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

Abstract

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.