ULTKASTRVCTURAL AND CHEMICAL STUDIES ON CARIES 189 



Oil carious enamel, lesions with and without break in surface con- 

 tinuity were selected. The ultrastructural studies were performed on 

 areas of advanced destruction in both types of lesions. For the chem- 

 ical analyses, lesions with intact surfaces were used for determina- 

 tion of major elements, carbonate, and fluoride; lesions showing 

 cavitation were studied for trace elements. In the study of dentinal 

 lesions all samples were obtained from soft carious dentin. 



For the electron microscopic work whole teeth or specimens of 

 sound and carious tissue were fixed immediately after operative re- 

 moval in cold (4°C) 1 per cent osmic acid solution containing 

 sucrose (0.22 m) and buffered to pH 7.2-7.4 with 0.025 m veronal 

 acetate buffer; or in neutral 10 per cent formalin or in 70 per cent 

 alcohol. After fixation some of the material was dehvdrated in al- 

 cohol and embedded in butyl methacrylate catalvzed with 1 per 

 cent benzoyl peroxide. Some material was stored in 70 per cent al- 

 cohol and sectioned without further treatment. A few observations 

 were also made on fresh untreated tissues. 



Obser\'ations on the inorganic phase of enamel and dentin were 

 made on ultrathin sections cut with a Porter-Blum microtome 

 equipped with a glass knife, and an LKB Ultratome equipped with 

 a diamond knife. Samples of enamel gentlv triturated in distilled 

 water were studied without embedding. The collagen-crvstallite re- 

 lationships in dentin were examined in tissue samples homogenized 

 in distilled water emploving a Dounce ground-glass homogenizer. 



For study of the organic phase the tissues were usuallv demineral- 

 ized prior to embedding. To protect the enamel matrix, the tissue 

 samples were maintained within a dialysis bag dining demineraliza- 

 tion with EDTA (0.46 m, pH 7.2-7.4) (Burrows, 1961), and also 

 during the subsequent washing, dehydration, and infiltration pro- 

 cedures. After embedding in butyl methacrylate and sectioning, the 

 tissue was stained with a lead preparation (Watson, 1958). Dentin 

 samples were demineralized in EDTA or in formic acid-sodium 

 citrate decalcification solution (Morse, 1945) prior to embedding. 

 Sections of dentin were stained with 10 per cent phosphotungstic 

 acid solutions for periods from 10 minutes to 2 hours. Micrographs 

 were taken with a Siemens Elmiskop la operated at 60 or 80 kv. 

 (For details of procedures, see Johansen and Parks, 1961, 1962.) 



