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The effect of pH on sucrose metabolism in vitro of sucrose- and saline-exposed experimental dental plaques in man
0003.9Y69 8, 020153-05302.00!0 Pcrpamon Press Ltd
Arch orul Bwl. Vol.26. pp. 153 to 157, 1981 Printed in Great Britain
SHORT
COMMUNICATION
THE EFFECT
OF pH ON SUCROSE METABOLISM IN VITRO OF SUCROSEAND SALINE-EXPOSED EXPERIMENTAL DENTAL PLAQUES IN MAN G. E. MINAH, MARIA MATHEUS and J. P. FINNEY
Departments of Microbiology and Pediatric Dentistry, Dental School, University of Maryland at Baltimore, Baltimore, MD 21201, U.S.A. Summary-Sucrose metabolism of sucrose-exposed (test) and saline-exposed (control) experimental dental plaques on removable enamel surfaces worn on oral appliances were compared in reaction mixtures of different hydrogen-ion concentrations. At pH 7.2, test plaques formed proportionally more lactic acid than controls, while at pH 6.0 and to a greater extent at pH 5.0 the proportional and quantitative production of lactic acid was higher in test plaques.
Removable tooth enamel surfaces worn intraorally in either acrylic appliances (Thott, Folke, and Sveen, 1974; Ostrom et al., 1977; Minah, Lovekin and Finney, 1981) or as insertions into molar teeth (Theilade et al., 1974, 1978; Sv,anberg and Loesche, 1977) have been utilized in investigations of human dental plaque. The technique permits control of environmental and experimental variables which is not possible for plaque on natural oral surfaces in man. The microbial composition (Thott et al., 1974; Theilade et al., 1974, 1978; Minah et al., 1981) and metabolic activity (Minah et nl., 1981) of dental plaques which developed on removable enamel surfaces have been generally similar to microbial specimens from natural enamel surfaces. We have employed a type of removable model system to investigate microbial composition, caries activity and metabolic changes in experimental dental plaques to variations in exposure to sucrose (Minah et al., 1981). We demonstrated that microbial population changes occurred in sucrose-exposed plaques when compared to saline-exposed control plaques developing simultaneously on the same appliances in 20 subjects. The sucrose-exposed plaques decreased the microhardness of enamel to a greater extent than control plaques. The enamel demineralization correlated with plaque levels of either Streptococcus mutuns or
total lactobacilli. Rates of sucrose consumption, lactic and volatile acid production, soluble polysaccharide formation, and cell-bound and insoluble product formation were not significantly different in test and control plaques at pH 7.2. To test the hypothesis that increased hydrogen ion concentration would increase the acid production of sucrose-exposed plaques compared to saline-exposed plaques, we have measured lactic acid levels of the experimental plaques in 10 of the 20 subjects at pH 7.2, 6.0 and 5.0. The removable dental model (RDM), which has been described in other reports (Minah ef al., 1981) was a horseshoe-shaped, acrylic appliance containing 6 sterile bovine enamel discs of known microhardness in removable stainless steel cylinders (3.0 mm diameter, 5.0 mm length). The enamel surfaces were 2.0 mm below the orifice of the cylinders which were covered with acrylic covers on stainless-steel wire springs. Ten subjects wore the appliance for 14 days during which period they immersed half of the enamel inserts 6 times daily for 30 min in a 10 per cent wt/vol sucrose in normal saline solution (test plaques) and half simultaneously in normal saline (control plaques). At 7 and 14 days, one cylinder from each side of the appliance was removed, the predominant plaque bacteria identified and quantitated and the enamel micro-
Table 1. Sequence of metabolic
procedures
1. Enamel cylinder placed in 1.0 ml RTF. 2. Plaque dispersed by sonication for 15 s. 3. 100 ~1 of dispersed sample was added to 200 ~1 of RTF containing 0.0876 per cent sucrose. 2 &i of [14C]-sucrose in 50 ~1 of RTF was added (final sucrose concentration, 0.05 per cent). 4. Mixture incubated for 60 min at 37°C anaerobically. 5. 2 ~1 was :spotted on TLC sheets for sucrose and lactic acid isolation and quantitation. 6. 50~1 war, filtered and washed on 0.1 pm filters. The retentate was hydrolyzed and counted as a measure of cell-bound and insoluble products. were precipitated on the disc 7. The filtra.te was dried on a glass fibre disc. Soluble polysaccharides by methanol and quantitated. 8. 100~1 was placed in the outer well of a Conway microdiffusion disc. Carbon dioxide (CO,) and total v’olatile products other than CO2 were quantitated. 153
15.5 (2-46)
4.6 (1.4-11.4)
18.4 (2.9.-50.3)
Cell-bound and insoluble products
Soluble polysaccharide
Volatile products (except CO*)
2 (0.3-s)
24 (2.574)
9 (0.8-53.5)
12.9 (2.4-40)
17.8 (240.7)
0.9 (0.06-3.7)
17 (2641.5)
9 (3.2-41.7)
8.9 (2.5-130)
26.2 (4.6-78.4)
62.2 (13.5-152.3)
Sucrose exposed plaques
at 37”C/2 x 10s particles.
