Although acidic bismuth nitrate stains the polysaccharide in the unfixed bovine epiphysial plate, it fails to do so in that of the rabbit. After fixation in glutaraldehyde, however, Araldite sections stained with bismuth nitrate exhibit 3-nm spots which are interpreted as polysaccharide chains in coiled conformation.
In the matrix of the proliferative zone, and in the large Golgi vesicles of the associated chondrocytes, the polysaccharide spots are arranged in closely packed groups. Lead citrate and uranyl acetate staining shows the non-fibrillar material in these situations as a continuous network of particles. The centre of each particle is translucent, and double staining with phosphotungstic acid and bismuth indicates that these central regions contain the groups of polysaccharide spots.
It is suggested that in the unfixed tissue bismuth binding is prevented by screening of the polysaccharide sulphates by positively charged non-collagenous protein, and that fixation, by precipitating this protein as a network around groups of proteinpolysaccharide molecules, frees the sulphate groups to react with bismuth.
Proteinpolysaccharide molecules appear to be tangentially attached to each period of the small collagen fibrils of the matrix. Fixation precipitation of the associated protein tends to aggregate these molecules into groups which are often somewhat eccentric to their original attachment sites, and consequently the fibrillar attachment of proteinpolysaccharide usually appears to be rather irregular.
In the zone of early calcification single mineral crystals are associated with dense bodies of unknown nature and function. Many are also associated with short linear rows of polysaccharide spots. This and the similarity in the lengths of the two structures suggest that initial mineral crystals may grow along a proteinpolysaccharide molecule from one end to the other. Absence of polysaccharide spots in relation to crystal clusters suggest that these clusters are formed as a result of the seeding effect of the initial single crystals.