Combined pH ratiometry and fluorescence lectin-binding analysis (pH-FLBA) for microscopy-based analyses of biofilm pH and matrix carbohydrates.

Bacterial biofilms have a complex and heterogeneous three-dimensional architecture that is characterized by chemically and structurally distinct microenvironments. Confocal microscopy-based pH ratiometry and fluorescence lectin-binding analysis (FLBA) are well-established methods to characterize pH developments and the carbohydrate matrix architecture of biofilms at the microscale. Here, we developed a combined analysis, pH-FLBA, to concomitantly map biofilm pH and the distribution of matrix carbohydrates in bacterial biofilms while preserving the biofilm microarchitecture. As a proof of principle, the relationship between pH and the presence of galactose- and fucose-containing matrix components was investigated in dental biofilms grown with and without sucrose. The pH response to a sucrose challenge was monitored in different areas at the biofilm base using the ratiometric pH-sensitive dye C-SNARF-4. Thereafter, the fucose- and galactose-specific fluorescently labeled lectins Aleuria aurantia lectin (AAL) and Morus nigra agglutinin G (MNA-G) were used to visualize carbohydrate matrix components in the same biofilm areas and their immediate surroundings. Sucrose during growth significantly decreased biofilm pH (P < 0.05) and increased the amounts of both MNA-G- and AAL-targeted matrix carbohydrates (P < 0.05). Moreover, it modulated the biofilm composition towards a less diverse community dominated by streptococci, as determined by 16S rRNA gene sequencing. Altogether, these results suggest that the production of galactose- and fucose-containing matrix carbohydrates is related to streptococcal metabolism and, thereby, pH profiles in dental biofilms. In conclusion, pH-FLBA using lectins with different carbohydrate specificities is a useful method to investigate the association between biofilm pH and the complex carbohydrate architecture of bacterial biofilms.IMPORTANCEBiofilm pH is a key regulating factor in several biological and biochemical processes in environmental, industrial, and medical biofilms. At the microscale, microbial biofilms are characterized by steep pH gradients and an extracellular matrix rich in carbohydrate components with diffusion-modifying properties that contribute to bacterial acid-base metabolism. Here, we propose a combined analysis of pH ratiometry and fluorescence lectin-binding analysis, pH-FLBA, to concomitantly investigate the matrix architecture and pH developments in microbial biofilms, using complex saliva-derived biofilms as an example. Spatiotemporal changes in biofilm pH are monitored non-invasively over time by pH ratiometry, while FLBA with lectins of different carbohydrate specificities allows mapping the distribution of multiple relevant matrix components in the same biofilm areas. As the biofilm structure is preserved, pH-FLBA can be used to investigate the in situ relationship between the biofilm matrix architecture and biofilm pH in complex multispecies biofilms.

The Effect of Enzymatic Treatment with Mutanase, Beta-Glucanase, and DNase on a Saliva-Derived Biofilm Model.

INTRODUCTION: The dental biofilm matrix is an important determinant of virulence for caries development and comprises a variety of extracellular polymeric substances that contribute to biofilm stability. Enzymes that break down matrix components may be a promising approach to caries control, and in light of the compositional complexity of the dental biofilm matrix, treatment with multiple enzymes may enhance the reduction of biofilm formation compared to single enzyme therapy. The present study investigated the effect of the three matrix-degrading enzymes mutanase, beta-glucanase, and DNase, applied separately or in combinations, on biofilm prevention and removal in a saliva-derived in vitro-grown model. METHODS: Biofilms were treated during growth to assess biofilm prevention or after 24 h of growth to assess biofilm removal by the enzymes. Biofilms were quantified by crystal violet staining and impedance-based real-time cell analysis, and the biofilm structure was visualized by confocal microscopy and staining of extracellular DNA (eDNA) and polysaccharides. RESULTS: The in vitro model was dominated by Streptococcus spp., as determined by 16S rRNA gene amplicon sequencing. All tested enzymes and combinations had a significant effect on biofilm prevention, with reductions of >90% for mutanase and all combinations including mutanase. Combined application of DNase and beta-glucanase resulted in an additive effect (81.0% ± 1.3% SD vs. 36.9% ± 21.9% SD and 48.2% ± 14.9% SD). For biofilm removal, significant reductions of up to 73.2% ± 5.5% SD were achieved for combinations including mutanase, whereas treatment with DNase had no effect. Glucans, but not eDNA decreased in abundance upon treatment with all three enzymes. CONCLUSION: Multi-enzyme treatment is a promising approach to dental biofilm control that needs to be validated in more diverse biofilms.