66 (20-170.5)
Saline exposed plaques
* Values expressed as nmol sucrose equivalents/l h incubation t Range given in parentheses. $ Difference significant at p < 0.05 (paired t-test).
1 (0.24.9)
19.8 (3.5-31)
COZ
pH 7.2
59.4* (10.22131.6)t
Lactic acid
Sucrose utilized
Sucrose exposed plaques
pH 6.0
0.6 (0.1-2.2)
26.7 (5.6-l 11)
6.7 (1.6-17)
13 (544.6)
17.2 (2.9-42)
64.4 (17.6-112.5)
Saline exposed plaques
0.6 (0.05-1.5)
15 (2.651)
8.8 (3.3-26)
12 (1.3-29.5)
19 (2.3-42)$
55.5 (9.6152)
Sucrose exposed plaques
pH 5.0
0.5 (0.8-1.5)
12.5 (5.2-36.2)
5 (M-18.8)
9.7 (3.8-28)
12.8 (2.2-39)
40.6 (18-123.6)
Saline exposed plaques
Table 2. Effect of pH on the metabolism of radiolabelled sucrose by 7 day sucrose-exposed and saline-exposed experimental dental plaque
9 (3.7-11)
28 (13-38)
1.7 (0.68)
Soluble polysaccharides
Volatile products (except COJ
CO1
pH 7.2
3 (0.68)
34.4 (1656)
10 (531)
20.3 (12-33.6)
28.2 (9-58)
Saline exposed plaques
of by-products pH 6.0
1.2 (0.244)
35 (184)
12.4 (9-25)
21.7 (1 l-34.6)
29.5 (1 l-57)
Saline exposed plaques
by 7 day sucrose-exposed
for 1 h at 37°C.
1 (0.346)
26.4 (17-38)
15 (628)
18 (9-36.7)
39 (31-58)
Sucrose exposed plaques
of sucrose metabolism
* Values are expressed as percent of total sucrose utilized during incubation t Range given in parentheses. $ Differences significant at p < 0.05 paired r-test.
22 (1%39)
39* (21-68)7$
Cell-bound and insoluble products
Lactic acid
Sucrose exposed plaques
Table 3. Effect of pH on the proportion
0.9 (0.16-3.1)
22.6 (1333.4)
17.5 (9.534.6)
23.6 (13-53)
35 (18-47.7)$
Sucrose exposed plaques
and saline-exposed pH 5.0
1.1 (0.33.6)
31 (2640)
12 (8-19)
26 (1244)
29.5 (11.540)
Saline exposed plaques
dental plaques
s 8 ‘c) F CI ti 3 ?! 3 o5 J
2 B 4
16
-4
4
26
pH 1.2
4
2
No. of subjects
2
19
5
pH 6.0
2
10
2
pH 5.0
22
32
46
Change in enamel microhardness rA)t
0.003
0.017
1o.m
testf
0.005
0.04
0.035
control11
Strep. mutans (%)
0.015
12.0
0.003
test
0.001
0.003
3 x 10-6
control
Lactobacilli (%)
of Strep.
