Adsorption from salivary fractions at solid/liquid and air/liquid interfaces.

Ellipsometry and the drop-volume technique were used to study the interfacial behaviour of fractions obtained from unstimulated whole saliva. Fractionation was by gel filtration on a Superdex 200 Hiload column equilibrated with 10 mM potassium phosphate buffer, pH 6.8, containing 0.15 M NaCl. The fractions were reconstituted to have the same absorbance at 215 nm (estimated molecular-weight range, F1 greater than 760-460 K, F2 205-39 K, F3 14-4.5 K, F4 4.5-2.5 K, F5 1.5-0.85 K, F6 0.85 less than or equal to 0.5 K). The fractions were analysed for amino acid composition and studied by hydrophobic interaction chromatography on a Phenyl-Superose column. Fraction 3 contained the largest amounts of proline, followed by fractions 4 and 2. Fraction 3 showed the highest relative hydrophobicity. Ellipsometric measurements on negatively charged silica surfaces and methylated hydrophobic surfaces revealed that larger amounts of material adsorbed on hydrophobic than on hydrophilic surfaces. On hydrophilic surfaces the largest amounts were adsorbed from the high molecular-weight fraction 1. Fractions 4 and 6 did not give any adsorption at all on these surfaces. Fraction 3 gave the largest amounts adsorbed on the hydrophobic surfaces. Drop-volume measurements showed distinct differences in the ability of the salivary fractions to lower the surface tension. Fractions 2 and 3 showed the greatest reduction in surface tension. It was concluded that the adsorption behaviour of salivary proteins showed a wide variation among the different fractions and that it is influenced by the physicochemical characteristics of the interfaces present in the mouth.     

In vitro interactions of anionic and cationic surfactants with salivary fractions on well-defined solid surfaces.

Ellipsometry was used to study the interaction of one anionic (SDS) and one cationic (CTAB) surfactant with films adsorbed from six different salivary fractions obtained after fractionation of whole unstimulated saliva on a Superdex 200 Hiload gel filtration column. Experiments were performed on both hydrophilic silica and hydrophobic methylated silica surfaces. The results of this study indicate that the adhesive and cohesive properties of the films adsorbed from the individual fractions were strongly dependent on the surface characteristics of the substrates and that the outcome of protein/surfactant interactions was dependent on factors such as protein composition, surfactant charge, and substrate characteristics. These interactions probably involve replacement of the adsorbed proteins by surfactants or protein/surfactant complex formation. The anionic surfactant seemed to be more efficient in removing adsorbed salivary proteins than the cationic one.     

An in vitro study of salivary film formation at solid/liquid interfaces

The aim of this study was to use the technique of in situ null ellipsometry to study some aspects of salivary film formation at solid/liquid interfaces. Experiments were performed in a fused quartz cell, and hydrophilic plasma cleaned silica and methylated hydrophobic silica surfaces were used as substrates. Samples of unstimulated whole saliva were allowed to adsorb on the test surfaces for 30 min. After the adsorption period, rinsing was performed for 5 min. Recordings were continued for another 30 min, and then new saliva samples were added in the cell. The results showed that statistically significant higher amounts were adsorbed on the hydrophobic than on the hydrophilic surfaces. The adsorbed films on both types of tested substrates consisted of loosely associated parts which were removed after rinsing and of more tightly adsorbed fractions. A significantly larger fraction was desorbed from the films adsorbed on the hydrophobic than on the hydrophilic surfaces. When saliva was introduced again in the cell, it was shown that the amounts adsorbed attained the values obtained before the rinsings. Increase in the concentration of saliva in the cell without previous rinsing did not cause any further increase in the mass of the adsorbed film.     

On the adsorption behaviour of saliva and purified salivary proteins at solid/liquid interfaces

