Effects of xylitol on survival of mutans streptococci in mixed-six-species in vitro biofilms modelling supragingival plaque.

Xylitol has been claimed to reduce mutans streptococci (MS) in dental plaque by energy-consuming futile metabolic cycles. This study aimed to investigate the effects of xylitol on MS in an in vitro 6-species oral biofilm model. Each multispecies biofilm contained either a laboratory reference strain, a fresh isolate, a xylitol-sensitive or a xylitol-resistant strain of Streptococcus mutans or Streptococcus sobrinus. Biofilms, grown on pellicle-coated hydroxyapatite discs, were fed with a glucose/sucrose-supplemented medium 3 times daily for 45 min and incubated in saliva between feedings. Before or after feeding, biofilms were exposed to either 7.5% xylitol, 7.5% sorbitol or to saliva (control) for 20 min. After 64.5 h, biofilms were harvested and the microbial composition was analysed by non-selective and selective culturing. Strain variability in the ability to colonize biofilms was observed. However, the response patterns in the biofilms to the 4 polyol treatments were similar. None of the MS were inhibited by xylitol provided either before or after feeding. Sorbitol given before feeding did not affect microbial growth whereas sorbitol provided after feeding showed a slight, albeit statistically significant increase in MS counts for some of the tested strains. It did so at the expense of Streptococcus oralis, which decreased in numbers. The present findings do not support the contention that xylitol reduces MS in plaque by futile metabolic cycles.

Cariogenicity of soluble starch in oral in vitro biofilm and experimental rat caries studies: a comparison.

AIMS: Common belief suggests that starch is less cariogenic than sugar; however, the related literature is quite controversial. We aimed to compare cariogenic and microbiological effects of soluble starch in both a standard animal model and an oral biofilm system, and to assess the possible substitution of the animal model. METHODS AND RESULTS: Six-species biofilms were grown anaerobically on enamel discs in saliva and medium with glucose/sucrose, starch (average molecular weight of 5000, average polymerization grade of 31), or mixtures thereof. After 64.5 h of biofilm formation, the microbiota were quantitated by cultivation and demineralization was measured by quantitative light-induced fluorescence. To assess caries incidence in rats, the same microbiota as in the biofilm experiments were applied. The animals were fed diets containing either glucose, glucose/sucrose, glucose/sucrose/starch or starch alone. Results with both models show that demineralization was significantly smaller with starch than sucrose. CONCLUSIONS: The data demonstrate that soluble starch is substantially less cariogenic than glucose/sucrose. SIGNIFICANCE AND IMPACT OF THE STUDY: By leading to the same scientific evidence as its in vivo counterpart, the described in vitro biofilm system provides an interesting and valuable tool in the quest to reduce experimentation with animals.

Effects of Streptococcus mutans gtfC deficiency on mixed oral biofilms in vitro.

The aim of this study was to examine the influence of glucosyltransferase-gene-negative (gtf-) Streptococcus mutans strains unable to synthesize water-insoluble or soluble glucan on the structure and macromolecular diffusion properties of in vitro grown mixed oral biofilms. Biofilms modeling supragingival plaque consisted of Actinomyces naeslundii OMZ 745, Candida albicans OMZ 110, Fusobacterium nucleatum KP-F2, Streptococcus oralis SK 248, Veillonella dispar ATCC 17748T and one of the S. mutans strains UA159, OMZ 966, OMZ 937 or OMZ 977. Biofilms were grown anaerobically on sintered hydroxyapatite disks for 64.5 h at 37 degrees C. To perform confocal laser scanning microscopy analyses, microorganisms were stained with Syto 13 and extracellular polysaccharides (EPS) with Calcofluor. Macromolecular diffusion properties were measured following timed biofilm exposure to Texas-Red-labeled 70-kDa dextran. Results showed that replacing wild-type S. mutans by a gtfC- mutant led to an increase in the volume fraction occupied by cells from 29 to 48% and a decrease of the EPS volume fraction from 51 to 33%. No such changes were observed when the S. mutans wild-type strain was replaced by a gtfB- or gtfD- mutant. The diffusion coefficient of 70-kDa dextran in biofilms containing the gtfC- S. mutans was 16-fold higher than in biofilms with the wild-type strain indicating a strong macromolecular sieving effect of GTF C-generated glucans. Our data demonstrate the influence of EPS on the structure and macromolecular diffusion properties of an oral biofilm model and uncover our still limited knowledge of the function of EPS in biofilms and plaque.

