Biofilm pH Research Articles

Calcium-Phosphate-Osteopontin Particles Reduce Biofilm Formation and pH Drops in in situ Grown Dental Biofilms

This 2-period crossover study investigated the effect of calcium-phosphate-osteopontin particles on biofilm formation and pH in 48-h biofilms grown in situ. Bovine milk osteopontin is a highly phosphorylated glycoprotein that has been shown to interfere with bacterial adhesion to salivary-coated surfaces. Calcium-phosphate-osteopontin particles have been shown to reduce biofilm formation and pH drops in a 5-species laboratory model of dental biofilm without affecting bacterial viability. Here, smooth surface biofilms from 10 individuals were treated ex vivo 6 times/day for 30 min with either calcium-phosphate-osteopontin particles or sterile saline. After growth, the amount of biofilm formed was determined by confocal microscopy, and pH drops upon exposure to glucose were monitored using confocal-microscopy-based pH ratiometry. A total of 160 biofilms were analysed. No adverse effects of repeated ex vivo treatment with calcium-phosphate-osteopontin particles were observed. Particle treatment resulted in a 32% lower amount of biofilm formed (p < 0.05), but large inter-individual differences could be observed. Biofilm pH was significantly higher upon particle treatment, both shortly after the addition of glucose and after 30 min of incubation with glucose (p < 0.05). Calcium-phosphate-osteopontin particles may represent a new therapeutic approach to caries control and aim at directly targeting virulence factors involved in the caries process. Further studies are required to determine the effect of particle treatment on more acidogenic/aciduric biofilms as well as the remineralizing potential of the particles.

Breastfeeding, Dental Biofilm Acidogenicity, and Early Childhood Caries

Aim: This study evaluated the acidogenicity of human milk by the dental biofilms of children with and without early childhood caries (ECC). Method: Biofilms of 16 children (7 with ECC; 9 caries free) were exposed to human milk or 10% sucrose solution in the crossover design, and the biofilm pH was determined. Results: Breastfeeding did not provoke a decrease in biofilm pH, irrespective of the children's caries status, whereas sucrose decreased the pH for both groups. In addition, higher ΔpH_5min (pH variation occurring at 5 min) was observed in the biofilms of ECC children (p < 0.05). The results suggest that breastfeeding may not contribute to ECC.

Candida albicans in oral biofilms could prevent caries.

Streptococcus mutans is a Gram-positive bacterium involved in development to caries, the most common infectious disease of our time. Streptococcus mutans interacts with other microbes, like the fungus Candida albicans and both are commonly isolated from patients with caries. Since the role of C. albicans in caries remains unknown, our aim was to unravel this using an in vitro dual-species cariogenic oral biofilm model. Biofilms were grown for 24-72 h on glass cover slips or hydroxyapatite (HA) disks to mimic the surface of teeth. Medium pH, lactic acid production capacity and calcium release from HA disks were determined. All 24-h biofilms had external pH values below the critical pH of 5.5 where enamel dissolves. In contrast, 72-h dual-species biofilms had significantly higher pH (above the critical pH) and consequently decreased calcium release compared to single-species S. mutans biofilms. Counter intuitively, lactic acid production and growth of S. mutans were increased in 72-h dual-species biofilms. Candida albicans modulates the pH in dual-species biofilms to values above the critical pH where enamel dissolves. Our results suggest that C. albicans is not by definition a cariogenic microorganism; it could prevent caries by actively increasing pH preventing mineral loss.

Ratiometric Imaging of Extracellular pH in Dental Biofilms.

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 µm thickness, but is limited to the pH range between 4.5 and 7.0.

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 µm thickness, but is limited to the pH range between 4.5 and 7.0.