Salivary proteinaceous substances are known to play important roles in the formation of the salivary pellicle. The aim of this study was to investigate some aspects of the interfacial behaviour of selected purified salivary proteins, as well as human saliva secretions, using time-resolved in situ ellipsometry. Hydrophobic methylated silica and hydrophilic pure silica were used as test substrates. Experiments were performed in vitro, preferentially in the low concentration range, with samples of fresh human whole resting saliva, parotid resting saliva and submandibular/sublingual resting saliva. The protein fractions investigated were human MUC5B, PRP-1, PRP-3 and statherin, as well as bovine submaxillary mucin (BSM). The results indicate that the amount of material adsorbed was strongly related to the protein concentration in the range investigated for both pure proteins and secretions. Generally, a larger amount of material was adsorbed onto hydrophobic surfaces than onto hydrophilic ones. However, pure PRP-1 adsorbed in similar amounts to both hydrophilic and hydrophobic surfaces in the concentration range investigated and BSM adsorbed in larger amounts at high concentrations on hydrophilic surfaces. Comparison of the observed adsorption rates for salivary secretions and calculated diffusion rates for individual proteins suggested initial adsorption of low-molecular-weight proteins/peptides. On hydrophilic surfaces the data indicate adsorption of proteins with diffusion rates corresponding to those of statherin, PRP-3 and PRP-1. MUC5B adsorbs at a later stage from both HWS and the individual secretions, which can be explained by a "Vroman effect"-like phenomenon. On hydrophobic surfaces, adsorption rates were found to be faster than those calculated for any of the proteins, and thus smaller proteins/peptides appear to be involved. The similar adsorption behaviour of PRP-1 and parotid saliva (HPS) on hydrophilic surfaces may suggest that long aPRPs account for a substantial portion of the film-forming capacity of HPS. Effects of added electrolyte could be explained by general screening effects and specific Ca2+ binding to serine phosphates in aqueous solutions, but were complex in phosphate buffer. Inter-individual differences in amounts adsorbed from HWS, HPS and HSMSLS, respectively, were not found to be statistically significant.     

Adsorption of chlorhexidine and black tea onto in vitro salivary pellicles, as studied by ellipsometry

The adsorption from 0.2% (w/w) chlorhexidine and black tea solutions onto an in vitro pellicle from whole unstimulated saliva on hydroxyapatite discs was studied by ellipsometry. It was found that chlorhexidine adsorbed to the pellicle and caused a modification of the pellicle properties, leading to a subsequent increase in adsorption of salivary and black tea components. There was a distinct order-of-addition effect, whereby chlorhexidine followed by black tea gave an overall greater adsorption of components compared with black tea followed by chlorhexidine. This increase in adsorption resulted in a concomitant increase in color or stain, as measured by a reflectance chromameter. The increase in adsorbed amounts and stain was modified, in part, by the adsorption of salivary fractions between the chlorhexidine and black tea treatments. In all cases, the chlorhexidine and black tea-modified pellicles were not readily removed by either phosphate or sodium dodecyl sulfate rinses. Thus, following exposure to chlorhexidine, the accelerated adsorption of salivary and black tea components can ultimately lead to increased staining of the pellicle.     

Initial studies on the behavior of salivary proteins at liquid/air interfaces.

The mode of adsorption of salivary proteins at air/liquid interfaces was studied by using the drop volume technique to measure the kinetics of surface tension decay of aqueous salivary solutions. Adsorption of salivary proteins from whole saliva was fast, with a plateau value of the surface tension of 43 (+/- 2) mNm-1. As the concentration of saliva was reduced, the plateau value of surface tension increased and was achieved more slowly. The reduction in surface tension of aqueous solutions was larger for salivary proteins than for many other proteins reported.     

Adsorption of whole saliva onto hydrophilic and hydrophobic solid surfaces: influence of concentration, ionic strength and pH

The influence of the concentration of salivary proteinaceous material from solutions of whole saliva on the kinetics of in vitro pellicle formation were studied together with the effects of ionic strength, pH and certain substrate characteristics. The pellicle formation was monitored by an automated Rudolph ellipsometer, equipped with a He-Ne laser (wavelength 632.8 nm). The substrates compared in the study were hydrophilic negatively charged silica surfaces and hydrophobic methylated silica surfaces. The results show that the adsorption of salivary proteins is a very rapid process on both types of surfaces. Part of the formed biofilm, however, desorbed upon rinsing, indicating that the proteinaceous material was adsorbed with varying binding strengths. Larger adsorbed amounts were recorded on hydrophobic than on hydrophilic surfaces. Increase of ionic strength caused larger amounts to be adsorbed on both types of surfaces but change of pH did not affect the adsorption on either of the studied surfaces. Ellipsometry was found to be a suitable technique to monitor the adsorption of salivary proteins at solid/liquid interfaces.     

Adsorption of whole saliva onto hydrophilic and hydrophobic solid surfaces: influence of concentration, ionic strength and pH.