An in vitro oral biofilm model for comparing the efficacy of antimicrobial mouthrinses.

The ability of commercial mouthrinses to reduce total viable counts of mixed microbial populations was examined using a previously developed in vitro model of supragingival plaque. Exploratory experiments aimed at fine-tuning the model indicated that optimal correspondence between in vitro and clinical results for chlorhexidine-containing formulations were obtained at a saliva:medium ratio of 70:30 (v/v); moreover, expanding the microbial population from 5 bacterial species to 5 bacterial species + Candida albicans had no noticeable impact on overall results. The efficacies of 12 different mouthrinse proprietary products containing chlorhexidine, hexetidine, octenidine, Triclosan, plant extracts, or aminefluoride/stannous fluoride vis-à-vis biofilm clearance were compared. All mouthrinses promoted a statistically significant reduction in microbial load compared to distilled water. The herbal- and phenolic-based products were substantially less effective than most chlorhexidine-containing mouthrinses, or mouthrinses containing hexetidine or octenidine. No significant difference between the plaque-clearing plaque-clearing abilities of Listerine and Meridol was observed. This polyspecies biofilm model can be a valuable tool for preclinical testing of antiplaque formulations, particularly during the product development stage.

Validation of an in vitro biofilm model of supragingival plaque.

The study of biofilm structure and function mandates the use of model systems for which a host of environmental variables can be rigorously controlled. We describe a model of supragingival plaque containing Actinomyces naeslundii, Veillonella dispar, Fusobacterium nucleatum, Streptococcus sobrinus, and Streptococcus oralis wherein cells are cultivated anaerobically in a saliva-based medium on hydroxyapatite discs coated with a salivary pellicle, with material and pieces of apparatus common to all microbiology laboratories. After 0.5 hr, 16.5 hrs, 40.5 hrs, and 64.5 hrs, the composition of adherent biofilms was analyzed by culture techniques, live/dead fluorescence staining, and confocal laser scanning microscopy. Repeated independent trials demonstrated the repeatability of biofilm formation after 40.5 hrs and 64.5 hrs. Brief exposures of biofilms to chlorhexidine or Triclosan produced losses in viability similar to those observed in vivo. This biofilm model should prove very useful for pre-clinical testing of prospective anti-plaque agents at clinically relevant concentrations.

Automated fluorescent in situ hybridization for the specific detection and quantification of oral streptococci in dental plaque.

Our aim was to develop a rapid fluorescent in situ hybridization (FISH) assay for the identification of different oral groups of streptococci in dental plaque and to combine it with digital image analysis for the automated enumeration of target cells. Cy3-labeled oligonucleotide probes specific for 16S rRNA gene sequences of the anginosus, mitis, mutans, and salivarius groups of streptococci were hybridized under stringent conditions with bacterial cultures or supragingival plaque samples that had been permeabilized with lysozyme. Probe specificity was determined with strains from 30 different species, mainly of oral origin. Results showed that probes ANG541, MIT447, SSP001, and SAL090 with specificity for the anginosus, mitis, mutans, and salivarius groups, respectively, the pan-reactive streptococcal probe STR405, the S. mutans specific probe MUT590, and the S. sobrinus specific probe SOB174 were well-suited for the identification of cultured streptococci. Probes STR405, MIT447 and SSP001 were then successfully applied to enumerate automatically bacteria of the recognized taxa in 144 supragingival plaque samples. On the average, total streptococci accounted for 8.2%, streptococci of the mitis and mutans groups for 3.9 and 1.7%, respectively, of the plaques. The combined application of FISH and automated image analysis provides an objective time-saving alternative to culture or PCR for the enumeration of selected oral streptococci in dental plaque.

Automated immunofluorescence for enumeration of selected taxa in supragingival dental plaque.

The present study investigated a recently developed automated image analysis technique for its applicability to the enumeration of selected bacteria in supragingival dental plaque. Following initial calibration, the system is capable to count fluorescence-labeled target cells in up to 48 samples without user interference. Test samples contained a characteristic mixture of planktonic bacteria, small almost planar bacterial aggregates, and large, virtually indisruptable clumps with cells from multiple species. Due to their complex composition, these samples provided a challenging validation step for the image analysis system. Automated enumeration of target bacteria was compared with visual counting of the fluorescence-labeled bacteria. Results are shown for six taxa (Actinomyces naeslundii, Fusobacterium nucleatum, Prevotella intermedia/Prev. nigrescens, Streptococcus gordonii/Strep. oralis/Strep. sanguis, Strep. sobrinus, and Veillonella dispar/ V. parvula) with characteristic differences in abundance, cell morphology and aggregation behavior. Results revealed good correspondence between the two enumeration techniques (correlation coefficients ranging from 0.77 to 0.92) provided that the portion of target bacteria exceeded 0.05% of the total bacterial cell number. This work demonstrates the applicability and usefulness of fully automated immunofluorescence to analyze such complex ecosystems as supragingival dental plaque.