Calcium-phosphate-osteopontin particles for caries control

Caries is caused by acid production in biofilms on dental surfaces. Preventing caries therefore involves control of microorganisms and/or the acid produced. Here, calcium-phosphate-osteopontin particles are presented as a new approach to caries control. The particles are made by co-precipitation and designed to bind to bacteria in biofilms, impede biofilm build-up without killing the microflora, and release phosphate ions to buffer bacterial acid production if the pH decreases below 6. Analysis of biofilm formation and pH in a five-species biofilm model for dental caries showed that treatment with particles or pure osteopontin led to less biofilm formation compared to untreated controls or biofilms treated with osteopontin-free particles. The anti-biofilm effect can thus be ascribed to osteopontin. The particles also led to a slower acidification of the biofilm after exposure to glucose, and the pH always remained above 5.5. Hence, calcium-phosphate-osteopontin particles show potential for applications in caries control.

Monitoring of extracellular pH in young dental biofilms grown in vivo in the presence and absence of sucrose

Background and objectivepH in dental biofilms is of central importance for the development of caries. We used the ratiometric pH-sensitive dye C-SNARF-4 in combination with digital image analysis to monitor extracellular pH in dental biofilms grown in situ with and without sucrose supply.DesignDental biofilms (48 h) from 10 individuals were collected on glass slabs mounted on intra-oral appliances. During growth, appliances were immersed extra-orally in either physiological saline or 4% sucrose for 2 min, eight times per day. Fluorescence emissions of C-SNARF-4 in deep layers of the biofilms were recorded ex vivo with confocal microscopy for 15 min or for 1 h after exposure to 0.4% glucose. Extracellular pH was determined ratiometrically using digital image analysis.ResultsExtracellular pH dropped rapidly in most examined sites after addition of glucose. Distinct pH microenvironments were observed within single biofilms. The variation in pH was similar between sites within the same biofilm and sites from different individuals. pH drop patterns did not differ between biofilms exposed to sucrose-free and sucrose-rich environments.ConclusionThe present study is the first of its kind to apply the combination of pH ratiometry and digital image analysis to systematically record extracellular pH in intact dental biofilms from several individuals for up to 1 h. We observed highly heterogeneous pH landscapes and the presence of acidogenic microenvironments – ‘acidogenic hotspots’ – within the biofilms. The data suggest that pH drops in young (48 h) dental biofilms are independent of the sucrose supply during growth.

Microscale profiling of photosynthesis-related variables in a highly productive biofilm photobioreactor.

In the present study depth profiles of light, oxygen, pH and photosynthetic performance in an artificial biofilm of the green alga Halochlorella rubescens in a porous substrate photobioreactor (PSBR) were recorded with microsensors. Biofilms were exposed to different light intensities (50-1,000 μmol photons m(-2) s(-1) ) and CO2 levels (0.04-5% v/v in air). The distribution of photosynthetically active radiation showed almost identical trends for different surface irradiances, namely: a relatively fast drop to a depth of about 250 µm, (to 5% of the incident), followed by a slower decrease. Light penetrated into the biofilm deeper than the Lambert-Beer Law predicted, which may be attributed to forward scattering of light, thus improving the overall light availability. Oxygen concentration profiles showed maxima at a depth between 50 and 150 μm, depending on the incident light intensity. A very fast gas exchange was observed at the biofilm surface. The highest oxygen concentration of 3.2 mM was measured with 1,000 μmol photons m(-2) s(-1) and 5% supplementary CO2. Photosynthetic productivity increased with light intensity and/or CO2 concentration and was always highest at the biofilm surface; the stimulating effect of elevated CO2 concentration in the gas phase on photosynthesis was enhanced by higher light intensities. The dissolved inorganic carbon concentration profiles suggest that the availability of the dissolved free CO2 has the strongest impact on photosynthetic productivity. The results suggest that dark respiration could explain previously observed decrease in growth rate over cultivation time in this type of PSBR. Our results represent a basis for understanding the complex dynamics of environmental variables and metabolic processes in artificial phototrophic biofilms exposed to a gas phase and can be used to improve the design and operational parameters of PSBRs.