The influence of the concentration of salivary proteinaceous material from solutions of whole saliva on the kinetics of in vitro pellicle formation were studied together with the effects of ionic strength, pH and certain substrate characteristics. The pellicle formation was monitored by an automated Rudolph ellipsometer, equipped with a He-Ne laser (wavelength 632.8 nm). The substrates compared in the study were hydrophilic negatively charged silica surfaces and hydrophobic methylated silica surfaces. The results show that the adsorption of salivary proteins is a very rapid process on both types of surfaces. Part of the formed biofilm, however, desorbed upon rinsing, indicating that the proteinaceous material was adsorbed with varying binding strengths. Larger adsorbed amounts were recorded on hydrophobic than on hydrophilic surfaces. Increase of ionic strength caused larger amounts to be adsorbed on both types of surfaces but change of pH did not affect the adsorption on either of the studied surfaces. Ellipsometry was found to be a suitable technique to monitor the adsorption of salivary proteins at solid/liquid interfaces.     

Chlorhexidine Uptake and Release From Modified Titanium Surfaces and Its Antimicrobial Activity

Background: Decontamination by adjunctive antiseptic agents such as chlorhexidine (CHX) is often recommended for the treatment of peri‐implant infections. However, its action on the titanium implant surface needs further research. This study is designed to evaluate the ability of modified titanium surfaces to release chlorhexidine after periodic CHX exposure. Methods: Four titanium surfaces were prepared: 1) no surface treatment control (machined surface [MA]); 2) an acid mix of 10% HNO₃ and 5% HF (HNF); 3) resorbable blast media (RBM); and 4) sandblasting and acid etching (SLA). Each surface was analyzed using a confocal laser scanning microscope and a scanning electron microscope. Each sample was incubated with whole saliva or phosphate‐buffered saline for 2 hours. Measurements of CHX release were performed using spectrometry on days 1, 2, and 5 after 1‐minute exposure to 0.5% chlorhexidine digluconate solution during a 5‐day cycle. CHX‐releasing experiments were repeated three consecutive times for 15 days. The antimicrobial activity of CHX‐adsorbed disks was determined by a disk diffusion test using Streptococcus gordonii. Results: The CHX‐adsorbed titanium surfaces exhibited a short‐term release of CHX, and CHX levels dropped rapidly within 3 days. SLA and RBM with smaller and narrower depressions released more CHX than HNF and MA, specifically in the saliva‐coated group. The disk diffusion test revealed that after CHX uptake, saliva‐coated SLA and RBM showed the highest antimicrobial activity. Conclusion: This study suggests that CHX release is significantly influenced by titanium surface modifications and that SLA and RBM might provide effective CHX uptake capacity in the saliva‐filled oral cavity.     

Delmopinol hydrochloride- and chlorhexidine digluconate-induced precipitation of salivary proteins of different molecular weights

Gel electrophoresis was used to analyze precipitates formed of delmopinol hydrochloride or chlorhexidine digluconate mixed with unstimulated whole saliva samples from five test subjects. Final concentrations of delmopinol (6.4 mM) or chlorhexidine (6.4 mM, 2.2 mM) mixed with whole saliva were incubated for 10 min at 37°C. The precipitates were pelleted by centrifugation and resuspended to a similar protein density. The protein patterns in the pellets were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using 12.5% gels. The amount of pellet protein was determined by densitometry in four molecular weight ranges (10-21.5, 21.5-26, 26-45, and 45-300). The results indicated that high molecular weight (45-300) proteins dominated in the precipitate and that 2.2 mM chlorhexidine precipitated more salivary protein than 6.4 mM. At equimolar concentration (6.4 mM) delmopinol precipitated more high molecular weight salivary proteins than chlorhexidine.     

Determination of chlorhexidine in saliva and in aqueous solutions.

A new method is presented for the determination of chlorhexidine in centrifuged saliva and in aqueous solutions by means of fluorescence spectroscopy. The method relies on complex formation between chlorhexidine and eosin. The fluorescence value of the chlorhexidine-eosin system decreases with increasing chlorhexidine concentrations. In centrifuged saliva a linear relation was found between the fluorescence at 541 nm and the chlorhexidine content up to about 15 ppm; the reproducibility of the method was found to be better than 1 ppm chlorhexidine. The biological spreading for centrifuged saliva from different participants as well as the spreading due to the period of saliva collection are about 3%. In whole uncentrifuged saliva the fluorescence method has been a detection limit of about 8 ppm chlorhexidine, due to the binding of the compound to salivary constituents. Above 8 ppm chlorhexidine, the biological variation in saliva is such that chlorhexidine determinations by means of fluorescence can be done only with a reproducibility of +/- 10 ppm. Advantages and disadvantages of the ultraviolet spectroscopy method, the radioactive labelling technique, and the fluorescence method for chlorhexidine determinations are discussed.     