Microbiological aspects of an in situ model to study effects of antimicrobial agents on dental plaque ecology.

This study validates an in situ model for ecological studies of dental plaque exposed to various antimicrobial agents with different modes of action on plaque bacteria. Eleven subjects wore two acrylic appliances, each containing two bovine enamel discs, during two 1-wk test periods. Using a split-mouth crossover design, the appliances were dipped twice daily for 1 min into water (control; treatment A), fluoride (26.3 mM NaF; B), zinc acetate (20.0 mM; C), or fluoride plus zinc acetate (D). Four of the subjects used also chlorhexidine diacetate (2.2 mM; E) and chlorhexidine plus fluoride (F). At the end of each period, plaque was collected from the discs, after which the microbiota were analyzed by culture, automated quantitative immunofluorescence, and a viability fluorescence stain. As compared to control, treatments B, C, and D resulted in a significant reduction of individual taxa as detected by immunofluorescence, whereas similar bacterial viability and total bacterial numbers were observed. In contrast, chlorhexidine significantly reduced bacterial viability, total cell numbers, and the abundance of most of the enumerated taxa. We conclude that this in situ model is well suited to study effects of antimicrobial agents on dental plaque ecology. Combined with viability testing, immunofluorescence is obviously superior to culture in detecting taxa-specific shifts caused by antimicrobial agents.

Effects of mouth rinses with xylitol and fluoride on dental plaque and saliva.

The aim of this study was to test the hypothesis that xylitol, alone and in combination with fluoride, affects the salivary flow rate and micro-biota, dental plaque accumulation, gingivitis development, and the acidogenic potential of plaque. Three groups, each of 10 subjects, rinsed for 1 min 3 times daily over two 4-week periods, first with 10 ml water (control), and thereafter with either 0.05% NaF, 40% xylitol, or with 0.025% NaF plus 20% xylitol according to a double-blind controlled design. They performed habitual mechanical tooth cleaning during the first 2 weeks of each period but abstained from interdental cleaning during the final 2 weeks. While mouth rinsing was continued, all mechanical oral hygiene was discontinued the last 2 days of each period to permit plaque accumulation. The last mouth rinse was administered in the clinic before the final examination. The following parameters were assessed: (1) unstimulated and paraffin-stimulated salivary secretion rates; (2) salivary micro-biota; (3) plaque index; (4) papillar bleeding; (5) plaque pH response to sucrose, and (6) lactate formation by dental plaque. No statistically significant differences in any of the parameters were found. In conclusion, three daily mouth rinses with fluoride and xylitol, separately or in combination, did not affect the salivary flow rate or micro-biota, dental plaque accumulation, gingivitis development, or the acidogenic potential of plaque.

Intra-oral variations in total plaque fluoride related to plaque pH. A study in orthodontic patients.

The aim of the present investigation was to study intra-oral variations in total plaque fluoride, and to examine whether such variations were related to plaque pH. Five orthodontic patients abstained from oral hygiene and daily fluoride rinsing for 2 days. Resting and fermenting plaque pH was measured with a touch micro-electrode at 14-21 localized sites on bonded vestibular tooth surfaces in each subject. Plaque samples from the same sites were analysed with a fluoride micro-electrode. A wide range of plaque pH values and fluoride concentrations were observed. In all subjects plaque pH and total fluoride levels were lower at upper than at lower front teeth. A direct log-linear relationship existed between total fluoride and fermenting plaque pH. In 4 of the 5 subjects this relationship was statistically significant (p < 0.05) and quite strong (adjusted R2 0.2-0.5, Beta 0.5-0.7). The study shows significant intra-oral variations in total plaque fluoride related to plaque pH.

Effects of mouthrinses with chlorhexidine and zinc ions combined with fluoride on the viability and glycolytic activity of dental plaque.

