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Scanning Electron Microscopic Studies of the Extracellular Polysaccharides (EP) Synthesized in Colonies of Streptococcus mutans: Development of EP and the Effect of Dextranase on Them

It has been known for years that certain species of streptococci often isolated from dental plaque of humans produce extracellular polysaccharides (3). It has been suggested that among the extracellular polysaccharides (EP) the water-insoluble forms play an important role in the initiation of dental caries (3-5). Whittaker and Drucker (8) first reported the colonial morphology of Streptococcus mutans as revealed by scanning electron microscopy but mentioned little about the chemical nature and morphology of the EP. Guggenheim and Schroeder ( 1) examined S. mutans electron microscopically and reported that globular forms of EP were sensitive to a-1 ,6-dextranase while Hamada et a! (3) showed that the fibrillar material was sensitive to their dextranase preparation and concluded that the material was mainly composed of a-1 ,6-glucosidic linkages. However, there is little information about the development of the morphological structures of EP or the developmental relationship between the different morphological forms of EP. The present paper describes in detail the initial observation by scanning electron microscopy of the extracellular polysaccharides synthesized in the colonies of S. mutans and shows the morphological relationship between the different structures of EP as well as the effects of dextranase on them. Streptococcus mutans, HS-1 (serotype a), FA-I (serotype b), LM (serotype c), SEll (serotype f), and OMZ 65 (serotype g) were used. All the strains were kindly provided by Dr. Shigeyuki Hamada (National Institute of Health of Japan, Tokyo). They were grown on Mitis-Salivarius agar (Difco) plates for 48 hr at 37 C under an atomosphere of 5% C02 and 95% air, as reported previously (7). Strains of each serotype were also grown similarly except that agar plates lacking sucrose but otherwise having the same formula as that of the Mitis-Salivarius agar were used. Blocks of agar (2 mm X 2 mm X 2 mm) containing colonies were cut, washed three times in 0.01 M phosphate buffer (pH 7.2) and fixed with 2% glutaraldehyde in the buffer for 2 hr at 4 C. After being washed again in the buffer solution the agar blocks were treated in micro titer plates with dextranase ( a-1 ,6glucan, 6-glucanohydrolase, Sigma Chemical Co., St. Louis, Mo., U.S.A.) for 48 hr at 37 C. Reaction was carried out in a total volume of 0.5 ml with a final con-