Microbial Populations Growing in the Presence of Fluoride at

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1 INFECTION AND IMMUNITY, Apr. 1982, p Vol. 36, No /82/ $02.00/0 Microbial Populations Growing in the Presence of Fluoride at Low ph Isolated from Dental Plaque of Children Living in an Area with Fluoridated Water G. H. W. BOWDEN,'* 0. ODLUM,2 N. NOLETTE,1 AND I. R. HAMILTON1 Department of Oral Biology' and Department of Preventive Dental Science,2 Faculty of Dentistry, The University of Manitoba, Winnipeg, Manitoba R3E 0W3, Canada Received 14 September 1981/Accepted 24 December 1981 Longitudinal microbiological examinations have been made of dental plaque from a site approximal to the upper central incisors of 10 8-year-old children living in an area with water fluoridation. Differential counts of viable bacteria, made using a selective medium containing various levels of fluoride (0 to 100,ug/ml) at ph levels of 7.0 to 5.5, demonstrated an effect of both ph and fluoride on the numbers and types of bacteria isolated. Strains of Streptococcus and Neisseria grew after only 16 h of incubation at ph levels as low as 6.0 with fluoride levels up to 50,ug/ml. The most commonly isolated streptococci were Streptococcus mitior and S. salivarius. S. mutans was isolated less frequently and was inhibited by 20 and 50,ug of fluoride per ml at ph 6.0 and 6.5, respectively. Veillonella strains were the most resistant isolates, being isolated after 16 h of incubation on media at ph 6.0 with 100 p.g of fluoride per ml. Despite their known fluoride resistance, Actinomyces spp. were often only detected on the selective media after 72 h of incubation. The ph of the medium had a definite selective effect, as the number of colonies growing on the fluoride-free basal media at ph 6.0 was only 30% of that at ph 7.0. Representative strains of S. mutans, S. mitior, S. sanguis, and S. milleri were tested for their ability to utilize glucose at the ph and fluoride levels of the medium on which they were initially isolated. Fluoride reduced the initial glycolytic rate of the cells, but in 5 of the 13 strains tested the final amount of glucose used after 2 h of incubation was the same in the presence or absence of fluoride. The isolation of bacteria capable of growth in the presence of fluoride over a significant portion of the ph range that occurs in plaque in vivo could explain in part the finding that fluoride does not have a dramatic effect on the plaque community. Fluoride in plaque may reduce the ecological advantage afforded to aciduric S. mutans strains by carbohydrate substances. In the in vivo situation this could mean that, even with high carbohydrate intake, fluoride may permit S. mitior to compete with S. mutans within the plaque ecosystem. Several mechanisms have been proposed to explain the cariostatic effects of fluoride. It may affect enamel by reducing the solubility or by promoting remineralizing of early carious lesions (9, 25, 28). A direct effect on microbial metabolism has been proposed, as fluoride reduces the capacity of bacteria to produce acid from carbohydrate (14, 20) and may also have an effect on plaque adhesion and development (17, 26). None of these mechanisms is exclusive, and it is likely that the collective effect of them produces a reduction in caries. However, the effects of fluoride on bacterial metabolism could vary, as bacteria might either be insensitive to fluoride or be able to adapt or mutate in its presence. Actinomyces spp. are insensitive to fluoride, and the capacity of members of the genus Streptococcus to adapt to 247 relatively high concentrations of fluoride is well known (1, 14). Both phenotypically adapted streptococcal strains and mutants resistant to fluoride have been produced in vitro (8, 14), although evidence for adaptation or the production of resistant mutants in vivo is scanty (29). Another consideration with regard to fluoride "resistance" is that adaptation or mutation to fluoride at ph levels below ph 7 has not been shown. A mutant of Streptococcus salivarius resistant to 2.4 mm fluoride at ph 7.2 was sensitive to this concentration at ph 5.8 (14). Cross sectional studies on the microbiology of plaque from subjects taking fluoride from natural sources or by supplement have shown that there are no obvious differences between plaque from these subjects and from those without fluoride (18, 30, 31). However, there are no data