* Percentage lactic acid (relative to sucrose consumed) in sucrose-exposed plaques minus percentage lactic acid in saline-exposed plaques in each individual. Mean values as presented. t Microhardness changes in enamel were calculated by the formula: Knoop hardness number (before-after,before) plaque exposure x 100. The data represents mean values for sucrose-exposed plaques minus mean values for saline-exposed plaques. $ Test; sucrose-exposed plaques. I/ Control; saline-exposed plaques. 7 Values represent mean percentage recovery relative to the total viable count.
High lactobacilh Low Strep. mutans and lactobacilli
Strep. mutans
High
Sucrose exposed plaques
Proportional differences in lactic acid production*
Table 4. Lactic acid production and difference in cariogenic potential of sucrose- and saline-exposed experimental plaques in relation to concentrations mutans and lactobacilli
157
Effect of pH on plaque metabolism hardness determined. The cultural and microhardness testing methods and findings are included in another report (Minah et al., 1981). Aliquots (100 ~1) of the test and control 14 day plaques which had been dispersed by low energy sonication (Kontes Cell Disruptor) in 1 ml of reduced tr,ansport fluid (RTF) pH 7.2 (Loesche, Hackett and Syed, 1972) in a Coy anaerobic chamber (Aranki et al., 1969) were introduced into three 1 dram vials containing 0.075 per cent wt/vol sucrose in 300~1 of RTF. The pH of the RTF had previously been adjustlsd with 1.0 M HCl in two of the vials to make the final pH value 6.0 in one vial and 5.0 in the other. The third vial was maintained at the pH of 7.2. Uniformly-labelled carbon-14 sucrose (New England Nuclear; 2.0 microcuries in 50 ~1 of RTF) were added to each vial1 and the mixtures incubated at 37°C for 60min in the anaerobic chamber. The final sucrose concentration of each mixture was 0.05 per cent wt/vol. At 60min the vials were removed from the anaerobic chamber and placed in crushed ice to stop the enzymic reactions. The rates of sucrose consumption and major metabolic by-product formation were then quantitatecl by techniques described previously (Minah and L,oesche, 1976, 1977). All values were expressed as either quantity of product formed in nmoles sucrose equivalents per 2 x 10s particles in the sample, or as the concentration of the product in per cent relative to the total quantity of sucrose consumed. The sequence of metabolic procedures is presented in Table 1. The quantity of sucrose consumed by test and control plaques did not differ significantly at pH 7.2 or 6.0 but sucrose was consumed at a significantly faster rate in test plaques at pH 5.0 (p < 0.05, paired t-test, Table 2). The mean sucrose equivalents of lactic acid produced was greater in test plaques at all pH levels, but was significantly greater only at pH 5.0. When the proportional values for products formed relative to the amount of sucrose consumed were compared (Table 3) the test plaques formed lactic acid in higher mean concentrations at all 3 pH values. The difference was significant at pH 7.2 and 5.0 and barely not significant at pH 6.0 (p < 0.055, paired t-test). The increased quantitative and proportional production of lactic acid at the lower pH levels suggests that the bacteria involved were not affected by an increased hydrogen ion concentration as readily as those in the control plaq.tes. The ability to metabolize sucrose and produce lactic acid as the pH of the environment decreases might explain, :m part, why certain plaques are more destructive to an enamel surface than others. In 2 of the 10 subjects, Strep. mutans levels of the test plaques increased to greater than 100 times that of the controls while Lactobacihs levels were either absent or negligible (Table 4). These plaques were associated with the highest degree of demineralisation, as well as the highest lactic acid production. In 4 of the 10 subjects, total Lactobacihs levels were over 100 times greater in test plaques versus controls, but Strep. mutans levels were negligible or absent. The plaques with a high Lactobacillus content showed dis-