Performance of stress resistant variants of Listeria monocytogenes in mixed species biofilms with Lactobacillus plantarum

Population diversity and the ability to adapt to changing environments allow Listeria monocytogenes to grow and survive under a wide range of environmental conditions. In this study, we aimed to evaluate the performance of a set of acid resistant L. monocytogenes variants in mixed-species biofilms with Lactobacillus plantarum as well as their benzalkonium chloride (BAC) resistance in these biofilms. L. monocytogenes LO28 wild type and acid resistant variants were capable of forming mixed biofilms with L. plantarum at 20°C and 30°C in BHI supplemented with manganese and glucose. Homolactic fermentation of glucose by L. plantarum created an acidic environment with pH values below the growth boundary of L. monocytogenes. Some of the variants were able to withstand the low pH in the mixed biofilms for a longer time than the WT and there were clear differences in survival between the variants which could not be correlated to (lactic) acid resistance alone. Adaptation to mild pH of liquid cultures during growth to stationary phase increased the acid resistance of some variants to a greater extent than of others, indicating differences in adaptive behaviour between the variants. Two variants that showed a high level of acid adaptation when grown in liquid cultures, showed also better performance in mixed species biofilms. There were no clear differences in BAC resistance between the wild type and variants in mixed biofilms. It can be concluded that acid resistant variants of L. monocytogenes show diversity in their adaptation to acidic conditions and their capacity to survive in mixed cultures and biofilms with L. plantarum.

Characterization of the Arginolytic Microflora Provides Insights into pH Homeostasis in Human Oral Biofilms

A selected group of oral bacteria commonly associated with dental health is capable of producing alkali via the arginine deiminase system (ADS), which has a profound impact on the pH of human oral biofilms. An increased risk for dental caries has been associated with reduced ADS activity of the bacteria in oral biofilms. Arginolytic bacterial strains from dental plaque samples of caries-free and caries-active adults were isolated and characterized to investigate the basis for differences in plaque ADS activity between individuals. Fifty-six ADS-positive bacterial strains were identified by 16S rRNA gene sequencing, and their ADS activity levels were compared under standard growth conditions. The spectrum of bacterial ADS activity ranged from 45.2 to 688.0 units (mg protein)^-1. Although Streptococcus sanguinis was the most prevalent species, other Streptococcus sp. were also represented. Biochemical assays carried out using 27 ADS-positive strains under conditions known to induce or repress ADS gene expression showed substantial variation in arginolytic activity in response to pH, oxygen and the availability of carbohydrate or arginine. This study reveals that the basis for the wide spectrum of arginolytic expression observed among clinical strains is, at least in part, attributable to differences in the regulation of the ADS within and between species. The results provide insights into the microbiological basis for intersubject differences in ADS activity in oral biofilms and enhance our understanding of dental caries as an ecologically driven disease in which arginine metabolism moderates plaque pH and promotes dental health.

Plaque pH in Caries-Free and Caries-Active Young Individuals before and after Frequent Rinses with Sucrose and Urea Solution

Objective: To examine pH in the approximal dental biofilm after acid and alkali formation from sucrose and urea, after an adaptation period to these substances, in caries-free (CF) and caries-active (CA) individuals. Saliva flow and buffer capacity, and aciduric bacteria in saliva and plaque were also examined. Material and Methods: Twenty adolescents and young adults (15-21 years) with no caries (n = 10, D_{m + i}MFS = 0) or ≥1 new manifest lesions/year (n = 10, D_mMFS = 3.4 ± 1.8) participated. After plaque sampling, interproximal plaque pH was measured using the strip method before (baseline) and up to 30 min (final pH) after random distribution of a 1-min rinse with 10 ml of 10% sucrose or 0.25% urea. This procedure was repeated after a 1-week adaptation period of rinsing 5 times/day with 10 ml of the selected solution. After a 2-week washout period the second solution was similarly tested. Mutans streptococci, lactobacilli and pH 5.2-tolerant bacteria were analyzed by culturing. Results: In the CF group, acid adaptation resulted in lowering of baseline and final plaque pH values after a sugar challenge, and in increased numbers of bacteria growing at pH 5.2, which was increased also after alkali adaptation. In the CA group, the final pH was decreased after acid adaptation. No clear effects of alkali adaptation were seen in this group. Conclusion: One-week daily rinses with sucrose and urea had the most pronounced effect on the CF group, resulting in increased plaque acidogenicity from the sugar rinses and increased number of acid-tolerant plaque bacteria from both rinses.