2 248 BOWDEN ET AL. available to suggest a reason for the above finding or how fluoride could affect the dynamics of some bacterial populations in plaque. This study was undertaken to determine whether children living in an area where water is fluoridated harbored bacterial populations in their plaque which would (i) grow rapidly in the presence of relatively high levels of fluoride and (ii) demonstrate this ability at ph levels below ph 7.0. In addition, longitudinal sampling could demonstrate an effect on the ability of specific microorganisms to persist at a given site on the tooth surface in the presence of fluoride. MATERIALS AND METHODS Subjects for sampling. Ten 8-year-old children were selected from a school in the central area of Winnipeg. Eight of the children had been born in Winnipeg, whereas two had lived in the city since age 2. The water supply in Winnipeg has been fluoridated since 28 December The children were selected into two groups, those with caries and those who were cariesfree. The children with caries had a relatively high number of decayed, missing, and filled teeth (DMFT) (mean DMFT, 4.6) for children in the city. (The mean DMFT for Winnipeg [age 9] was 1.4.) Thus, the children with caries represented an unusual group of children born in the city. Clinical examination of the oral health of the subjects was made at the commencement of the study and after the final sample had been taken. Sampling site. The most susceptible sites for caries attack in those children born in Winnipeg are the occlusal surfaces of the first permanent molars. However, when the occlusal surfaces have become carious the sites most at risk are the approximal areas of the central incisors. These areas are readily accessible for sampling in the field. The above considerations guided the selection of the mesial surface of tooth no. 21 as the sampling site for plaque removal. Sampling period. Samples were taken at 3-month intervals over a period of 12 months. Six subjects provided five samples, whereas only four were obtained from four of the caries-free children. The intervals between sampling were, in the main, dictated by the availability of the subjects in the school. Sampling method. Samples were taken by using an abrasive strip device (5). This method has proved very effective in previous studies which have examined the microflora of approximal areas. Immediately after sampling, the abrasive strip was placed into a reduced transport fluid (23) and taken to the laboratory. The period between sampling and cultivation of the plaque did not exceed 4 h. Cultivation of the sample. The abrasive strip in reduced transport fluid was sonicated for 30 s at setting 4 of a Kontes Sonifier, and the sample was diluted in reduced transport fluid. Sonication and dilution were carried out on the bench, and cultivation of the dilutions was made with media and techniques described previously (5, 6). A variety of media was inoculated to provide data on the flora for comparison to those bacteria on a selective fluoride medium. The media included: blood agar (no. 2, CM 271; Oxoid INFECT. IMMUN. Canada Ltd., Toronto, Ontario) supplemented with hemin and menadione and containing 5% (vol/vol) sheep blood (Atlas Laboratories, Winnipeg, Manitoba); blood agar base (no. 2, CM 271; Oxoid Canada Ltd.) with 0.5% (vol/vol) laked horse blood (SR 48, Oxoid) and 7.5,ug of vancomycin per ml, (Eli Lilly & Co. [Canada] Ltd., Toronto, Ontario); TYC medium (6) with and without bacitracin, 100 U/ml (The Upjohn Co., Canada, Don Mills, Ontario); Rogosa agar (Difco Laboratories, Detroit, Mich.). In addition to these media, a selective medium containing fluoride at final concentrations of 0 to 100,ug/ml at different ph levels (ph 7.0 to 5.5) was inoculated. The basal medium composition was tryptic soy agar CM 131 (Oxoid, Canada) with 0.4% laked horse blood SR 48 (Oxoid, Canada), 0.1% (wt/vol) glucose, and 2 mg of bromophenol blue per 100 ml (BDH, Terochem Laboratories, Winnipeg, Manitoba). The ph level of