A New Device for In Situ Dental Biofilm Collection Additively Manufactured by Direct Metal Laser Sintering and Vat Photopolymerization.

Dental biofilms are complex medical biofilms that cause caries, the most prevalent disease of humankind. They are typically collected using handcrafted intraoral devices with mounted carriers for biofilm growth. As the geometry of handcrafted devices is not standardized, the shear forces acting on the biofilms and the access to salivary nutrients differ between carriers. The resulting variability in biofilm growth renders the comparison of different treatment modalities difficult. The aim of the present work was to design and validate an additively manufactured intraoral device with a dental bar produced by direct metal laser sintering and vat photopolymerized inserts with standardized geometry for the mounting of biofilm carriers. Additive manufacturing reduced the production time and cost, guaranteed an accurate fit of the devices and facilitated the handling of carriers without disturbing the biofilm. Biofilm growth was robust, with increasing thickness over time and moderate inter- and intraindividual variation (coefficients of variance 0.48-0.87). The biofilms showed the typical architecture and composition of dental biofilms, as evidenced by confocal microscopy and 16S rRNA gene sequencing. Deeper inserts offering increased protection from shear tended to increase the biofilm thickness, whereas prolonged exposure to sucrose during growth increased the biofilm volume but not the thickness. Ratiometric pH imaging revealed considerable pH variation between participants and also inside single biofilms. Intraoral devices for biofilm collection constitute a new application for medical additive manufacturing and offer the best possible basis for studying the influence of different treatment modalities on biofilm growth, composition, and virulence. The Clinical Trial Registration number is: 1-10-72-193-20.

Semi-Automated Planimetric Quantification of Dental Plaque Using an Intraoral Fluorescence Camera.

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI), which measures the relative area of a tooth that is covered by plaque deposits. Compared to clinical indices, the PPI has a higher discriminatory power, but traditional planimetry is a time-consuming analysis, as the plaque-covered and clean tooth areas have to be determined manually for each image using image-processing software. Here, we present a method for the semi-automated planimetric quantification of dental plaque, which allows for the rapid processing of up to 1,000 images simultaneously. The method exploits the enhanced contrast between disclosed plaque, sound tooth surfaces, and soft tissues in fluorescence images acquired with an intraoral camera. Careful execution of the clinical procedures and accurate image acquisition are crucial steps for the successful semi-automated identification of the plaque-covered areas. The method is suitable for planimetry on sound facial and oral tooth surfaces, on most composite resin restorations, and on teeth with orthodontic brackets, but not on metallic restorations. Compared to traditional PPI recordings, semi-automated planimetry considerably reduces the amount of time spent on the analysis, as well as the subjective human input, thus increasing the reproducibility of planimetric measurements.

A fast and reliable method for semi-automated planimetric quantification of dental plaque in clinical trials.

