Journal of Environmental Health Sciences (한국환경보건학회지)

Korean Society of Environmental Health (한국환경보건학회)
권호 표지
DOI QR코드

DOI QR Code

Determining the Proportions of Bone and Cartilage Growth in the Crucian Carp (carassius auratus) Using the Modified Simultaneous Differential Staining Technique

  • Lee, Jin-Heon (Department of Environmental Education, Kongju National University)
  • Received : 2010.04.25
  • Accepted : 2001.08.20
  • Published : 2010.08.30
Download PDF
⟨ Previous Next ⟩

Abstract

The modified simultaneous differential staining technique, which enables double staining of cartilage and bones, needs to be improved to prevent soft tissues from being damaged during the staining process. Key factors influencing the extent to which soft tissues are damaged include the fixative used, macerating time, potassium hydroxide concentration, incubation temperature and the removal of skin from specimens. Here we describe a protocol that enables the hardening of tissues during bleaching and maceration. We also describe a method for objectively measuring rates of cartilage and bone growth. The use of formalin as a fixative rendered soft tissues more rigid due to the resulting chemical bonds formed between proteins. Blotted specimens were immersed in 1% potassium hydroxide (KOH) and incubated at $37^{\circ}C$ for 1 day (smaller specimens) or 2-3 days (larger specimens). The 1% KOH solution was also used as the diluent solution for the subsequent immersion in a graded series of 30%, 50%, 70%, 90%, 100% glycerol solutions, a procedure that made soft tissues even more transparent and hardened. It was not necessary to remove the skin of specimens shorter than 2 cm, since the macerating solution could easily penetrate their thin skin layer and continuously remove those pigments hindering visibility. Since excessive osmosis is another factor that can damage soft tissues in the macerating process by causing the rupture of those cells not able to withstand the osmotic pressure, here it was minimized by balancing the salt concentration between the interior and exterior of cells with the addition of 0.9% sodium chloride (NaCl) in the macerating solution. Finally, to determine the proportions of cartilage and bone growth, photographs of the stained specimens were taken with a dissecting microscope and sections corresponding to the cartilage and bones were cut out from the printed pictures and weighed. Our results show that this method is suitable for the objective evaluation of bone and cartilage growth.

Keywords

References

  1. Al-Saraj, A. : Use of saturated sodium chloride solution as a tissue fixative. Iraqi Journal of Veterinary Sciences, 24(1), 53-58, 2010.
  2. Baker, J. R. : Principles of biological microtechnique. London: Methuen. 1-368, 1958.
  3. Borg, F. G. : What is osmosis? Explanation and understanding of a physical phenomenon: General Physics, Jyvaskyla University, Chydenius Institute, Karleby, Finland, 1-39, 2003.
  4. Crary, D. D. : Modified benzyl alcohol clearing of alizarin stained specimens without loss of flexibility. Stain Technology, 37, 124-125, 1962.
  5. Dawson, A. B. : A note on the staining of the skeleton of cleared specimens with alizarin red S. Stain Technology, 1, 123-124, 1926.
  6. Dingerkus, G. and Uhler, L. D. : Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage. Stain Technolology, 51, 229-232, 1977.
  7. Green, M. C. : A rapid method for clearing and staining specimens for the demonstration of bone. Ohio Journal of Science, 52, 21-33, 1952.
  8. Inouye, M. : Differential staining of cartilage and bone in fetal mouse skeleton by alcian blue and alizarin red S. Congenital Anomalies, 16, 171-173, 1976.
  9. Kelly, W. L. and Bryden, M. M. : A modified differential stain for cartilage and bone in whole mount preparations of mammalian fetus and small vertebrates. Stain Technology, 58, 131-134, 1983.
  10. Kimmel, C. A. and Trammell, C. : A rapid procedure for routine double staining of cartilage and bone in fetal and adult animals. Stain Technology, 56(5), 271-273, 1981.
  11. Lee, J. H., Park, K. L. and Kim, D. S. : Protective effects of Korean garlic juice against the toxicity of methyl mercuric chloride in relation to fetal ossification in pregnant fischer-344 rats. Korea Journal of Environmental Health Sciences, 34(2), 161-169, 2008. https://doi.org/10.5668/JEHS.2008.34.2.161
  12. Mcleod, M. J. : Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology 22, 299-301, 1980. https://doi.org/10.1002/tera.1420220306
  13. Park, E. H. and Kim, D. S. : A procedure for staining cartilage and bone of whole vertebrate larvae while rendering all other tissues transparent. Stain Technology, 59(5), 269-172, 1984.
  14. Skoog, D. A. : Chromatography: Principles of instrumental analysis, 3rd. Saunders College publishing, 653-679, 1991.
  15. Staples, R. E. and Schnell, V. L. : Refinements in rapid clearing technique in KOH-alizarine red S method for fetal bone. Stain Technology, 39, 61-63, 1964.
  16. Wassersug, R. J. : A procedure for differential staining of cartilage and bone in whole formalinfixed verterates. Stain Technolology, 51(2), 131-134, 1976.
  17. Webb, G. N. and Byrd, R. A. : Simultaneous differential staining of cartilage and bone in rodent fetuses: an alcian blue and alizarin red S procedure without glacial acetic acid. Biotechnic and Histochemistry, 69(4), 181-185, 1994. https://doi.org/10.3109/10520299409106284
  18. Whitaker, J. and Dix, K. M. : Double staining techniques for rat fetus skeletons in teratological studies. Lab Animals, 13, 309-310, 1979. https://doi.org/10.1258/002367779780943233
  19. Wikipedia : Cytolysis, the free encyclopedia, http://en.wikipedia.org, 2010.