Inhibition of plaque acidogenicity by a mouthrinse with chlorhexidine (CHX) or zinc ions has been ascribed to a prolonged bacteriostasis due to substantive properties of the agents. The present aim was to study the effects of mouthrinses with CHX and Zn ions combined with fluoride on the viability and glycolytic activity of dental plaque in order to assess the bacteriostatic versus possible bactericidal effects. Following 2 d of plaque accumulation, 4 groups of 10 students rinsed with either 12 mM NaF (F), 0.55 mM CHX diacetate+F (F-CHX), 10 mM Zn acetate+F (F-Zn), or with the three agents in combination (F-CHX-Zn). Plaque samples were collected before and 90 min after mouthrinsing. Thereafter, the in vivo plaque pH response to sucrose was monitored in each student using touch microelectrodes. F-CHX and F-CHX-Zn reduced the in vivo pH fall significantly as compared with F, whereas F-Zn exerted a non-significant inhibition. Pooled pre- and post-rinse plaque samples were used to measure the pH fall during fermentation of [14C]-glucose, and the glycolytic profiles were analyzed by HPLC. Bacterial viability was assessed by counting the colony-forming units (CFU). All mouthrinses except F reduced glucose consumption and acid formation and thus the pH fall. F-CHX reduced the CFU equal to the reduction of glucose consumption, indicating that inhibition of plaque acidogenicity was due to a bactericidal rather than a bacteriostatic effect. F and F-Zn did not reduce the CFU, thus F-Zn decreased glucose metabolism without affecting plaque viability. F-CHX-Zn reduced both the CFU and glucose metabolism of surviving plaque microorganisms.

Effects of chlorhexidine-fluoride mouthrinses on viability, acidogenic potential, and glycolytic profile of established dental plaque.

Inhibition of dental plaque acidogenicity by chlorhexidine (CHX) mouthrinses has been ascribed to a long-lasting bacteriostatic effect due to binding of CHX to oral surface structures combined with a slow release rate from the binding sites. The present aims were to study the effects of CHX-containing mouthrinses on the viability and glycolytic activity of established plaque in order to assess the bactericidal versus the bacteriostatic effects. Following 2 days of plaque accumulation, three groups of 10 students rinsed with either 12.0 mM NaF, 0.55 mM CHX plus NaF, or with 2.2 mM CHX plus NaF. Plaque samples were collected before and 90 min after mouthrinsing. The pH in pooled pre- and post-rinse plaque samples was recorded before and up to 10 min after the addition of D-[U-14C]glucose. Total colony-forming units in each sample were determined. High-performance liquid chromatography analyses showed lactate to be the major extracellular glycolytic metabolite in all samples. CHX-NaF markedly reduced the colony-forming units, the pH fall from fermentation of glucose, as well as glucose consumption and lactate formation, whereas NaF alone exhibited no such effects. The reduction of glucose consumption by the CHX-NaF mouthrinses corresponded to the reduction of colony-forming units, indicating no bacteriostatic effect. The plaque pH in vivo was monitored in each student 90 min after mouthrinsing with the test solutions prior to and up to 1 h after a sucrose mouthrinse using touch microelectrodes. The CHX-NaF mouthrinses reduced the fall in pH significantly (p < 0.05) as compared with the NaF mouthrinse.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo effects of fluoride, chlorhexidine and zinc ions on acid formation by dental plaque and salivary mutans streptococcus counts in patients with irradiation-induced xerostomia.

Irradiation therapy including major salivary glands may result in xerostomia and enhanced susceptibility to dental caries. The present aim was to assess the ability of mouthrinses with F-, Zn2+, and chlorhexidine (CH), in various combinations, to reduce acidogenic potential of dental plaque and salivary mutans streptococcus counts (SMSC) in 7 patients with xerostomia secondary to irradiation. The patients rinsed twice daily for 3 weeks with the following test solutions: (1) 12 mmol/l NaF (F; control), (2) NaF + 20 mmol/l ZnCl2 (F-Zn), and (3) NaF + 1.1 mmol/l CH (F-CH). Resting periods (F) of varying lengths were incorporated. Acid formation by dental plaque was monitored as plaque pH response to a sucrose mouthrinse, at the end of each test period, 4 h after mouthrinsing with test solution. Plaque pH was measured repeatedly at 2-8 sites in each patient before, and up to 60 min after the sucrose mouthrinse using touch microelectrodes. SMSC were determined using Dentocult SM-Strip mutans. Compared with F, F-CH significantly (P < or = 0.02) reduced acid formation by plaque and SMSC, whereas F-Zn did not affect acid formation or SMSC significantly. Pilot experiments in 4 patients showed mouthrinses with NaF + 0.55 mmol/l CH + 10 mmol/l Zn2+ to be ineffective, whereas NaF + 2.2 mmol/l CH was highly effective, but no better than F-CH. Twice daily mouthrinses with 12 mmol/l NaF in combination with 1.1 mmol/l CH may be an effective regimen to prevent post-irradiation caries.