The Oral Microbiome in Dental Caries

Dental caries is one of the most common chronic and multifactorial diseases affecting the human population. The appearance of a caries lesion is determined by the coexistence of three main factors: acidogenic and acidophilic microorganisms, carbohydrates derived from the diet, and host factors. Socio-economic and behavioral factors also play an important role in the etiology of the disease. Caries develops as a result of an ecological imbalance in the stable oral microbiom. Oral microorganisms form dental plaque on the surfaces of teeth, which is the cause of the caries process, and shows features of the classic biofilm. Biofilm formation appears to be influenced by large scale changes in protein expression over time and under genetic control Cariogenic microorganisms produce lactic, formic, acetic and propionic acids, which are a product of carbohydrate metabolism. Their presence causes a decrease in pH level below 5.5, resulting in demineralization of enamel hydroxyapatite crystals and proteolytic breakdown of the structure of tooth hard tissues. Streptococcus mutans, other streptococci of the so-called non-mutans streptococci group, Actinomyces and Lactobacillus play a key role in this process. Dental biofilm is a dynamic, constantly active metabolically structure. The alternating processes of decrease and increase of biofilm pH occur, which are followed by the respective processes of de- and remineralisation of the tooth surface. In healthy conditions, these processes are in balance and no permanent damage to the tooth enamel surface occurs.

Effect of separator and inoculum type on electricity generation and microbial community in single-chamber microbial fuel cells

Single-chamber microbial fuel cell (SMFC)-I consisted of 4 separator-electrode assemblies (SEAs) with two types of cation exchange membrane (CEM: Nafion and CMI 7000) and an anion exchange membrane (AEM: AMI 7001). SMFC-II consisted of 4 SEAs with Nafion and three types of nonwoven fabric. SMFC-I and -II were inoculated with anaerobic digested and activated sludge, respectively, and operated under fed-batch mode. In SMFC I, AEM-SEA showed a maximum power density (PDmax). Nafion-SEA showed a PDmax in SMFC II, which was similar to that of Nafion-SEA of SMFC I. Although different bacteria were developed in SMFC-I (Deltaproteobacteria and Firmicutes) and SMFC-II (Gammaproteobacteria, Betaproteobacteria and Bacteroidetes), the inoculum type little affects electricity generation. Variations of pH and oxygen in biofilm have influenced microbial community structure and electricity generation according to the electrode and separator material. Although the electricity generation of non-woven fabric-SEA was less than that of Nafion-SEA, the use of non-woven fabrics is expected to reduce the construction and operating costs of MFCs.

Microscale gradients of oxygen, hydrogen peroxide, and pH in freshwater cathodic biofilms.

