Skip to main content
SpringerLink
Log in

Comparison of the Ruthenium hexammine trichloride method to other methods of chemical fixation for preservation of avian physeal cartilage

  • Papers
  • Published:
The Histochemical Journal Aims and scope Submit manuscript

Summary

Several methods of chemical fixation of avian physeal cartilage were compared. The Ruthenium hexammine trichloride method was compared to isotonic glutaraldehyde and neutral buffered formalin for light microscopy and paraffin embedment, and to two osmium-ferrocyanide methods and a combination of 1% glutaraldehyde and 4% formaldehyde for electron microscopy. Only the Ruthenium hexammine trichloride method prevented the loss of matrix proteoglycans and shrinkage of chondrocytes. In undecalcified paraffin-embedded cartilage, preservation of matrix and cellular detail was excellent, but Ruthenium hexammine trichloride interfered with Haematoxylin and Eosin staining. Glutaraldehyde gave more intense eosinophilia than neutral buffered formalin. Ultrastructurally, the Ruthenium hexammine trichloride method was the most consistent and gave the best overall fixation. Matrix elements and cellular and nuclear membranes were well preserved. It did result in vacuolation of the cytoplasm and mitochondria, and it increased granularity of the cytoplasm, chromatin, and rough endoplasmic reticulum. Other fixatives produced minimal vacuolation and finer granularity, but preservation was less consistent, cell/matrix contrast was often excessive, and they caused shrinkage of all chondrocytes. Large dilatations of the rough endoplasmic reticulum that appear to be cytoplasmic inclusions by light microscopy are described for the first time in avian cartilage.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Akisaka, T., Kawaguchi, H., Subita, G. P., Shigenaga, Y. &Gay, C. V. (1988a) Ultrastructure of matrix vesicles in chick growth plate as revealed by quick freezing and freeze substitution.Calcif. Tissue Int. 42, 383–93.

    PubMed  Google Scholar 

  • Akisaka, T., Subita, G. P. &Shigenaga, Y. (1988b) Surface modifications at the periosseus region of chick osteoclast as revealed by freeze-substitution.Anat. Rec. 222, 323–32.

    PubMed  Google Scholar 

  • Björnsson, S. &Heinegård, D. (1981) Assembly of proteoglycan aggregates in cultures of chondrocytes from bovine tracheal cartilage.Biochem. J. 199, 17–29.

    PubMed  Google Scholar 

  • Bone, Q. &Ryan, K. P. (1972) Osmolarity of osmium tetroxide and glutaraldehyde fixatives.Histochem. J. 4, 331–47.

    PubMed  Google Scholar 

  • Carlson, C. S., Hilley, H. D. &Meuten, D. J. (1989) Degeneration of cartilage canal vessels associated with lesions of osteochondrosis in swine.Vet. Pathol. 26, 47–54.

    PubMed  Google Scholar 

  • Engfeldt, E. &Hjertquist, S. O. (1968) Studies on the epiphyseal growth zone. I. The preservation of acid glycosaminoglycans in tissues in some histotechnical procedures for electron microscopy.Virchows Arch. [B] 1, 222–9.

    Google Scholar 

  • Farnum, C. E. &Wilsman, N. J. (1983) Pericellular matrix of growth plate chondrocytes: a study using postfixation with osmium-ferrocyanade.J. Histochem. Cytochem. 31, 765–75.

    PubMed  Google Scholar 

  • Farnum, C. E. &Wilsman, N. J. (1986) Ultrastructural histochemical evaluation of growth plate cartilage matrix from healthy and osteochondritic swine.Am. J. Vet. Res. 47, 1105–15.

    PubMed  Google Scholar 

  • Farnum, C. E. &Wilsman, N. J. (1987) Morphologic stages of the terminal hypertrophic chondrocyte of growth plate cartilage.Anat. Rec. 219, 221–32.

    PubMed  Google Scholar 

  • Fell, H. B. (1925) The histogenesis of cartilage and bone in the long bones of the embryonic fowl.J. Morphol. Physiol. 40, 417–59.

    Google Scholar 

  • Hargest, T. E., Gay, C. V., Schraer, H. &Wasserman, A. J. (1985a) Vertical distribution of elements in cells and matrix of epiphyseal growth plate cartilage determined by quantitative electron probe analysis.J. Histochem. Cytochem. 33, 275–86.

    PubMed  Google Scholar 

  • Hargest, T. E., Leach, R. M. &Gay, C. V. (1985b) Avian tibial dyschondroplasia. I. Ultrastructure.Am J. Pathol. 119, 175–90.

    PubMed  Google Scholar 

  • Haynes, J. S. &Walser, M. M. (1986) Ultrastructure ofFusarium-induced tibial dyschondroplasia in chickens: a sequential study.Vet. Pathol. 23, 499–505.