AIM: To develop a simple and reproducible method for semi-automated planimetric quantification of dental plaque. MATERIALS AND METHODS: Plaque from 20 healthy volunteers was disclosed using erythrosine, and fluorescence images of the first incisors, first premolars, and first molars were recorded after 1, 7, and 14 days of de novo plaque formation. The planimetric plaque index (PPI) was determined using a semi-automated threshold-based image segmentation algorithm and compared with manually determined PPI and the Turesky modification of the Quigley-Hein plaque index (TM-QHPI). The decrease of tooth autofluorescence in plaque-covered areas was quantified as an index of plaque thickness (TI). Data were analysed by analysis of variance (ANOVA) and Pearson correlations. RESULTS: The high contrast between teeth, disclosed plaque, and soft tissues in fluorescence images allowed for a fast threshold-based image segmentation. Semi-automated PPI is strongly correlated with manual planimetry (r = 0.92; p < .001) and TM-QHPI recordings (r = 0.88; p < .001), and may exhibit a higher discriminatory power than TM-QHPI due to its continuous scale. TI values corresponded to optically perceived plaque thickness, and no differences were observed over time (p > .05, ANOVA). CONCLUSIONS: The proposed semi-automated planimetric analysis based on fluorescence images is a simple and efficient method for dental plaque quantification in multiple images with reduced human input.

Prevention of Initial Bacterial Attachment by Osteopontin and Other Bioactive Milk Proteins.

A considerable body of work has studied the involvement of osteopontin (OPN) in human physiology and pathology, but comparably little is known about the interaction of OPN with prokaryotic cells. Recently, bovine milk OPN has been proposed as a therapeutic agent to prevent the build-up of dental biofilms, which are responsible for the development of caries lesions. Bioactive milk proteins are among the most exciting resources for caries control, as they hamper bacterial attachment to teeth without affecting microbial homeostasis in the mouth. The present work investigated the ability of OPN to prevent the adhesion of three dental biofilm-forming bacteria to saliva-coated surfaces under shear-controlled flow conditions in comparison with the major milk proteins α-lactalbumin, ß-lactoglobulin, αs1-casein, ß-casein and κ-casein, as well as crude milk protein. OPN was the most effective single protein to reduce the adhesion of Actinomyces naeslundii, Lactobacillus paracasei subsp. paracasei and Streptococcus mitis. ß-casein and crude milk protein also had a pronounced effect on all three species, which suggests binding to different microbial surface structures rather than the blocking of a specific bacterial adhesin. Bioactive milk proteins show potential to delay harmful biofilm formation on teeth and hence the onset of biofilm-related oral disease.

Fluorescence lectin binding analysis of carbohydrate components in dental biofilms grown in situ in the presence or absence of sucrose.

Carbohydrate components, such as glycoconjugates and polysaccharides, are constituents of the dental biofilm matrix that play an important role in biofilm stability and virulence. Exopolysaccharides in Streptococcus mutans biofilms have been characterized extensively, but comparably little is known about the matrix carbohydrates in complex, in situ-grown dental biofilms. The present study employed fluorescence lectin binding analysis (FLBA) to investigate the abundance and spatial distribution of glycoconjugates/polysaccharides in biofilms (n = 306) from 10 participants, grown in situ with (SUC) and without (H2O) exposure to sucrose. Biofilms were stained with 10 fluorescently labeled lectins with different carbohydrate specificities (AAL, ABA, ASA, HPA, LEA, MNA-G, MPA, PSA, VGA and WGA) and analyzed by confocal microscopy and digital image analysis. Microbial composition was determined by 16S rRNA gene sequencing. With the exception of ABA, all lectins targeted considerable matrix biovolumes, ranging from 19.3% to 194.0% of the microbial biovolume in the biofilms, which illustrates a remarkable variety of carbohydrate compounds in in situ-grown dental biofilms. MNA-G, AAL, and ASA, specific for galactose, fucose, and mannose, respectively, stained the largest biovolumes. AAL and ASA biovolumes were increased in SUC biofilms, but the difference was not significant due to considerable biological variation. SUC biofilms were enriched in streptococci and showed reduced abundances of Neisseria and Haemophilus spp., but no significant correlations between lectin-stained biovolumes and bacterial abundance were observed. In conclusion, FLBA demonstrates the presence of a voluminous biofilm matrix comprising a variety of different carbohydrate components in complex, in situ-grown dental biofilms.

A comparison of video-based and slide-based teaching before hands-on rubber dam application: A quantitative and qualitative study.