the medium was adjusted before autoclaving and checked just before the plates were poured. Fluoride was added to the molten base as a filter-sterilized solution of sodium fluoride. A single dilution (1:100) of the sonicated sample was used to inoculate the fluoride plates. Initial studies had shown that this dilution produced the optimum numbers of colonies (50 to 200) from sonicated plaque samples. An important consideration in the use of this medium was that it was incubated anaerobically for only 16 h. This short incubation was selected for two reasons: (i) the chances of adaptation of the bacteria during growth were reduced; and (ii) only those bacterial populations which were capable of relatively rapid growth under the environmental conditions imposed would be isolated Ċounting and selection of isolates. The methods followed techniques described previously (6). Differential colony counts were made with the aid of a stereomicroscope (Olympus), and representative colonies were subcultured onto blood agar for further identification. Comparisons were made between the numbers and types of bacteria isolated on standard media and those growing after 16 h on the selective fluoride medium. Counts on the various media were compared by using a factorial analysis of variance. Identification of isolates. Identification of the isolates was based on a variety of tests (6, 15). Some modifications were introduced: raffinose and melibiose were included in the tests for Streptococcus isolates (27); whole-cell agglutination with rabbit antisera (4; E. D. Fillery, personal communication) was used to assist in the identification of Actinomyces isolates; and Neisseria spp. were tested for the ability to produce acid from maltose and polysaccharide (3) from sucrose. Fluoride levels in the samples. Confirmation of the presence of fluoride in the plaque samples was sought by analysis of the plaque sample sonicated in 1.0 ml of reduced transport fluid. It was not possible to equate the fluoride to plaque weight, but fluoride could be assayed with a fluoride-specific ion electrode (12). Calculation of the persistence and numbers of bacterial species isolated. Comparisons were drawn between the two groups of children and between the results on standard media and the selective fluoride medium. Frequencies of isolation were calculated as the number of positive isolations of a species from the total plaque samples. The numbers of a given organism in the plaque samples were calculated as a percentage of

3 VOL. 36, 1982 BACTERIAL GROWTH IN PRESENCE OF FLUORIDE 249 TABLE 1. Frequency of isolation (IF) and mean percentages of viable counts of selected bacteria on standard nonfluoride media from approximal plaque from children in a water-fluoridated area Subject group 1, Subject group caries present 2, caries-free Bacterial species (n =25) (n =21) IF Mean IF Mean (no.)a %b (no.) % S. mutans "S. salivarius" S. mitior S. sanguis A. viscosus A. naeslundii A. odontolyticus Neisseria sp Veillonella sp a Number of samples showing positive isolations. b Means of the percentages of the organisms calculated from the total viable counts after 96-h incubation on standard media. the total viable count on blood agar after 96 h. Mean values for isolation frequency and percentage of viable counts were obtained for the isolates from each group of subjects. Calculation of the mean percentages of the viable counts of a species included those samples in which the organism was not detected. Thus, the percentage of an organism isolated from only one sample would be divided by the total number of samples. Glucose metabolism by resting cells. The ability of various bacterial strains to metabolize glucose under the conditions of their isolation was tested. Several isolates were picked directly from the selective medium and grown in tryptone-yeast extract broth at the same ph and fluoride concentration. The broth was frozen (-20 C) and used later to provide an inoculum for cells from the test. Thus, the strains inoculated into the test flasks had undergone only two subcultures. The isolates were identified while the glucose metabolism tests were being carried out. Cells for the test were grown in 250 ml of a medium containing (grams per liter): tryptone, 10; yeast extract, 5; with 50 mm phosphate buffer at either ph 6.0 or 6.5. Fluoride was added at either 20 or 50 ilg/ml as the fluoride ion. The growth of the cells was monitored by optical density measurements at intervals of 1 h after the initial inoculation. Cells were harvested in the exponential phase, washed once with saline, and resuspended at 25 mg/ml (dry weight) in saline. Glucose metabolism was assayed in a mixture containing 50 mm Na-K phosphate buffer at either ph 6.0 or 6.5, 50 mg (dry weight) of cells, and 0.5 ml of 0.2 M glucose to give a final volume of 5.0 ml (final concentration, 20 mm glucose). The reactions were carried out anaerobically in stoppered 25-ml Erlenmeyer flasks as previously described (13). Two flasks were prepared for each strain at the given ph; one was a control without fluoride and a second contained fluoride at the appropriate level. When the solutions had equilibrated to 37 C the glucose was added. Samples (0.5 ml) were removed every 15 min for 2 h and immediately added to 50,ul of 25% ZnSO4 to stop the reaction. After neutralization and centrifugation (13), the sample was assayed for glucose by the method of Kingsley and Getchell (19). RESULTS Forty-six plaque samples from a possible total of 50 were collected. The presence of fluoride in the plaque samples was confirmed, as levels of 0.1 to 0.42,ug were detected in 1.0 ml of the original sonicate. TABLE 2. Bacteria isolated at various levels of fluoride and ph from children's plaque after 16-h incubation Fluoride basal medium Species Blood agar, 0 4Lg/ml 20,ug/ml 50 flg/ml 100 Fg/ml anaerobic ph 6.5 ph 6.0 ph 6.5 ph 6.0 ph 6.5 ph 6.0 ph 6.5 ph 6.0 Streptococcus S. sanguis (4)a S. mitior (+)b S. mutans S. salivariusc (+) Neisseria sp Actinomyces A. viscosus A. naeslundii A. odontolyticus Veillonella sp Leptotrichia Sp.d a (-), Not detected. b (+), Only isolated on two (S. mitior) and five (S. salivarius) occasions. c "S. salivarius" represents a group of strains which varied in their biochemical reactions. d Leptotrichia buccalis was isolated consistently from only one subject.

4 250 BOWDEN ET AL. Persistence and percentage of viable counts of selected bacterial populations at the sample site. Table 1 shows the frequency of isolation and the percentage of the viable counts of selected organisms on the standard media. The presence of fluoride in the plaque environment did not produce any obvious effect on the presence or persistence of several genera and species. The colonization of the approximal area by S. mutans in the children with caries (Table 1) was the most striking difference between the two groups. Neisseria and Veillonella spp. also showed a higher isolation frequency in the children with caries. The frequency of isolation of the other organisms. and their numbers were similar to those described for other longitudinal studies of approximal plaque from children (5, 6, 17), and there were no obvious differences between the two groups. Effects of fluoride and ph levels on the numbers of bacte'ria isolated. A marked additive effect of short incubation time, high fluoride concentration, and low ph was demonstrated when the counts on fluoride media at 16 h were compared with those on blood agar incubated for 96 h (Fig. 1). The effect of the incubation time was seen when the selective medium base at ph 7.0 or 6.5, without fluoride, was incubated further for a total of 72 h. The total counts on this medium after 16 h of incubation were only 33% (mean) of the counts on blood agar after 96 h of incubation. However, after 72 h of incubation, the counts on the selective medium base increased to 78 to 100% of the blood agar count. Basal medium at the lower ph levels (ph 6.0) without fluoride did not show such a dramatic change on 72-h incubation. Comparison of growth on the basal selective medium with that on the same medium containing fluoride demonstrated that, although the ph level alone was an effective selective pressure, it was enhanced by the addition offluoride, Figure 1 shows that the effect of ph was at least as great as that produced by fluoride. Differences in counts produced by fluoride or ph alone were significant (P < 0.01); the