Mar. 2021, doi: 10.1038/s41598-021-86257-4.
[7] K. Bogoevski, A. Woloszyk, K. Blackwood, M. A. Woodruff, and V. Glatt, “Tissue Morphology and Antigenicity in Mouse and Rat Tibia: Comparing 12 Different Decalcification Conditions,” J. Histochem. Cytochem., vol. 67, no. 8, pp. 545–561, Aug. 2019, doi: 10.1369/0022155419850099.
[8] R. T. Basting, M. H. Napimoga, J. M. de Lima, N. S. de Freitas, and J. T. Clemente-Napimoga, “Fast and accurate protocol for histology and immunohistochemistry reactions in temporomandibular joint of rats,” Arch. Oral Biol., vol. 126, p. 105115, Jun. 2021, doi: 10.1016/j.archoralbio.2021.105115.
[9] R. Permatasari, F. V. Putra, and B. Yoharli, “Pengaruh Konsentrasi Asam Nitrat Sebagai Reagen Dekalsifikasi Terhadap Kualitas Pewarnaan Safranin O Pada Tulang,” BIOMA J. Biol. Makassar, vol. 10, no. 2, pp. 53–63, 2025, doi: 10.20956/bioma.v10i2.44183.
[10] F. M. Savi, G. I. Brierly, J. Baldwin, C. Theodoropoulos, and M. A. Woodruff, “Comparison of Different Decalcification Methods Using Rat Mandibles as a Model,” J. Histochem. Cytochem., vol. 65, no. 12, pp. 705–722, Dec. 2017, doi: 10.1369/0022155417733708.
[11] E. Miquelestorena-Standley et al., “Effect of decalcification protocols on immunohistochemistry and molecular analyses of bone samples,” Mod. Pathol., vol. 33, no. 8, pp. 1505–1517, Aug. 2020, doi: 10.1038/s41379-020-0503-6.
[12] H. Liu et al., “Evaluation of Decalcification Techniques for Rat Femurs Using HE and Immunohistochemical Staining,” Biomed Res. Int., vol. 2017, pp. 1–6, 2017, doi: 10.1155/2017/9050754.
[13] D. H. Chow, L. Zheng, L. Tian, K.-S. Ho, L. Qin, and X. Guo, “Application of ultrasound accelerates the decalcification process of bone matrix without affecting histological and immunohistochemical analysis,” J. Orthop. Transl., vol. 17, pp. 112–120, Apr. 2019, doi: 10.1016/j.jot.2018.08.001.