Cathodic reactions in biofilms employed in sediment microbial fuel cells is generally studied in the bulk phase. However, the cathodic biofilms affected by these reactions exist in microscale conditions in the biofilm and near the electrode surface that differ from the bulk phase. Understanding these microscale conditions and relating them to cathodic biofilm performance is critical for better-performing cathodes. The goal of this research was to quantify the variation in oxygen, hydrogen peroxide, and the pH value near polarized surfaces in river water to simulate cathodic biofilms. We used laboratory river-water biofilms and pure culture biofilms of Leptothrix discophora SP-6 as two types of cathodic biofilms. Microelectrodes were used to quantify oxygen concentration, hydrogen peroxide concentration, and the pH value near the cathodes. We observed the correlation between cathodic current generation, oxygen consumption, and hydrogen peroxide accumulation. We found that the 2 e(-) pathway for oxygen reduction is the dominant pathway as opposed to the previously accepted 4 e(-) pathway quantified from bulk-phase data. Biofouling of initially non-polarized cathodes by oxygen scavengers reduced cathode performance. Continuously polarized cathodes could sustain a higher cathodic current longer despite contamination. The surface pH reached a value of 8.8 when a current of only -30 μA was passed through a polarized cathode, demonstrating that the pH value could also contribute to preventing biofouling. Over time, oxygen-producing cathodic biofilms (Leptothrix discophora SP-6) colonized on polarized cathodes, which decreased the overpotential for oxygen reduction and resulted in a large cathodic current attributed to manganese reduction. However, the cathodic current was not sustainable.

Relationship among Salivary Carbonic Anhydrase VI Activity and Flow Rate, Biofilm pH and Caries in Primary Dentition

This study aimed to determine the activity of carbonic anhydrase isoenzyme VI (CAVI) in the saliva of preschool children with caries and to investigate the relationship between caries and salivary CAVI activity, salivary flow rate and biofilm pH before and after a 20% sucrose rinse. Thirty preschool children aged 45.3–80.3 months were divided into two groups: a caries-free group and a caries group. Clinical examinations were conducted by one examiner (ĸ = 0.95) according to WHO criteria (dmfs) and early caries lesions. From each subject, CAVI activity, salivary flow rate and plaque pH were determined before and after a sucrose rinse. The results were submitted to Wilcoxon, Mann-Whitney and Spearman correlation tests (α = 0.05). The results showed that prerinse CAVI activity and its variation were higher in the saliva from caries children than from caries-free children. No difference was found between the two groups in postrinse salivary CAVI activity. After rinsing, biofilm pH differences were lower in both groups (p = 0.0012 and p = 0.0037 for the caries and caries-free groups, respectively). Also, after the sucrose rinse, salivary flow rate significantly increased in caries and caries-free groups (p = 0.0003, p = 0.0037). The variation of salivary CAVI activity was negatively correlated with caries (r = –0.501, p = 0.005). Child’s age showed a positive correlation with caries (r = 0.456, p = 0.011). These results suggest that variation of salivary CAVI activity and child’s age are associated with dental caries in preschool children.

PH and Redox Potential Variations in an Anodic Biofilm Located in a Three-Electrode Bioreactor and a Microbial Fuel Cell

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PH Landscapes in a Novel Five-Species Model of Early Dental Biofilm

BackgroundDespite continued preventive efforts, dental caries remains the most common disease of man. Organic acids produced by microorganisms in dental plaque play a crucial role for the development of carious lesions. During early stages of the pathogenetic process, repeated pH drops induce changes in microbial composition and favour the establishment of an increasingly acidogenic and aciduric microflora. The complex structure of dental biofilms, allowing for a multitude of different ecological environments in close proximity, remains largely unexplored. In this study, we designed a laboratory biofilm model that mimics the bacterial community present during early acidogenic stages of the caries process. We then performed a time-resolved microscopic analysis of the extracellular pH landscape at the interface between bacterial biofilm and underlying substrate.Methodology/Principal FindingsStrains of Streptococcus oralis, Streptococcus sanguinis, Streptococcus mitis, Streptococcus downei and Actinomyces naeslundii were employed in the model. Biofilms were grown in flow channels that allowed for direct microscopic analysis of the biofilms in situ. The architecture and composition of the biofilms were analysed using fluorescence in situ hybridization and confocal laser scanning microscopy. Both biofilm structure and composition were highly reproducible and showed similarity to in-vivo-grown dental plaque. We employed the pH-sensitive ratiometric probe C-SNARF-4 to perform real-time microscopic analyses of the biofilm pH in response to salivary solutions containing glucose. Anaerobic glycolysis in the model biofilms created a mildly acidic environment. Decrease in pH in different areas of the biofilms varied, and distinct extracellular pH-microenvironments were conserved over several hours.Conclusions/SignificanceThe designed biofilm model represents a promising tool to determine the effect of potential therapeutic agents on biofilm growth, composition and extracellular pH. Ratiometric pH analysis using C-SNARF-4 gives detailed insight into the pH landscape of living biofilms and contributes to our general understanding of metabolic processes in in-vivo-grown bacterial biofilms.