interaction between ph and fluoride was also significant (P < 0.01). Species isolated in the presence of fluoride. The species isolated were governed to some extent by the use of a single dilution (i.e., 1 in 100) on the initial plates. Thus, an organism would have to be present in levels at or above 5 x 103 in the original sonicate to be detected. Therefore, the strains isolated represent those which were in relatively high concentration and able to grow rapidly under the conditions imposed. Table 2 shows those species detected on media with fluoride at ph 6.5 and 6.0. A wide range of bacteria could be isolated from the plates up to a level of 50,ug of fluoride per ml at ph 6.5; ( INFECT. IMMUN. ph 7.0 ph 6.0 ph 5.0 FIG. 1. Mean counts of bacteria from dental plaque cultivated anaerobically for 16 h at different ph and fluoride levels, expressed as a percentage of the mean counts on blood agar cultivated anaerobically for 96 h. beyond this level of fluoride and at ph 6.0 the medium was more selective. Some species were isolated more frequently than others (Tables 3 and 4); S. mitior was the most commonly isolated of the streptococci, and specific strains of this species showed a relatively high level of resistance to fluoride. The basal medium at ph 7.0 did not become obviously selective until 100.g of fluoride per ml was added, when only Veillonella, Actinomyces, and Neisseria spp. were recovered. Some variations were noted in the reactions of strains designated as S. mitior in the identification tests. All produced H202 and were negative for arginine and esculin hydrolysis. However, some isolates fermented raffinose and melibiose, whereas others were negative. As both of the S. mitior types could produce hard or soft colonies on TYC medium, four subdivisions could be delineated in S. mitior growing on fluoride. The strains designated as S. salivarius were positive for esculin hydrolysis. However, some strains fermented a single carbohydrate and several of the isolates grew as hard, dry adherent colonies on TYC medium. S. salivarius strains have not been included in Tables 3 and 4 as the results of the biochemical tests did not permit the formation of a clearly defined group of strains. Strains of S. mutans isolated on standard and fluoride medium were examined at the laboratories of the National Caries Program, National Institutes of Health. The majority were serogroup c, with a few serogroup e, and no strains resembling serogroup b (arginine positive) or serogroup d were detected. The most resistant of the isolates were members of the genera Veillonella and Neisseria. Actinomyces spp. were not isolated regularly on the fluoride media after 16 h of incubation. Sixteen plaque samples were positive for Actinomyces cultured on the selective media at ph 6.5, 20,g of fluoride per ml; these strains represented a mean

5 VOL. 36, 1982 BACTERIAL GROWTH IN PRESENCE OF FLUORIDE 251 TABLE 3. Frequency of isolation (IF) and mean percentages of viable counts of selected bacteria from group 1 (caries present) plaque samples grown on fluoride-containing media at ph 6.5 and 6.0 after 16-h incubation' Fluoride Bacterial species 0 FLg/ml 20 slg/ml 50 1.g/ml IF (no.)b Mean %W IF (no.) Mean % IF (no.) Mean % ph 6.5 S. mutans S. mitior S. sanguis Veillonella sp Neisseria sp ph 6.0 S. mutans S. mitior S. sanguis Veillonella sp Neisseria sp a Group 1, caries present; n = 25. b Number of samples showing positive isolations. c Means of the percentages of the organisms calculated from the total viable counts after 96-h incubation on blood agar. of 6.16% of the total viable count on blood agar. Culture at ph 6.5, 50 ~,ug of fluoride per ml, reduced the positive isolations to nine, with a mean of 2.03%. Glucose utilization. Thirteen strains of streptococci were tested for glucose metabolism at the ph and fluoride concentrations of their initial isolation. All of the strains utilized glucose in the presence of fluoride (Table 5). Differences in initial glycolytic rates were demonstrated between controls (without fluoride) and tests (with fluoride). Fluoride reduced the initial glycolytic rate, although in five strains the amount of glucose used in the presence and absence of fluoride during a