tinct proportional differences in lactic acid production, especially at pH 6.0, compared to controls and caused marked enamel demineralization. The remaining 4 subjects harboured experimental plaques with low levels of, and only minor differences between Strep. mutans and total Lactobacillus levels (test versus control). These plaques exhibited the lowest experimental cariogenicity and the lowest proportional differences in lactic acid production of the three groups (Table 3). It was previously shown that Strep. mutans dominated natural plaques had greater active glycolysis than plaques from the same teeth harbouring low Strep. mutans levels (Minah and Loesche, 1977). The metabolic characteristics of Lactobacihsdominated plaques have not been investigated. The cariogenicity of plaques harbouring high levels of lactobacilli, a genus which does not produce lactic acid as rapidly from sucrose as do oral streptococci (Minah and Loesche, 1977) might be attributable to their ability to produce proportionally high concentrations of lactic acid from sucrose at low pH, while plaques low in lactobacilli might not be capable of sustaining acidogenesis at low pH levels. Acknowledgement-This project was supported, in part, by National Institute of Dental Research grant No. DE-04795. REFERENCES
Aranki A., Syed S. A., Kenney E. B. and Freter R. 1969. Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl. Microbial. 17, 568-576. Loesche W. J., Hackett R. N. and Syed S. A. 1972. The predominant cultivable flora of tooth surface plaque removed from institutionalized subjects. Arch oral Viol. 17, 1311-1326. Minah G. E. and Loesche W. J. 1976. Development of methods to analyze sucrose metabolism by small dental plaque suspensions. Microbial. Abstr. BII,.491-520. Minah G. E. and Loesche W. J. 1977. Sucrose metabolism in resting-cell suspensions of caries-associated and noncaries associated dental plaque. Infect. Immun.17, 43-54. Minah G. E.. Lovekin G. and Finnev J. P. 1981. The effect of a cariogenic substrate environment on microbial succession and metabolic activity in experimental dental plaques. Caries Rex In press. Ostrom C. A.. Koulourides T.. Hickman F. and McGhee J. R. 1977. Microbial characterization of an experimental cariogenic plaque in man. J. dent. Res. 56, 556558. Svanberg M. and Loesche W. J. 1977. The salivarv concentration of Streptococci mutans and Streptococh sanguis and their colonization of artificial tooth fissures in man. Arch oral Biol. 22, 441447. Theilade E., Fejerskov O., Prackyabrued W. and Kilian M. 1974. Microbiologic study on developing plaque in human fissures. Stand. J. dent. Res. 82, 42&427. Theilade E., Fejerskov O., Karring T. and Theilade J. 1978. A microbiological study of old plaque in occlusal fissures of human teeth. Caries Res. 12, 313-319. Thott E. K., Folke L. E. A. and Sveen 0. B. 1974. A microbiologic study of human fissure plaque. Stand. J. dent. Res. 82, 428436.
Arch orul Bwl. Vol.26. pp. 153 to 157, 1981 Printed in Great Britain
SHORT
COMMUNICATION
THE EFFECT
OF pH ON SUCROSE METABOLISM IN VITRO OF SUCROSEAND SALINE-EXPOSED EXPERIMENTAL DENTAL PLAQUES IN MAN G. E. MINAH, MARIA MATHEUS and J. P. FINNEY
Departments of Microbiology and Pediatric Dentistry, Dental School, University of Maryland at Baltimore, Baltimore, MD 21201, U.S.A. Summary-Sucrose metabolism of sucrose-exposed (test) and saline-exposed (control) experimental dental plaques on removable enamel surfaces worn on oral appliances were compared in reaction mixtures of different hydrogen-ion concentrations. At pH 7.2, test plaques formed proportionally more lactic acid than controls, while at pH 6.0 and to a greater extent at pH 5.0 the proportional and quantitative production of lactic acid was higher in test plaques.