The effect of chlorhexidine mouthrinses on the human oral flora

The purpose of the present study was to examine the effect of chlorhexidine mouthrinses on the oral flora. Four students rinsed, twice daily, with 10 ml of a 0.2 per cent solution of chlorhexidine gluconate, and four students served as controls on a no oral hygiene programme. The number of bacteria in saliva was estimated by a cultural technique and impression preparations were used for the study of the bacteria on the gingiva and tooth surface. The controls showed a 300 per cent increase in bacterial counts during the experiment. In the chlorhexidine group the number of bacteria per ml saliva was reduced by 85 per cent, after 24 hours, reaching a 95 per cent reduction on day 5. An 85–90 per cent reduction was maintained throughout the experimental period. Impression preparations of the gingival area in the controls showed a heavier accummulation of bacteria than in the chlorhexidine group. In addition an increasing bacterial colonization of the tooth surfaces occurred in the controls throughout the experiment, but was never observed in the chlorhexidine group. Although the number of bacteria in saliva was markedly reduced, large numbers still persisted. It, therefore, appears unlikely that the inhibition of plaque formation is primarily the result of a reduction of the salivary flora.     

Inhibition of adherence of Candida albicans and Candida dubliniensis to a resin composite restorative dental material by salivary secretory IgA and monoclonal antibodies

OBJECTIVE: The attachment of Candida to oral surfaces is a crucial step in the colonization of the oral cavity and the eventual development of oral diseases caused by this microorganism. Inhibition of adhesion is one of the strategies currently studied to prevent Candida infections. The main objective of this study was to investigate the inhibitory effect of the human salivary components on the adherence of Candida albicans and C. dubliniensis to Herculite, a widely used resin composite restorative dental material. We have also investigated the influence on the adherence of three monoclonal antibodies (mAbs) directed against C. albicans cell wall antigens. DESIGN: The adhesion of three strains of C. albicans and one strain of C. dubliniensis was studied by a visual method after incubating the fungus and the resin in presence and in absence of human whole saliva, secretory immunoglobulin A (sIgA) and three mAbs directed against C. albicans cell wall surface antigens. RESULTS: Adherence of C. albicans was inhibited by whole saliva (41.7%), salivary sIgA (55.7%) and the salivary components that bind to the cell wall (36.7%). Whole saliva significantly reduced the adhesion of C. dubliniensis to Herculite to 45.3% of the control level. Saliva previously adsorbed with fungal cells or sIgA depleted saliva had no effect on adherence. An inhibition in the adhesion of C. albicans and C. dubliniensis to Herculite similar to that shown by whole saliva was also observed when mAbs C7 and 26G7 were used. However, mAb 21E6 increased adhesion of all the strains to Herculite. CONCLUSIONS: The results suggest that sIgA, as well as whole saliva, are important in blocking adherence of C. albicans and C. dubliniensis to Herculite and that this effect can be reproduced with mAbs directed against the cell wall surface of C. albicans.     

The affinity of chlorhexidine for hydroxyapatite and salivary mucins

This study indicates that chlorhexidine gluconate adsorbs to hydroxyapatite, tooth surfaces and salivary mucins and that the adsorbed chlorhexidine is released when the concentration in the environment is low. The possible formation of reservoirs of chlorhexidine on the tooth surface in vivo from which chlorhexidine is slowly released could, therefore, prevent bacterial colonization and the development of dental plaque.     