period of 2 h was the same (ratio glucose used without/with fluoride, 0.9 to 1.1), whereas in four strains the ratio was 0.48 to The reasons for these differences in the initial rate of glucose metabolism versus the final amount of glucose used are not known. They may be related to changes in ph or the KS for glucose of the different strains. DISCUSSION Studies on the bacterial composition of dental plaque from subjects exposed to fluoride and those from fluoride-free areas have shown few differences between the two groups (11, 17, 29, 30). This suggests that the reduction in caries produced by fluoride is not the result of the elimination of specific populations from the community. However, topical applications of fluoride have been shown to reduce the levels of S. mutans in plaque (23). Furthermore, high levels of fluoride given prophylactically may produce adapted or mutated strains resistant to fluoride (8, 13, 29). Whether the levels of fluoride added to public water supplies are sufficient to produce such resistant strains is not known. Moreover, the nature of those oral bacteria which metabolize and grow in the presence of fluoride at the ph levels attained in plaque "in vivo" has never been determined. The results of the present study show that several plaque populations are capable of initial growth in fluoride at ph levels as low as 6.0. Isolation of Actinomyces, Veillonella, and Neisseria spp. might have been expected (1, 2). However, strains of S. mitior (10) and "S. salivarius" also demonstrated fluoride resistance. S. mutans was isolated less frequently on the fluoride medium, but this may be a reflection of the low numbers of this organism in some samples. Strains of S. mutans capable of in vitro growth at levels up to 1 mg of fluoride per ml at ph 7.2 have been reported (8, 13, 28; unpublished data). The relatively short incubation period (16 h) selected strains capable of rapid growth under the isolation conditions used. Thus, these strains could respond rapidly to carbohydrate added to the plaque in vivo. The results of the in vitro tests of glucose utilization by the Streptococcus isolates support this suggestion (Table 5). S. mitior, S. mutans, and S. salivarius were able to metabolize glucose in the presence of fluoride at the ph at which they were isolated. The differences in initial glycolytic rate produced by fluoride did not influence the total glucose utilized by several of the strains after 2 h of incubation. This suggests that fluoride may have only a

6 252 BOWDEN ET AL. INFECT. IMMUN. TABLE 4. Frequency of isolation (IF) and mean percentages of viable counts of selected bacteria from group 2 (caries absent) plaque samples grown on fluoride-containing media at ph 6.5 and 6.0 after 16-h incubationa Fluoride Bacterial species 0,ug/ml 20 11g/ml 50,ig/ml IF (no.)b Mean %C IF (no.) Mean % IF (no.) Mean % ph 6.5 S. mutans S. mitior S. sanguis Veillonella sp Neisseria sp ph 6.0 S. mutans S. mitior S. sanguis Veillonella sp Neisseria sp a Group 2, caries-free; n = 21. b Number of samples showing positive isolations. c Means of the percentages of the organisms calculated from the total viable count after 96-h incubation on blood agar. limited effect on the carbohydrate metabolism of cells growing in vitro. It would be expected, therefore, that these organisms could metabolize over a large part of the range of the fluctuations in ph likely to occur in plaque in vivo (16). This observation explains in part the fact that water fluoridation does not significantly alter the plaque community. Kilian et al. (17) found S. mitior to be the most numerous of the streptococci in plaque from children in Tanzania living in high- or lowfluoride areas. Such a finding suggests that S. mitior may be able to adapt and also compete effectively in both fluoride and nonfluoride ecological environments. Growth of an organism in the presence of fluoride at low ph, be it a natural or acquired characteristic, could impart an ecological advantage in a community exposed to fluoride. Such adaptation might allow strains to achieve dominance in a localized habitat. The results from the present study suggest that the level of fluoride used in water fluoridation is not sufficient