Role of a cell surface-associated protein in adherence and dental caries.

Insertional inactivation of the Streptococcus mutans spaP gene was used to construct an isogenic mutant (834) of strain NG8 (serotype c) which lacked the major cell surface-associated protein referred to as P1 (15). Results of several studies suggest that P1 is involved in the adherence of S. mutans to saliva-coated apatite surfaces. With an in vitro model system of hydroxyapatite (HA) beads coated with parotid saliva (PS) and additional HA surfaces coated with PS and in situ-formed glucan, it was observed that mutant 834 adhered poorly to the PS/HA surfaces. In contrast, both parent and mutant strains bound to the PS-glucan/HA surface. Groups of intact and desalivated rats were infected with each strain to determine relative capacities to induce dental caries. Rats were fed a highly cariogenic diet containing 56% sucrose for 3 to 5 weeks. Each strain colonized the rodent model and caused similar levels of smooth-surface caries under these dietary conditions. It was concluded that P1 influences the ability of organisms to adhere to saliva-coated surfaces and possibly affects primary colonization of the oral cavity in the absence of a glucan surface but has no effect on glucan-mediated adherence in vitro or in vivo.

Combined effects of Zn(2+)-chlorhexidine and Zn(2+)-cetylpyridinium chloride on caries incidence in partially desalivated rats.

The effects of Zn2+ combined with either chlorhexidine or cetylpyridinium chloride (CPC) on caries incidence in partially desalivated rats were investigated. Seven groups of 12 animals each received topical applications for 20 s with a saturated swab (0.2 ml) of the following aqueous solutions twice daily on weekdays (10 a.m. and 3 p.m.) and once daily during weekends (12 a.m.) for 5 wk: deionized water (placebo); 40 mM zinc acetate; 2.2 mM chlorhexidine diacetate; 4.4 mM CPC; 40 mM zinc acetate and 2.2 mM chlorhexidine diacetate; 40 mM zinc acetate and 4.4 mM CPC; and 20 mM NaF (positive control). Coronal caries was scored by the method of Keyes. All treatments except CPC alone resulted in significantly (P less than 0.05, ANOVA) less smooth-surface caries than did the placebo. NaF treatment resulted in significantly less smooth-surface caries than did Zn2+, chlorhexidine, CPC, and Zn(2+)-CPC. The inclusion of zinc ions did not significantly increase the caries-inhibitory efficacy of chlorhexidine (CH). The combination of Zn(2+)-CPC decreased smooth-surface scores significantly more than did CPC alone. Significant differences in sulcal-surface caries were not observed among the groups. Zn(2+)-CPC suppressed the Streptococcus sobrinus counts significantly more than did the separate agents. Animals treated with Zn(2+)-CH harbored the lowest populations of S. sobrinus.

Antimicrobial and antiplaque effects of a chlorhexidine and Triton X-100 combination.

This study aimed to assess the in vitro antimicrobial effect and in vivo plaque-inhibiting capacity of chlorhexidine (CH) combined with the nonionic detergent Triton X-100. Synergistic inhibition was observed by the combination of Triton X-100 and CH on in vitro growth of S. sobrinus OMZ 176 and of S. sanguis 10556. In the clinical experiments, 10 subjects rinsed twice daily with 10 ml aqueous solutions of 11.6 mM Triton X-100, and 0.55 mM CH, and with a combination of the two agents. All mechanical oral hygiene was suspended during the test periods. Sucrose enhanced plaque accumulations were recorded after 4 days of rinsing. The combination of CH-Triton X-100 decreased plaque accumulations significantly compared to placebo, but was less effective than CH alone (P less than 0.05). Thus no beneficial clinical effect was gained by combining the nonionic agent Triton X-100 with the cationic agent CH. The results also clearly demonstrate that addition which increase the antibacterial activity of CH do not necessarily coincide with improved in vivo antiplaque efficacy.

In vivo effects of zinc and chlorhexidine on dental plaque ureolysis and glycolysis.