PURPOSE/OBJECTIVES: Instructional videos may demonstrate the execution of complex clinical procedures and the cooperation between members of the dental team better than traditional slide-based teaching materials. The aim of the present study was to compare the effect of a procedural video on student ratings to a traditional still-image-based presentation in a course on rubber dam application. METHODS: In a randomized, double-blind, parallel arm design, participants (46 dental students) completed a seven-item, five-step Likert-scale questionnaire at baseline (t1), after a video-based or slide-based demonstration of rubber dam application (t2) and after hands-on training (t3). The students' judgement on the benefits of rubber dam (items 1-3), their motivation to use rubber dam (item 4), their self-efficacy (items 5-6) and their expected use of the teaching material (item 7) were assessed. Changes in the students' individual answers were analyzed for each item and comparison between intervention groups made. Moreover, the impact of the teaching format on in-class discussions was analyzed qualitatively using a thematic approach RESULTS: Both interventions arose comparable significant improvement in the students' Likert-scale ratings from t1 to t2, and again from t2 to t3. No significant differences between intervention groups were found in the students' ratings or in the qualitative analysis. CONCLUSIONS: Procedural videos have proven to be a valuable learning aid in a variety of teaching formats, but in the context of a live lecture, they may not constitute an improvement over traditional text- and still-image-based presentations.

Ratiometric imaging of extracellular pH in Streptococcus mutans biofilms exposed to different flow velocities and saliva film thicknesses.

Introduction: Fluid flow has a prominent influence on the metabolism of surface-attached biofilms. Dental biofilms are covered by a thin saliva film that flows at different rates in different locations under stimulated and unstimulated conditions. Methods:The present study employed pH ratiometry to study the impact of different flow velocities, saliva film thicknesses and saliva concentrations on microscale pH developments in Streptococcus mutans biofilms of different age. Results:While saliva flow at a velocity of 0.8 mm/min (unstimulated flow) had little impact on biofilm pH, stimulated flow (8 mm/min; 80 mm/min) affected vertical pH gradients in the biofilms and raised the average pH in 48-h biofilms, but not in 72-h and 168-h biofilms. The saliva film thickness had a strong impact on biofilm pH under both static and dynamic conditions. pH drops were significantly higher in biofilms exposed to a thin saliva film (≤ 50 µm) than a thick saliva film (> 50 µm). pH drops in the biofilms were also strongly dependent on the saliva concentration and thus the buffer capacity of the salivary medium. For 48-h and 72-h biofilms, but not for 168-h biofilms, pH drops in distinct microenvironments were more pronounced when the local biofilm thickness was high.

Reduction of dual-species biofilm after sonic- or ultrasonic-activated irrigation protocols: A laboratory study.

AIM: To evaluate the antibacterial effect of sonic- and ultrasonic-activated irrigation on bacterial reduction of a dual-species biofilm in root canals compared to nonactivated irrigation in a laboratory study. METHODOLOGY: Two hundred and forty extracted human single-rooted maxillary anterior teeth were divided into two main groups (G, n = 120) according to the initial preparation size of the root canal (G1: size 25, 0.06 taper, G2: size 40, 0.06 taper). Root canals were inoculated with Enterococcus faecalis and Streptococcus oralis. After 5 days, G1 received combined instrumentation (up to size 40, 0.06 taper) and irrigation/activation, whereas G2 received solely irrigation/activation protocols. In both groups, irrigation was performed with sodium hypochlorite (NaOCl 1%) or physiological saline (NaCl 0.9%), using nonactivated syringe irrigation, sonic activation (2 x 30 s) or ultrasonic activation (2 x 30 s). Logarithmic reduction factors (LRFs) of colony-forming units were analysed separately for dentine-adherent and planktonic bacteria immediately after irrigation/activation protocols (time-point 1) or after 5 days of further incubation (time-point 2) by analysis of variance (anova) and post hoc tests (Tukey's HSD, t-test). The significance level was set at 0.05. RESULTS: In G1 subgroups (combined instrumentation with irrigation/activation), LRFs were significantly affected by the applied irrigation solution (p < .0001), but not by the activation method (p > .05; anova). In G2 subgroups (solely irrigation/activation), both, irrigant solution and activation, significantly affected LRFs (p < .0001, anova). Sonic activation resulted in significantly higher LRFs than ultrasonic activation (p < .0001) which had significantly greater reductions than nonactivated irrigation (p < .05; Tukey's HSD). At T2, strong bacterial regrowth was observed in all groups; however, a significant bacterial reduction was detected for factors instrumentation, irrigant solution and activation (p < .0001; anova). Similar LRFs were found for dentine-adherent and planktonic bacterial cells in all groups (r = 0.91 at T1, r = 0.8 at T2). CONCLUSIONS: In this laboratory study on extracted maxillary anterior teeth high-frequency sonic activation resulted in a greater bacterial reduction compared to ultrasonic activation in groups receiving solely irrigation/activation protocols; however, irrigation using NaOCl and ultrasonic activation also contributed significantly to bacterial reduction compared to the control groups.