to provide an environment in which adapted species can dominate. A different situation could occur if very high levels of fluoride were given, which might cause elimination of populations (7, 24). High levels of fluoride could produce a situation closer to that of using an antibiotic, in which a resistant organism is released from the ecological restrictions of the community and can, therefore, grow to excessive levels. The level of fluoride used in drinking water could be considered optimal from this point of view as it does not place an extreme ecological pressure on the community. However, fluoride may modify the plaque environment and affect the community interactions. One of the significant characteristics of S. mutans is its ability to survive and grow at low ph (14a, 23, 24). Growth at low ph could provide S. mutans with an advantage over less aciduric organisms in an ecosystem with carbohydrate, leading to its dominance in the community. If fluoride reduced the ability of plaque to produce acid, this ecological advantage would be lost. In addition, other bacteria able to grow in fluoride at ph 6.0 would compete with S. mutans. Therefore, fluoride could stabilize the ph of the ecosystem and allow bacteria to grow and compete at carbohydrate levels which would, in the absence of fluoride, confer an advantage on acriduric organisms. Coupled to this, the genera capable of lactate utilization (e.g., Veillonella and Neisseria spp.) are resistant to fluoride at low ph. These organisms could help to reduce the levels of lactate produced within the plaque community. If the difference detected between the colonization of the approximal areas by S. mutans in the two groups is representative of other areas on the dentition, it could provide a valuable public health tool (21). However, occlusal colonization would be expected in both groups (22, 30). A study of a larger number of children, sampling approximal and occlusal surfaces, is being carried out. One part of this current study includes the examination of children living in an

7 VOL. 36, 1982 BACTERIAL GROWTH IN PRESENCE OF FLUORIDE 253 TABLE 5. Rate of glycolysis and residual glucose by resting cells of various streptococcal strains incubated at the ph and fluoride concentration at which they were isolated Fluode Initial glycolytic rate (nmol of Residual Strain ph uon;/ml) glucose utilized mg [dry wt] of glucose' Ratiob (MM) cells-' min-') S. salivarius S. mitior S. mitior S. mitior S. mitior S. mutans 43al S. mutans S. mutans S. salivarius S. mitior S. milleri S. salivarius S. mitior a Glucose remaining after incubation at 37 C for 2 h. b Ratio = residual glucose no fluoride/fluoride. area without water fluoridation. The results from this group should help in deciding whether the ability to grow in the presence of fluoride at low ph is a natural or acquired characteristic of the bacterial species described in this paper. ACKNOWLEDGMENTS We gratefully acknowledge the cooperation of D. Konyk, Health Department, City of Winnipeg and Winnipeg School Division 1, and, in particular, the students and staff at Wellington School. We also thank W. H. Bowen and W. Little of the National Caries Program, National Institutes of Health, Bethesda, Md., for assistance with the serotyping of the S. mutans isolates. TIhis research was supported by grants (DG-227 and MT- 3546) from the Medical Research Council of Canada. LITERATURE CITED 1. Beighton, D., and G. Colman A medium for the isolation and enumeration of oral Actinomycetaceae from dental plaque. J. Dent. Res. 55: Beighton, D., and W. A. McDougll The effects of fluoride on the percentage bacterial composition of dental plaque, on caries incidence, and on the in vitro growth of Streptococcus mutans, Actinomyces viscosus and Actinobacillus sp. J. Dent. Res. 56: Berger, U Neisseria animalis nov. spec. Z. Hyg. 147: Bowden, G. H., J. M. Hardie, and E. D. Fillery Antigens from Actinomyces species of value in identification. J. Dent. Res. Sp. Issue A 55: Bowden, G. H., J. M. Hardie, A. S. McKee, P. D. Marsh, E. D. Fllery, and G. L. Slack The microflora associated with developing carious lesions of the distal surfaces of the upper first premolars in year old children, p In