We assessed the in vivo effects of zinc and chlorhexidine (CH) on plaque ureolysis and glycolysis in five volunteers. We monitored plaque pH by a surface glass electrode on two teeth in each subject, after topical application of either 5% wt/vol urea or 5% wt/vol glucose solutions. The recordings were repeated 15 and 75 min after a single mouthrinse, with either 20 mmol/L zinc acetate or 0.33 mmol/L CH diacetate. Ureolytic activity decreased significantly (p less than 0.03) up to 75 min after a single mouthrinse with the zinc-containing test solution. CH had no effect on plaque ureolytic activity. Acid production by dental plaque decreased significantly (p less than 0.01) up to 75 min after a single mouthrinse with either the zinc- or the CH-containing test solution.

Dose-related effects of ZnCl2 on dental plaque formation and plaque acidogenicity in vivo.

The plaque-inhibiting capacity of 5-, 10-, 20-, and 100-mM ZnCl2 solutions was examined in 10 dental hygienist students. Twice daily mouthrinses with a 10-ml solution of 5, 10, and 20 mM ZnCl2 significantly inhibited plaque formation (p less than or equal to 0.05), whereas 100 mM ZnCl2 had only a negligible effect. Mouthrinses with 5 and 100 mM ZnCl2 were also tested in 6 dental students characterized as being heavy plaque formers. In this group, the antiplaque effect of 5 mM ZnCl2 was more pronounced, while 100 m ZnCl2 also significantly decreased plaque accumulation. The effect of a single mouthrinse with a 10-ml solution of 5 and 100 mM ZnCl2 on plaque acidogenicity was also assessed in 5 dental students. 5mM ZnCl2 significantly depressed acid production by plaque 30 min after a rinse, while 100 mM ZnCl2 inhibited acid production significantly for up to 4 h. 100 mM ZnCl2 was significantly more effective at depressing acid production than 5 mM ZnCl2 30 min after a rinse.

Plaque inhibition by a combination of zinc citrate and sodium lauryl sulfate.

Bacteriological tests demonstrated an additive inhibitory effect of ZnCl2 and sodium lauryl sulfate (SLS) on in vitro growth of Streptococcus sobrinus OMZ 176 and of Streptococcus sanguis ATCC 10556. As measured by atomic absorption spectrophotometry, the solubility of zinc citrate increased in the presence of SLS. After 48 h, the concentration of solubilized zinc from aqueous solutions of 5.0 mM zinc citrate was 12.0 mM versus 14.4 mM in the presence of 34.7 mM SLS. The plaque-inhibiting properties of aqueous solutions of 12.0 mM Zn2+ from zinc citrate, 34.7 mM SLS, and 14.4 mM Zn2+ from zinc citrate in combination with 34.7 mM SLS were examined in 7 volunteers. Plaque accumulations were assessed by using a method earlier described after 3 days of twice daily mouthrinses with 10 ml test solution, during which period no mechanical oral hygiene was performed. Compared to placebo, SLS and zinc citrate increased the frequency of plaque index score 0 by 52.9 and 98.3%, respectively, and SLS gave 36.9% and zinc citrate 55.7% less surfaces with scores 2 or 3 (p less than 0.05 in all cases). The combination of zinc citrate and SLS gave a threefold increase of plaque index score 0 and a 70.5% reduction of scores 2 or 3 (p less than 0.05).

Inhibition of plaque formation and plaque acidogenicity by zinc and chlorhexidine combinations.

Zinc ions and chlorhexidine (CH) were found to exhibit a synergistic inhibitory effect on in vitro growth of S. sobrinus OMZ 176 and of S. sanguis 10556. A clinical mouthrinsing experiment was performed in a group of 10 volunteers to assess the plaque-inhibiting capacity of this combination. Sucrose enhanced plaque accumulations were assessed (Plaque Index, Silness & Löe) after 4 days of twice daily mouthrinses with 10 ml aqueous solutions of either 10.0 mM zinc or 0.55 mM CH, or with a combination of zinc ions and CH, during which period no mechanical toothcleaning was performed. The Zn-CH combination showed improved inhibition properties compared to the individual agents. The effects on plaque acidogenicity of 8.0 mM zinc, 0.44 mM CH, and of zinc and CH in combination were also assessed in a test panel of five volunteers. The Zn-CH combination inhibited acid production by dental plaque significantly (P less than or equal to 0.05) more than the individual agents 1 h 30 min after a single rinse.