The impact of glass ionomer cement and composite resin on microscale pH in cariogenic biofilms and demineralization of dental tissues.

OBJECTIVE: Secondary caries is among the most frequent reasons for the failure of dental restorations. Glass ionomer cement (GIC) restorations have been proposed to protect the surrounding dental tissues from demineralization through the release of fluoride and by buffering the acid attack from dental biofilms. In contrast, the lack of buffering by composite resin (CR) restorations has been suggested as a promoting factor for the development of secondary caries. METHODS: The present study employed transversal microradiography and confocal microscopy based pH ratiometry to quantify mineral loss and map microscale pH gradients inside Streptococcus mutans biofilms grown on compound specimens consisting of enamel, dentin and either GIC or CR. RESULTS: Mineral loss in dentin was significantly lower next to GIC than next to CR, but no significant differences in local biofilm pH were observed between the two restorative materials. SIGNIFICANCE: The cariostatic effect of GIC relies predominantly on the provision of fluoride and not on a direct buffering action. The lack of buffering by CR did not affect local biofilm pH and may therefore be of minor importance for secondary caries development.

Root Caries Preventive Effect of Varnishes Containing Fluoride or Fluoride + Chlorhexidine/Cetylpyridinium Chloride In Vitro.

Caries preventive varnishes containing only fluoride might differ from those containing a combination of fluoride and antimicrobial components in terms of mineralization properties and their impact on the cariogenic biofilm. We compared a fluoride and a fluoride + chlorhexidine (CHX)/cetylpyridinium chloride (CPC) varnish on root caries formation in vitro. One hundred bovine root dentin samples were allocated to five groups (n = 20/group): (1) 7700 ppm fluoride varnish (Fluorprotector S (F)), (2) experimental placebo varnish for F (F-P), (3) 1400 ppm fluoride + 0.3% CHX/0.5% CPC varnish (Cervitec F (CF)), (4) experimental placebo varnish for CF (CF-P), (5) untreated control. Cariogenic challenge was provided using a multi-station, continuous-culture 3-species (Streptococcus mutans (SM), Lactobacillus rhamnosus (LR), Actinomyces naeslundii (AN)) biofilm model for 10 days. Mineral loss (ΔZ) was evaluated using transversal microradiography and bacterial counts in the biofilm assessed as colony-forming units. Fluorescence in situ hybridization (FISH) and confocal microscopy were performed to assess the three-dimensional biofilm architecture. Mean ± SD (vol% × µm) ΔZ was significantly lower for F (9133 ± 758) and CF (9835 ± 1677) compared to control (11362 ± 919) (p < 0.05), without significant differences between F and CF. SM counts were significantly lower and LR counts significantly higher in F- and CF-biofilms compared to control. AN counts were significantly higher in the F-biofilms than in all other groups. According to FISH, SM and LR invaded dentinal tubules only in the control-group. In the CF-group, the basal biofilm layer did not contain SM and AN. Both F and CF varnishes had similar caries-preventive effects and a considerable impact on biofilm structure and composition.

Hands-on live demonstration vs. video-supported demonstration of an aesthetic composite restoration in undergraduate dental teaching.