Effect of critical surface tension on retention of oral microorganisms

Abstract – The effect of critical surface tension on the initial retention of microorganisms from unstimulated human saliva was tested in a flow cell system. Prior to each experiment the total numbers and the morphotypes of microorganisms present in saliva were recorded. The test surfaces were prepared to display known increasing critical surface tensions, as verified and standardized by contact angle measurements. Surfaces of initially low (20–22 mN/m), medium (35–38 mN/m) and high (>50 mN/m) critical surface tension were exposed to saliva at a flow rate of 1 ml/min. Microbiota and biofilm material associated with the test surface after 15 min of salivary exposure, were then subjected to standard detachment forces, by introducing a cell-free rinsing fluid at two different shear rates. Both the attachment and the detachment phases were executed at room temperature or 37°C. The retained population was counted in three different zones of the test surfaces with a light microscope and statistically tested for correlation to the main variables (critical surface tension, flow rate and temperature) and interactions. Retention success was significantly dependent on the initial critical surface tension and the flow rate. Surfaces of medium critical surface tension, representative of human tooth surfaces and most restorative dental materials, retained the highest numbers of microorganisms in comparison with the other surfaces tested, with no statistically verified selectivity in proportions of retained coccoid and rodshaped microorganisms for any surface. A 30-fold increase of the flow rate resulted in a 70–80% reduction of the retention success, with a higher relative number of cocci present on all the test surfaces. These results demonstrate that initial retention of microorganisms to surfaces is non-specific with regard to morphotypes, but is strongly related both to the mechanical removal forces and the surface energetic state of the solid surface exposed. Retention of microbial populations at interfaces might, advance selection of the critical surface tensions and predicted if shear forces at given sites are known.     

Comparison of the in vivo and in vitro antibacterial properties of povidone iodine and chlorhexidine gluconate mouthrinses

Clinical and laboratory studies were carried out to compare the antibacterial properties of two antiseptic mouthwashes, namely 1% povidone iodine and 0.2% chlorhexidine gluconate. In a group of 10 subjects after a single rinse with povidone iodine, an immediate mean fall in total salivary aerobes and anaerobes occurred, followed by a return to normal levels by 1-hour postrinsing. With chlorhexidine gluconate a similar but greater reduction in salivary bacterial counts was observed, which was still present up to the 7-h postrinsing period. Saliva samples obtained from the subjects 2 min after rinsing with providone iodine produced little or no inhibition to the growth of a test organism in vitro, whereas following chlorhexidine gluconate, antibacterial activity was present in the saliva specimens up to the 3-h sampling time. Using culture media containing comparable levels of soluble protein to saliva, the minimum inhibitory concentrations of povidone iodine against several standard test organisms were much higher than those of chlorhexidine gluconate. The results suggest that povidone iodine, as a mouthwash, exerts only an immediate antibacterial effect and unlike chlorhexidine, is not retained at antibacterial levels within the oral cavity after expectoration. This lack of prolonged action of povidone iodine in the oral cavity would appear to be relevant to its reported lack of antiplaque activity.     

Inhibition of attachment of human gingival fibroblast-like cells in vitro by saliva and salivary-sulfated glycoprotein in the presence of serum

We have previously shown that human whole saliva and a high molecular weight sulfated glycoprotein (SGP) salivary component inhibits attachment of human gingival fibroblast-like cells to plastic substrata in serum-free conditions. The purpose of this study was to investigate the influence of saliva on attachment of these cells to tissue culture plastic in the presence of serum. Individual wells of multiwell dishes were coated with either sterile whole saliva or SGP, sequentially with fetal bovine serum followed by saliva or SGP, sequentially with the latter agents applied in the reverse order, with mixtures of saliva and serum or SGP and serum. Washed wells were seeded with 1.0 x 10(5) fibroblasts in alpha-MEM and numbers of adhering cells determined after 30 minutes. Saliva or SGP inhibited cell adherence as previously reported. Cell adherence in wells treated sequentially with saliva or SGP followed by serum, or with the latter followed by the salivary agents, was reduced significantly compared with that in untreated control wells. Wells treated with mixtures of serum and saliva or SGP exhibited progressive reduction in numbers of adhering cells as the concentration of the salivary agents increased. Significant suppression of attachment compared with controls also occurred when cells in alpha-MEM containing 15% serum were plated onto saliva- or SGP-treated wells. These results are consistent with the hypothesis that adsorbed salivary glycoprotein may bring about periodontal wound healing by repair rather than by regeneration by inhibiting fibroblast attachment to root surfaces in vivo.     