The activity of a small lytic peptide PTP-7 on Staphylococcus aureus biofilms

One of the most important features of bacterial biofilms is their resistance to antibiotics and to the host immune system. In this study, we have found that a small lytic peptide, PTP-7, is very potent to Gram-positive bacteria and is able to kill antibiotic sensitive and resistant Staphylococcus aureus indiscriminately. Further studies have revealed that despite being a cationic peptide, the antibacterial activity of PTP-7 was not affected by the negatively charged extracellular polymeric substance (EPS) of biofilms. PTP-7 could diffuse into the deep layer of S. aureus biofilms to kill bacteria inside biofilms efficiently and effectively. Neither the high concentrations of metal ions nor the acidic pH in biofilms affected the activity of peptide PTP-7. It seems that the unique sequence/structure together with the resistant bacteria killing ability of peptide PTP-7 confers its anti-biofilm activity. This study sheds new light on the treatment of bacterial biofilms, especially various biofilm related infections.

Redox and pH Microenvironments within Shewanella oneidensis MR-1 Biofilms Reveal an Electron Transfer Mechanism

The goal of this research was to quantify the variations in redox potential and pH in Shewanella oneidensis MR-1 biofilms respiring on electrodes. We grew S. oneidensis MR-1 on a graphite electrode, which was used to accept electrons for microbial respiration. We modified well-known redox and pH microelectrodes with a built-in reference electrode so that they could operate near polarized surfaces and quantified the redox potential and pH profiles in these biofilms. In addition, we used a ferri-/ferrocyanide redox system in which electrons were only transferred by mediated electron transfer to explain the observed redox potential profiles in biofilms. We found that regardless of the polarization potential of the biofilm electrode, the redox potential decreased toward the bottom of the biofilm. In a fully redox-mediated control system (ferri-/ferrocyanide redox system), the redox potential increased toward the bottom when the electrode was the electron acceptor. The opposite behavior of redox profiles in biofilms and the redox-controlled system is explained by S. oneidensis MR-1 biofilms not being redox-controlled when they respire on electrodes. The lack of a significant variation in pH implies that there is no proton transfer limitation in S. oneidensis MR-1 biofilms and that redox potential profiles are not caused by pH.

An in vitro microbial model associated with sucrose to produce dentin caries lesions

Abstract The complexity of the oral environment and ethical issues have prompted the development of an in vitro bacterial model to evaluate the effect of frequency of sucrose exposure on dentin caries formation, biofilm composition, and pH changes. In the experiment, dentin specimens (n=45) were randomly divided into four groups: control (C), negative control (0S), 3S (three sucrose baths), and 6S (six sucrose baths). The specimens then were inoculated with Streptococcus mutans and treated according to the protocol described below. Dentin demineralization and lesion depth were assessed by transverse microradiography. Extracellular polysaccharides that formed in the biofilm were analyzed and counts of microorganisms in the carious dentin were measured. After a 7-day period of growth, the biofilm pH was assessed before and after sucrose baths (n=5). The addition of sucrose led to dentin caries development regardless of the number of sucrose baths performed. The number of colony forming units (cfu) from the carious dentin did not differ among the treatment groups, though the extracellular polysaccharides from both 3S and 6S differed from 0S. The pH decreased immediately after the sucrose bath but increased again after 5 min. We demonstrate here that the in vitro microbial model for the study of dentin caries formation is reproducible and able to produce dentin caries, irrespective of the frequency of sucrose exposure.