PURPOSE/OBJECTIVES: Live hands-on demonstration of dental procedures is a central format in undergraduate dental teaching. It captures the immediacy of the clinical situation and allows for direct communication between instructor and students, but it also requires an experienced instructor who is able to handle both the performed treatment and its visualization alongside the actual teaching. The aim of the present work is to compare the hands-on demonstration of a class IV composite restoration to a teaching format where the instructor guided the students through a prerecorded procedural video of the same treatment. METHODS: The effect of both interventions on the students' self-perceived learning outcomes was analyzed by questionnaires (response rate 100%) in a randomized controlled double-blind (participants, outcome assessor) parallel group design (September 10 to October 3, 2019). In-class discussions were explored qualitatively by thematic analysis. RESULTS: Both teaching formats increased the students' self-reported motivation, self-efficacy, and patient-centeredness in a similar way, with no significant differences between interventions. During in-class discussions, both the instructor and the students were more active in the video group. In contrast to the hands-on group, discussions in the video group also involved patient-related topics, such as aesthetics and general health. The video-supported teaching format considerably reduced the amount of time spent on optimizing the visualization of the performed treatment. CONCLUSION: Video-supported instructor-guided demonstrations may represent a promising teaching format as an alternative to live hands-on demonstrations of dental procedures in undergraduate dental education.

The whitening effect of single brushing with blue-covarine containing toothpaste-A randomized controlled trial.

OBJECTIVE: To evaluate whether a blue covarine containing silica-based toothpaste promotes a whitening effect immediately after tooth brushing. METHODS: A triple-blind, parallel group, randomized design was used to compare the tooth color before and after brushing. Two silica-based toothpastes were chosen, one of them containing blue covarine (BCT) and the other one without pigment to serve as control (CT). Tooth color was measured using a spectrophotometer and expressed as CIEDE 2000 ΔE, CIELAB values, WIO index, WID index and VITA scale tooth shade. In addition, the participants' satisfaction with and perception of their own tooth color were registered using an aesthetic numeric analogue scale. RESULTS: No significant changes in color components or dimensions measured using CIEDE 2000 ΔE (CT: mean = 1.3, 95% CI 0.8, 1.7; BCT: mean = 1.6, 95% CI 1.2, 2.1), WIO, WID, VITA scale or CIELAB values were detected for the CT and BCT groups, comparing the results before and after brushing. Likewise, the participants' satisfaction with the treatment results (P = 0.31) and the participants' evaluation of tooth color (P = 0.71) did not differ significantly between groups. CONCLUSION: After a single application, no significant improvement in tooth whiteness by the presence of blue covarine in a silica-based toothpaste was detected in color dimensions or by assessing the participants' satisfaction and perception. CLINICAL SIGNIFICANCE: The present study does not show a clinically relevant increase in tooth whiteness after a single brushing with a blue covarine containing toothpaste, nor could the study participants observe a difference between treatment with blue covarine containing toothpaste and control treatment.

Probiotic Effects on Multispecies Biofilm Composition, Architecture, and Caries Activity In Vitro.

While probiotics have been tested for their anti-caries effect in vitro and also clinically, there is a lack of understanding of their effects on complex dental biofilms. We assessed two probiotics, Lactobacillus reuteri and Streptococcus oligofermentans, on a continuous-cultured model containing Streptococcus mutans, Lactobacillus rhamnosus and Actinomyces naeslundii. Cariogenic biofilms were grown on bovine enamel specimens and daily challenged with L. reuteri or S. oligofermentans whole culture (LC/SC) or cell-free supernatant (LS/SS) or medium only (negative control, NC) (n = 21/group) for 10 days. Biofilm was assessed via counting colony-forming units, quantitative polymerase chain reaction, and fluorescence in situ hybridization. Caries activity was determined by pH measurements and by assessing mineral loss (ΔZ) using transverse microradiography. Both LC and SC significantly reduced total and strain-specific cariogenic bacterial numbers (p < 0.05). ΔZ was reduced in LC (mean ± SD: 1846.67 ± 317.89) and SC (3315.87 ± 617.30) compared to NC (4681.48 ± 495.18, p < 0.05). No significant reductions in bacterial numbers and ΔZ was induced by supernatants. Biofilm architecture was not considerably affected by probiotic applications. Viable probiotics L. reuteri and S. oligofermentans, but not their culture supernatants, could reduce the caries activity of multi-species biofilms in vitro.