Removable tooth enamel surfaces worn intraorally in either acrylic appliances (Thott, Folke, and Sveen, 1974; Ostrom et al., 1977; Minah, Lovekin and Finney, 1981) or as insertions into molar teeth (Theilade et al., 1974, 1978; Sv,anberg and Loesche, 1977) have been utilized in investigations of human dental plaque. The technique permits control of environmental and experimental variables which is not possible for plaque on natural oral surfaces in man. The microbial composition (Thott et al., 1974; Theilade et al., 1974, 1978; Minah et al., 1981) and metabolic activity (Minah et nl., 1981) of dental plaques which developed on removable enamel surfaces have been generally similar to microbial specimens from natural enamel surfaces. We have employed a type of removable model system to investigate microbial composition, caries activity and metabolic changes in experimental dental plaques to variations in exposure to sucrose (Minah et al., 1981). We demonstrated that microbial population changes occurred in sucrose-exposed plaques when compared to saline-exposed control plaques developing simultaneously on the same appliances in 20 subjects. The sucrose-exposed plaques decreased the microhardness of enamel to a greater extent than control plaques. The enamel demineralization correlated with plaque levels of either Streptococcus mutuns or
total lactobacilli. Rates of sucrose consumption, lactic and volatile acid production, soluble polysaccharide formation, and cell-bound and insoluble product formation were not significantly different in test and control plaques at pH 7.2. To test the hypothesis that increased hydrogen ion concentration would increase the acid production of sucrose-exposed plaques compared to saline-exposed plaques, we have measured lactic acid levels of the experimental plaques in 10 of the 20 subjects at pH 7.2, 6.0 and 5.0. The removable dental model (RDM), which has been described in other reports (Minah ef al., 1981) was a horseshoe-shaped, acrylic appliance containing 6 sterile bovine enamel discs of known microhardness in removable stainless steel cylinders (3.0 mm diameter, 5.0 mm length). The enamel surfaces were 2.0 mm below the orifice of the cylinders which were covered with acrylic covers on stainless-steel wire springs. Ten subjects wore the appliance for 14 days during which period they immersed half of the enamel inserts 6 times daily for 30 min in a 10 per cent wt/vol sucrose in normal saline solution (test plaques) and half simultaneously in normal saline (control plaques). At 7 and 14 days, one cylinder from each side of the appliance was removed, the predominant plaque bacteria identified and quantitated and the enamel micro-
Table 1. Sequence of metabolic
procedures
1. Enamel cylinder placed in 1.0 ml RTF. 2. Plaque dispersed by sonication for 15 s. 3. 100 ~1 of dispersed sample was added to 200 ~1 of RTF containing 0.0876 per cent sucrose. 2 &i of [14C]-sucrose in 50 ~1 of RTF was added (final sucrose concentration, 0.05 per cent). 4. Mixture incubated for 60 min at 37°C anaerobically. 5. 2 ~1 was :spotted on TLC sheets for sucrose and lactic acid isolation and quantitation. 6. 50~1 war, filtered and washed on 0.1 pm filters. The retentate was hydrolyzed and counted as a measure of cell-bound and insoluble products. were precipitated on the disc 7. The filtra.te was dried on a glass fibre disc. Soluble polysaccharides by methanol and quantitated. 8. 100~1 was placed in the outer well of a Conway microdiffusion disc. Carbon dioxide (CO,) and total v’olatile products other than CO2 were quantitated. 153
15.5 (2-46)
4.6 (1.4-11.4)
18.4 (2.9.-50.3)
Cell-bound and insoluble products
Soluble polysaccharide
Volatile products (except CO*)
2 (0.3-s)
24 (2.574)
9 (0.8-53.5)
12.9 (2.4-40)
17.8 (240.7)
0.9 (0.06-3.7)
17 (2641.5)
9 (3.2-41.7)
8.9 (2.5-130)
26.2 (4.6-78.4)
62.2 (13.5-152.3)
Sucrose exposed plaques
at 37”C/2 x 10s particles.
66 (20-170.5)
Saline exposed plaques
* Values expressed as nmol sucrose equivalents/l h incubation t Range given in parentheses. $ Difference significant at p < 0.05 (paired t-test).
1 (0.24.9)
19.8 (3.5-31)
COZ
pH 7.2
59.4* (10.22131.6)t
Lactic acid
Sucrose utilized
Sucrose exposed plaques
pH 6.0
0.6 (0.1-2.2)
26.7 (5.6-l 11)
6.7 (1.6-17)
13 (544.6)
17.2 (2.9-42)
64.4 (17.6-112.5)
Saline exposed plaques
0.6 (0.05-1.5)
15 (2.651)
8.8 (3.3-26)
12 (1.3-29.5)
19 (2.3-42)$
55.5 (9.6152)
Sucrose exposed plaques
pH 5.0
0.5 (0.8-1.5)
12.5 (5.2-36.2)
5 (M-18.8)
9.7 (3.8-28)
12.8 (2.2-39)
40.6 (18-123.6)
Saline exposed plaques
Table 2. Effect of pH on the metabolism of radiolabelled sucrose by 7 day sucrose-exposed and saline-exposed experimental dental plaque
9 (3.7-11)
28 (13-38)
1.7 (0.68)
Soluble polysaccharides
Volatile products (except COJ
CO1
pH 7.2
3 (0.68)
34.4 (1656)
10 (531)
20.3 (12-33.6)
28.2 (9-58)
Saline exposed plaques
of by-products pH 6.0
1.2 (0.244)
35 (184)
12.4 (9-25)
21.7 (1 l-34.6)
29.5 (1 l-57)
Saline exposed plaques
by 7 day sucrose-exposed
for 1 h at 37°C.