Protein and mucin retention on oral mucosal surfaces in dry mouth patients

Pramanik R, Osailan SM, Challacombe SJ, Urquhart D, Proctor GB. Protein and mucin retention on oral mucosal surfaces in dry mouth patients. Eur J Oral Sci 2010; 118: 245–253. © 2010 The Authors. Journal compilation©2010 Eur J Oral Sci Oral homeostasis depends largely on proteins and mucins present in saliva that coat all oral surfaces. The present study compared the protein composition of residual fluid on mucosal surfaces in subjects with normal salivary flow with that of patients with dry mouth caused by salivary hypofunction. Samples of residual mucosal fluid were collected using paper strips and then analysed by protein electrophoresis and immunoblotting. In both patients and controls, residual fluids on mucosal surfaces (except the anterior tongue in control subjects) had higher protein concentrations than unstimulated whole‐mouth saliva. High‐molecular‐weight mucin (MUC5B) was present in greater amounts on the anterior tongue than on other surfaces in control subjects. In dry mouth patients who were unable to provide a measurable saliva sample, MUC5B was often still present on all mucosal surfaces but in reduced amounts on the anterior tongue. The membrane‐bound mucin, MUC1, was prominent on buccal and labial surfaces in patients and controls. Statherin was still present on surfaces that were dried to remove salivary fluid, suggesting that it may be adsorbed as a protein pellicle. It is concluded that oral mucosal surfaces in dry mouth patients can retain MUC5B and other salivary proteins, although the functional integrity of these proteins is uncertain.     

Rinsing with delmopinol 0.2% and chlorhexidine 0.2%: short-term effect on salivary microbiology, plaque, and gingivitis.

The aim of this short-term study was to compare the effect of delmopinol HCl 0.2% and chlorhexidine digluconate 0.2% rinses on the development of dental plaque, the healing of experimental gingivitis, and the salivary microbiology. As part of a larger study protocol, 45 healthy males enrolled in an oral hygiene program to upgrade their oral health. For this portion of the study, participants had their teeth professionally cleaned on day 0. The participants then abstained from standard mechanical oral hygiene procedures, but applied a placebo solution twice daily for 2 weeks. At the end of this period the subjects received a second professional cleaning and were then assigned to 2 treatment groups: Group 1 rinsed with 10 ml of delmopinol HCl 0.2% and Group 2 rinsed with 10 ml of chlorhexidine digluconate 0.2% for 1 minute twice daily for the next 2 weeks and continued to refrain from mechanical oral hygiene procedures. At the end of the placebo and active treatment periods 1) saliva samples were taken and cultivated on a series of media; 2) the degree of gingivitis was assessed with gingival crevicular fluid (GCF) and gingivitis index (GI); and 3) the plaque index was assessed and the stainable buccal plaque extension was analyzed planimetrically. No changes in the salivary microbiological counts were detected for the subjects rinsing with delmopinol. Subjects rinsing with chlorhexidine showed significant reductions of anaerobes, aerobes, and S. mutans in saliva. The amounts of GCF and GI were reduced largely to the same extent in both treatment groups. Mean plaque extension was reduced by 52% after delmopinol and 88% after chlorhexidine rinsing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of highly concentrated stannous fluoride and chlorhexidine regimes on human dental plaque flora

The aim of this study was to determine the effect of an intensive antimicrobial treatment on the number of Streptococcus mutans, Streptococcus sanguis, Actinomyces viscosus/Actinomyces naeslundii, and the total Colony-forming Units (CFU) in plaque. The dentition of human volunteers was treated in a dental office with either chlorhexidine (5%) or stannous fluoride (8%). Following the office treatment with chlorhexidine, selected volunteers rinsed daily at home for seven or 49 days with chlorhexidine solution (0.2%), while another group flossed daily at home for seven days with dental floss impregnated with chlorhexidine. On days one, seven, 21, 35, and 49 after the local applications, we took saliva samples and plaque samples from fissures, smooth surfaces, and approximal areas. Chlorhexidine and stannous fluoride suppressed S. mutans and the Actinomyces species on all surfaces and in saliva. S. mutans on tooth surfaces was suppressed for approximately seven days and returned to the baseline level at day 21. A. viscosus/naeslundii was suppressed for more than seven days on the teeth. S. sanguis and the total CFU returned to the baseline level within seven days on all surfaces and in saliva. Rinsing or flossing with chlorhexidine suppressed S. mutans during the period of time that these supplements were used. Brushing for seven days with chlorhexidine gel (1%) without a preceding intensive chlorhexidine treatment had virtually no effect on S. mutans in approximal areas and in saliva, but suppressed S. mutans in fissures and on smooth surfaces.