H. M. Stiles, W. D. Loesche, and T. C. O'Brien (ed.), Proceedings, Microbial Aspects of Dental Caries. Sp. Suppl. Microbiol. Abstr., vol. 1. Information Retrieval, Inc. 6. Bowden, G. H., J. M. Hardie, and G. L. Slack Microbial variations in approximal dental plaque. Caries Res. 9: Brown, L. R., S. Dreizen, and S. Handler Effects of selective caries preventive regimens on microbial changes following irradiation-induced xerostomia in cancer patients, p In H. M. Stiles, W. D. Loesche, and T. C. O'Brien (ed.), Proceedings, Microbial Aspects of Dental Caries. Sp. Suppl. Microbiol. Abstr., vol. 1. Information Retrieval, Inc. 8. Brown, L. R., S. F. Handler, I. M. Horton, J. L. Streckfuss, and S. Dreizen Effect of sodium fluoride on the viability and growth of Streptococcus mutans. J. Dent. Res. 59: Brown, W. E., T. M. Gregory, and L. C. Chow Effects of fluoride on enamel solubility and cariostasis. Caries Res. 11(Suppl. 1): Colman, G., and R. E. 0. WUlIams Taxonomy of some human viridans streptococci, p In L. Wannamaker and J. M. Matson (ed.), Streptococci and

8 254 BOWDEN ET AL. streptococcal diseases. Academic Press, Inc., New York. 11. De Stoppelar, J. D., J. Van Houte, and 0. Backer-Dirks The relationship and smooth surface caries in 13- year old children. Caries Res. 3: Frant, M. S., and J. W. Ross Electrode for sensing fluoride ion in solution. Science 154: Hamilton, I. R Synthesis and degradation of intracellular polyglucose in Streptococcus salivarius. Can. J. Microbiol. 14: Hamilton, I. R Effects of fluoride on enzymic regulation of bacterial carbohydrate metabolism. Caries Res. 11(Suppl. 1): a.Hamilton, I. R., and G. H. Bowden Response of freshly isolated strains of Streptococcus mutans and Streptococcus mitior to change in ph in the presence and absence of fluoride during growth in continuous culture. Infect. Immun. 36: Hardie, J. M., and G. H. Bowden Physiological classification of oral viridans streptococci. J. Dent. Res. Sp. Issue A 55: Imfeld, T Evaluation of the cariogenicity of confectionary by intra-oral wire telemetry. Helv. Odontol. Acta 21: Kilian, M., M. J. Larsen, 0. Fejerskov, and A. Thylstrup Effects of fluoride on the initial colonisation of teeth in vivo. Caries Res. 13: Kilian, M., A. Thylatrup, and 0. Fejerskov Predominant plaque flora of Tanzanian children exposed to high and low water fluoride concentrations. Caries Res. 13: KIngsley, G. R, and G. Getchell Direct ultramicro glucose oxidase method for determination of glucose in biological fluids. Clin. Chem. 6: Kleinberg, I., R. Chatterjee, J. Reddy, and D. Craw Effects of fluoride on the metabolism of the mixed oral flora. Caries Res. 11(Suppl. 1): INFECT. IMMUN. 21. Klock, B., and B. Krasse Microbial and salivary conditions in 9-12 year old children. Scand. J. Dent. Res. 85: Loesche, W. J., and L. H. Straffon Longitudinal investigation of the role of Streptococcus mutans in human fissure decay. Infect. Immun. 26: Loesche, W. J., and S. A. Syed The predominant cultivable flora of carious plaque and carious dentine. Caries Res. 1: Loesche, W. J., S. A. Syed, R. J. Murray, and J. R. Mellberg Effect of topical acidulated phosphate fluoride on percentage of Streptococcus mutans and Streptococcus sanguis in plaque. Caries Res. 9: Moreno, E. C., M. Kresak, and R. T. Zahradnik Physiochemical aspects of fluoride-apatite systems relevant to the study of dental caries. Caries Res. 11(Suppl. 1): Rolla, G Effects of fluoride on initiation of plaque formation. Caries Res. 11(Suppl. 1): Shklalr, J. L., and H. J. Keene A biochemical scheme for the separation of the five varieties of Streptococcus mutans. Arch. Oral Biol. 19: Silverstone, L. M Remineralization phenomena. Caries Res. 11(Suppl 1): Streckfuss, J. L., D. Perkins, I. M. Horton, L. R. Brown, S. Dreizen, and L. Graves Fluoride resistance and adherence of selected strains of Streptococcus mutans to smooth surfaces after exposure to fluoride. J. Dent. Res. 59: Van Houte, J., R. Aasenden, and T. C. Peebles Oral colonisation of Streptococcus mutans in human subjects with low caries experience given fluoride supplements from birth. Arch. Oral Biol. 23: Van Houte, J., R. Aasenden, and T. C. Peebles Lactobacilli in human plaque and saliva. J. Dent. Res. 60:2-5.