1 (0.346)
26.4 (17-38)
15 (628)
18 (9-36.7)
39 (31-58)
Sucrose exposed plaques
of sucrose metabolism
* Values are expressed as percent of total sucrose utilized during incubation t Range given in parentheses. $ Differences significant at p < 0.05 paired r-test.
22 (1%39)
39* (21-68)7$
Cell-bound and insoluble products
Lactic acid
Sucrose exposed plaques
Table 3. Effect of pH on the proportion
0.9 (0.16-3.1)
22.6 (1333.4)
17.5 (9.534.6)
23.6 (13-53)
35 (18-47.7)$
Sucrose exposed plaques
and saline-exposed pH 5.0
1.1 (0.33.6)
31 (2640)
12 (8-19)
26 (1244)
29.5 (11.540)
Saline exposed plaques
dental plaques
s 8 ‘c) F CI ti 3 ?! 3 o5 J
2 B 4
16
-4
4
26
pH 1.2
4
2
No. of subjects
2
19
5
pH 6.0
2
10
2
pH 5.0
22
32
46
Change in enamel microhardness rA)t
0.003
0.017
1o.m
testf
0.005
0.04
0.035
control11
Strep. mutans (%)
0.015
12.0
0.003
test
0.001
0.003
3 x 10-6
control
Lactobacilli (%)
of Strep.
* Percentage lactic acid (relative to sucrose consumed) in sucrose-exposed plaques minus percentage lactic acid in saline-exposed plaques in each individual. Mean values as presented. t Microhardness changes in enamel were calculated by the formula: Knoop hardness number (before-after,before) plaque exposure x 100. The data represents mean values for sucrose-exposed plaques minus mean values for saline-exposed plaques. $ Test; sucrose-exposed plaques. I/ Control; saline-exposed plaques. 7 Values represent mean percentage recovery relative to the total viable count.
High lactobacilh Low Strep. mutans and lactobacilli
Strep. mutans
High
Sucrose exposed plaques
Proportional differences in lactic acid production*
Table 4. Lactic acid production and difference in cariogenic potential of sucrose- and saline-exposed experimental plaques in relation to concentrations mutans and lactobacilli
157
Effect of pH on plaque metabolism hardness determined. The cultural and microhardness testing methods and findings are included in another report (Minah et al., 1981). Aliquots (100 ~1) of the test and control 14 day plaques which had been dispersed by low energy sonication (Kontes Cell Disruptor) in 1 ml of reduced tr,ansport fluid (RTF) pH 7.2 (Loesche, Hackett and Syed, 1972) in a Coy anaerobic chamber (Aranki et al., 1969) were introduced into three 1 dram vials containing 0.075 per cent wt/vol sucrose in 300~1 of RTF. The pH of the RTF had previously been adjustlsd with 1.0 M HCl in two of the vials to make the final pH value 6.0 in one vial and 5.0 in the other. The third vial was maintained at the pH of 7.2. Uniformly-labelled carbon-14 sucrose (New England Nuclear; 2.0 microcuries in 50 ~1 of RTF) were added to each vial1 and the mixtures incubated at 37°C for 60min in the anaerobic chamber. The final sucrose concentration of each mixture was 0.05 per cent wt/vol. At 60min the vials were removed from the anaerobic chamber and placed in crushed ice to stop the enzymic reactions. The rates of sucrose consumption and major metabolic by-product formation were then quantitatecl by techniques described previously (Minah and L,oesche, 1976, 1977). All values were expressed as either quantity of product formed in nmoles sucrose equivalents per 2 x 10s particles in the sample, or as the concentration of the product in per cent relative to the total quantity of sucrose consumed. The sequence of metabolic procedures is presented in Table 1. The quantity of sucrose consumed by test and control plaques did not differ significantly at pH 7.2 or 6.0 but sucrose was consumed at a significantly faster rate in test plaques at pH 5.0 (p < 0.05, paired t-test, Table 2). The mean sucrose equivalents of lactic acid produced was greater in test plaques at all pH levels, but was significantly greater only at pH 5.0. When the proportional values for products formed relative to the amount of sucrose consumed were compared (Table 3) the test plaques formed lactic acid in higher mean concentrations at all 3 pH values. The difference was significant at pH 7.2 