    PubMed  Google Scholar 

  • Haynes, J. S., Walser, M. M. &Lawler, E. M. (1985) Morphogenesis ofFusarium sp-induced tibial dyschondroplasia in chickens.Vet. Pathol. 22, 629–36.

    PubMed  Google Scholar 

  • Hopwood, D. (1973) Theoretical and practical aspects of glutaraldehyde fixation. InFixation in Histochemistry (edited byStoward, P. J.) pp. 47–83. London: Chapman and Hall.

    Google Scholar 

  • Howlett, C. R. (1973) The fine structure of the proximal growth plate of the avian tibia.J. Anat. 128, 377–99.

    Google Scholar 

  • Howlett, C. R. (1980) The fine structure of the proximal growth plate and metaphysis of the avian tibia: endochondral osteogenesis.J. Anat. 130, 745–68.

    PubMed  Google Scholar 

  • Hunziker, F. B. &Schenk, R. K. (1984) Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. II. Intercellular matrix ultrastructure-p-preservation of proteoglycans in their native state.J. Cell Biol. 98, 277–82.

    PubMed  Google Scholar 

  • Hunziker, E. B. &Schenk, R. K. (1987) Structural organization of proteoglycans in cartilage. InBiology of Proteoglycans (edited byWight, T. N. &Mecham, R. P.) pp. 155–85. Orlando, San Diego, New York, Austin, Boston, London, Sydney, Tokyo, Toronto: Academic Press, Inc.

    Google Scholar 

  • Hunziker, E. B., Herrmann, W. &Schenk, R. K. (1982) Improved cartilage fixation by ruthenium hexammine trichloride (RHT): a prerequisite for morphometry in growth cartilage.J. Ultrastruct. Res. 81, 1–12.

    PubMed  Google Scholar 

  • Hunziker, E. B., Herrmann, W. &Schenk, R. K. (1983) Ruthenium hexammine trichloride (RHT)-mediated interaction between plasmalemmal components and pericellular matrix proteoglycans is responsible for the preservation of chondrocytic plasma membranesin situ during cartilage fixation.J. Histochem. Cytochem. 31, 717–27.

    PubMed  Google Scholar 

  • Hunziker, E. B., Herrmann, W., Schenk, R. K., Mueller, M. &Moor, H. (1984) Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. I. Chondrocyte ultrastructure-implications for the theories of mineralization and vascular invasion.J. Cell Biol. 98, 267–76.

    PubMed  Google Scholar 

  • Hwang, W., McQueen, D., Monson, R. C. &Reed, M. H. (1982) The significance of cytoplasmic chondrocyte inclusions in multiple osteochondromatosis, solitary osteochondromas, and chondrodysplasias.Am. J. Clin. Pathol. 78, 89–91.

    PubMed  Google Scholar 

  • Kashiwa, H. K., Luchtel, D. I. &Park, H. Z. (1975) Chondroitin sulfate and electron lucent bodies in the pericellular rim about unshrunken hypertrophied chondrocytes of chick long bone.Anat. Rec. 183, 359–72.

    PubMed  Google Scholar 

  • Kimura, J. H., Hardingham, T. E., Hascall, V. C. &Solrush, M. (1979) Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the Swarm rat chondrosarcoma.J. Biol. Chem. 254, 2600–9.

    PubMed  Google Scholar 

  • Lawler, E. M., Fletcher, T. F. &Walser, M. M. (1985) Chondroclasts inFusarium-induced tibial dyschondroplasia: a histomorphometric study.Am. J. Pathol. 120, 276–81.

    PubMed  Google Scholar 

  • Lewinson, D. (1989) Application of the ferrocyanidereduced osmium method for mineralizing cartilage: further evidence for the enhancement of intracellular glycogen and visualization of matrix components.Histochem. J. 21, 259–70.

    PubMed  Google Scholar 

  • Luft, J. H. (1965) The fine structure of hyaline cartilage matrix following ruthenium red fixative and staining.J. Cell Biol. 27, 61A.

    Google Scholar 

  • Lutfi, A. M. (1971) The fate of chondrocytes during cartilage erosion in the growing tibia in the domestic fowl (Gallus domesticus).Acta Anat. (Basel) 79, 27–35.

    Google Scholar 

  • Lutfi, A. M. (1974) The ultrastructure of cartilage cells in the epiphyses of long bones in the domestic fowl.Acta Anat. (Basel) 87, 12–21.

    Google Scholar 

  • McDowell, E. M. &Trump, B. F. (1976) Histologic fixatives suitable for diagnostic light and electron microscopy.Arch. Pathol. Lab. Med. 100, 405–14.

    PubMed  Google Scholar 

  • Paulsson, M. &Heinegård, D. (1981) Furification and structural characterization of a cartilage matrix protein.Biochem. J. 197, 367–75.

