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Immunization of rabbits or monkeys with walls prepared from Streptococcus mutans by a procedure including extraction with SDS at room-temperature induced antibodies to three antigens (A, B and C) detectable by crossed immunoelectrophoresis. Antigens A and B have previously been characterized as proteins of molecular weight 29 000 and 190 000, respectively. Antigen C was characterized as having a molecular weight of 70 000 and was purified by immunosorbent affinity chromatography and hydrophobic interaction chromatography. Another wall protein, antigen D, of molecular weight 13 000, was extracted from walls with Triton X-100. Immunization of monkeys with walls prepared from cultures of S. mutans grown at a high (D = 0.5 h-1) or low (D = 0.05 h-1) dilution rate in a chemostat showed that only the latter induced protection against dental caries. There was a positive correlation between levels of antibody to antigens A and C and induction of protection and a negative correlation between protection and the level of antibody to antigen B. No antibody to antigen D was detected in protected monkeys and an experiment in which monkeys were immunized with pure antigen D confirmed that it does not induce protection.

DIET has long been assumed to be one of the major determinants of the faecal flora (Dudgeon, 1926). Studies with experimental animals provide support for this hypothesis (Smith, 1965); however, studies with human faeces have provided less satisfactory evidence (Moore, Cat0 and Holdeman 1969).In a study of faecal specimens from different dietary groups in various parts of the world, we demonstrated differences in the numbers of bacteroides organisms present in faeces (Hill et al., 1971). In the present paper we report on the more detailed classification of the non-sporing anaerobes isolated in that study and during subsequent extensions of the study. MATERIALS AND METHODSSource of faeces. Stool samples were obtained from groups of people consuming a mixed western diet, in England, Scotland and the USA, and from people living on diets with a high carbohydrate content in India, Uganda and Japan. The characteristics of these groups are shown in table I. The groups contained both men and women. All were healthy individuals, mostly aged between 20 and 50. Samples from the USA came from both black and white persons.Transport and storage of specimens. Fresh samples of faeces were diluted 1 in 10 in 10% glycerol broth and immediately frozen in solid carbon dioxide or in liquid nitrogen for transport to London (Crowther, 1971). The samples were stored at -50°C.Isolation of non-sporing anaerobic bacteria. Isolation plates were seeded in an anaerobic cabinet. During the initial stages of this investigation a rigid Perspex cabinet filled with oxygen-free nitrogen (N2) containing 5% carbon dioxide (C02) was employed (Drasar, 1967). Later studies were carried out in a flexible polyvinyl cabinet filled with 5% C02 in a mixture of 90% N2 and 10% hydrogen (H2) (Aranki et al., 1969). Ten-fold dilutions of the specimens were prepared in Brain-Heart-Infusion (BHI) broth (Oxoid) containing 0.05% (w/v) cysteine hydrochloride and 0.03% sodium formaldehyde sulphoxylate as reducing agents. The diluent was heated to 100°C before being introduced into the cabinet, where it was dispensed and allowed to cool in the anaerobic environment. 0.1-ml samples of appropriate dilutions were spread on the surface of plates of Reinforced Clostridial Medium (Oxoid) with 1 % glucose (w/v), 1 % liver digest (Panmede, Paines and Byme, Greenford, England) and 10% defibrinated horse blood. In order to maintain the media in a reduced state, the plates were poured and dried in the Perspex cabinet, or stored for 3 days in the flexible cabinet. Seeded plates were packed into anaerobic jars and on removal from the cabinet the jars were re-evacuated and filled with a gas mixture containing 30% C02 and 70% H2 and incubated at 37°C for 3 days. Cold " D " catalyst was used in the anaerobic jars (Englhard Industries, Cindeford, Gloucestershire).

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