and 5.0 and barely not significant at pH 6.0 (p < 0.055, paired t-test). The increased quantitative and proportional production of lactic acid at the lower pH levels suggests that the bacteria involved were not affected by an increased hydrogen ion concentration as readily as those in the control plaq.tes. The ability to metabolize sucrose and produce lactic acid as the pH of the environment decreases might explain, :m part, why certain plaques are more destructive to an enamel surface than others. In 2 of the 10 subjects, Strep. mutans levels of the test plaques increased to greater than 100 times that of the controls while Lactobacihs levels were either absent or negligible (Table 4). These plaques were associated with the highest degree of demineralisation, as well as the highest lactic acid production. In 4 of the 10 subjects, total Lactobacihs levels were over 100 times greater in test plaques versus controls, but Strep. mutans levels were negligible or absent. The plaques with a high Lactobacillus content showed dis-
tinct proportional differences in lactic acid production, especially at pH 6.0, compared to controls and caused marked enamel demineralization. The remaining 4 subjects harboured experimental plaques with low levels of, and only minor differences between Strep. mutans and total Lactobacillus levels (test versus control). These plaques exhibited the lowest experimental cariogenicity and the lowest proportional differences in lactic acid production of the three groups (Table 3). It was previously shown that Strep. mutans dominated natural plaques had greater active glycolysis than plaques from the same teeth harbouring low Strep. mutans levels (Minah and Loesche, 1977). The metabolic characteristics of Lactobacihsdominated plaques have not been investigated. The cariogenicity of plaques harbouring high levels of lactobacilli, a genus which does not produce lactic acid as rapidly from sucrose as do oral streptococci (Minah and Loesche, 1977) might be attributable to their ability to produce proportionally high concentrations of lactic acid from sucrose at low pH, while plaques low in lactobacilli might not be capable of sustaining acidogenesis at low pH levels. Acknowledgement-This project was supported, in part, by National Institute of Dental Research grant No. DE-04795. REFERENCES
Aranki A., Syed S. A., Kenney E. B. and Freter R. 1969. Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl. Microbial. 17, 568-576. Loesche W. J., Hackett R. N. and Syed S. A. 1972. The predominant cultivable flora of tooth surface plaque removed from institutionalized subjects. Arch oral Viol. 17, 1311-1326. Minah G. E. and Loesche W. J. 1976. Development of methods to analyze sucrose metabolism by small dental plaque suspensions. Microbial. Abstr. BII,.491-520. Minah G. E. and Loesche W. J. 1977. Sucrose metabolism in resting-cell suspensions of caries-associated and noncaries associated dental plaque. Infect. Immun.17, 43-54. Minah G. E.. Lovekin G. and Finnev J. P. 1981. The effect of a cariogenic substrate environment on microbial succession and metabolic activity in experimental dental plaques. Caries Rex In press. Ostrom C. A.. Koulourides T.. Hickman F. and McGhee J. R. 1977. Microbial characterization of an experimental cariogenic plaque in man. J. dent. Res. 56, 556558. Svanberg M. and Loesche W. J. 1977. The salivarv concentration of Streptococci mutans and Streptococh sanguis and their colonization of artificial tooth fissures in man. Arch oral Biol. 22, 441447. Theilade E., Fejerskov O., Prackyabrued W. and Kilian M. 1974. Microbiologic study on developing plaque in human fissures. Stand. J. dent. Res. 82, 42&427. Theilade E., Fejerskov O., Karring T. and Theilade J. 1978. A microbiological study of old plaque in occlusal fissures of human teeth. Caries Res. 12, 313-319. Thott E. K., Folke L. E. A. and Sveen 0. B. 1974. A microbiologic study of human fissure plaque. Stand. J. dent. Res. 82, 428436.