    PubMed  Google Scholar 

  • Pearse, A. G. E. (1980)Histochemistry: Theoretical and Applied. Vol. 1, 4th edn. pp. 97–158. Edinburgh, London, New York: Churchill Livingstone.

    Google Scholar 

  • Pottenger, L. A., Webb, J. E. &Lyon, N. B. (1985) Kinetics of extraction of proteoglycans from human cartilage.Arthritis Rheum 28, 323–30.

    PubMed  Google Scholar 

  • Reynolds, E. S. (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy.J. Cell Biol. 17, 208–12.

    PubMed  Google Scholar 

  • Riddell, C. (1975) The development of tibial dyschondroplasia in broiler chickens.Avian Dis. 19, 443–62.

    PubMed  Google Scholar 

  • Schofield, B. H., Williams, B. R. &Doty, S. B. (1975) Alcian blue staining of cartilage for electron microscopy. Application of the critical electrolyte concentration principle.Histochem. J. 7, 139–49.

    PubMed  Google Scholar 

  • Scott, J. E., Quintarelli, G. &Dellovo, M. C. (1964) The chemical and histochemical properties of alcian blue. I. The mechanism of alcian blue staining.Histochemie 4, 73–85.

    PubMed  Google Scholar 

  • Sheehan, D. C. &Hrapchak, B. B. (1980)Theory and Practice of Histotechnology, 2nd edn. pp. 46, 172–3. St Louis, Toronto, London: C. V. Mosby Company.

    Google Scholar 

  • Shepard, N. &Mitchell, N. (1976a) The localization of proteoglycan by light and electron microscopy using safrinin o.J. Ultrastruct. Res. 54, 451–60.

    PubMed  Google Scholar 

  • Shepard, N. &Mitchell, N. (1976b) Simultaneous localization of proteoglycans by light and electron microscopy using toluidine blue o: a study of epiphyseal cartilage.J. Histochem. Cytochem. 24, 621–9.

    PubMed  Google Scholar 

  • Sorrell, J. M. &Weiss, L. (1980) A light and electron microscopic study of the region of cartilage resorption in the embryonic chick femur.Anat. Rec. 198, 513–30.

    PubMed  Google Scholar 

  • Spurr, A. R. (1969) A low viscosity epoxy resin embedding medium for electron microscopy.J. Ultrastruct. Res. 26, 31–43.

    PubMed  Google Scholar 

  • Szirmai, J. A. (1963) Quantitative approaches in the histochemistry of mucopolysaccharides.J. Histochem. Cytochem. 11, 24–34.

    Google Scholar 

  • Thyberg, J., Lohmander, S. &Friberg, U. (1973) Electron microscopic demonstration of proteoglycans in guinea pig epiphyseal cartilage.J. Ultrastruct. Res. 45, 407–27.

    PubMed  Google Scholar 

  • White, D. L., Mazurkiewicz, J. E. &Barrnett, R. J. (1979) A chemical mechanism for tissue staining by osmium tetroxide-ferrocyanide mixtures.J. Histochem. Cytochem. 27, 1084–91.

    PubMed  Google Scholar 

  • Wilsman, N. J., Farnum, C. E., Hilley, H. D. &Carlson, C. S. (1981) Ultrastructural evidence of a functional heterogeneity among physeal chondrocytes in growing swine.Am. J. Vet. Res. 42, 1547–53.

    PubMed  Google Scholar 

  • Wise, D. R. &Jennings, A. R. (1973) The development and morphology of the growth plates of two long bones of the turkey.Res. Vet. Sci. 14, 161–6.

    PubMed  Google Scholar 

  • Wolbach, S. B. &Hegsted, D. M. (1952) Endochondral bone growth in the chick.Arch. Pathol. 54, 1–12.

    Google Scholar 

Download references

Author information

Author notes

  1. L. P. Nuehring

    Present address: C. S. Roberts Veterinary Diagnostic Laboratory, P.O. Box 2209, 36831-2209, Auburn, AL, USA

Authors and Affiliations

  1. Department of Veterinary Pathology College of Veterinary Medicine, The University of Georgia, 30602, Athens, GA, USA

    L. P. Nuehring & G. N. Rowland

  2. Department of Anatomy and Radiology College of Veterinary Medicine, The University of Georgia, 30602, GA, Athens, USA

    W. L. Steffens

  3. Department of Avian Medicine, College of Veterinary Medicine, The University of Georgia, 30602, Athens, GA, USA

    G. N. Rowland

Authors
  1. L. P. Nuehring
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. W. L. Steffens
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. G. N. Rowland
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nuehring, L.P., Steffens, W.L. & Rowland, G.N. Comparison of the Ruthenium hexammine trichloride method to other methods of chemical fixation for preservation of avian physeal cartilage. Histochem J 23, 201–214 (1991). https://doi.org/10.1007/BF01462242

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01462242

Keywords

Search

Navigation