A 3D printed microfluidic flow-cell for microscopy analysis of in situ-grown biofilms.

Biofilm phenomena ranging from metabolic processes to attachment, detachment and quorum sensing are influenced by the fluid flow across the biofilm. A number of commercially available flow-cells allow for microscopy analysis of laboratory biofilms under flow, but there is a lack of shear controlled microfluidic devices that accommodate biofilms grown in situ on carriers or tissue samples. Therefore, we developed a flow-cell with adjustable geometry for microscopy analysis of in situ-grown biofilm samples under shear-controlled flow. The flow-cells were designed as one-piece disposable models, 3D-printed in resin and sealed with a coverslip after insertion of the biofilm sample. As a proof of concept, we studied the impact of stimulated saliva flow on pH developments in in situ-grown dental biofilms exposed to sucrose. Under static conditions, pH dropped in the biofilms, with pronounced differences between individual biofilms, but also between different microscopic fields of view within one biofilm. pH in the top layer of the biofilms tended to be lower than pH in the bottom layer. Under conditions of stimulated saliva flow (5 mm/min), pH rose to neutral or slightly alkaline values in all biofilms, and the vertical gradients were reversed, with the biofilm bottom becoming more acidic than the top. Hence, the present work demonstrates the importance of flow for the study of pH in dental biofilms.

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy.

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 µm or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

Improved pH-ratiometry for the three-dimensional mapping of pH microenvironments in biofilms under flow conditions.

Confocal microscopy-based monitoring of pH in biofilms is gaining increasing interest, as it allows for a quick assessment of horizontal pH gradients without mechanically perturbing the biofilm. Ratiometric monitoring of pH with the fluorescent dye C-SNARF-4 has been used to reliably map extracellular pH in the basal layers of biofilms, but only under static conditions. Here, we expand this methodology to measurements of vertical gradients in multispecies in vitro-grown and in situ-grown dental biofilms of different age, and to pH measurements in in vitro-grown biofilms under flow conditions. After static incubation with glucose, young in vitro-grown biofilms (30h) were more acidogenic than older biofilms (120h). However, under dynamic conditions mimicking the oral salivary flow, low pH was only preserved in older biofilms. As both types of biofilm were of similar thickness (~20 µm), these findings highlight the importance of cell density and biofilm matrix maturation for pH developments. In both in vitro-grown and in in situ-grown biofilms, horizontal and vertical pH gradients were observed. Under static conditions, the surface layer of the biofilms tended to be more acidic, whereas the bottom layer became more acidic under dynamic conditions. Compared to in vitro-grown biofilms, 120 h in situ-grown biofilms showed higher acidogenicity during static incubation. This study shows that pH ratiometry with C-SNARF-4 is well-suited to monitor extracellular pH in thin biofilms in all three dimensions. The different pH dynamics observed under static and dynamic conditions argue for the implementation of flow during real-time assessment of biofilm pH.

A confocal microscopy based method to monitor extracellular pH in fungal biofilms.

pH in fungal biofilms is important for a variety of fungal infections and industrial applications involving fungal biofilms, but to date, it has never been measured directly inside the biofilm matrix. In the present study, a new methodology was developed allowing for confocal microscopy based monitoring of extracellular pH inside fungal biofilms. Monospecies biofilms of Aspergillus fumigatus, Candida albicans, Candida dubliniensis and Cryptococcus neoformans were stained with the pH dependent ratiometric probe C-SNARF-4, imaged with a confocal microscope, and a digital image analysis procedure was developed to determine pH in the extracellular matrix. As a proof of concept, pH developments at the biofilm-substratum interface were monitored for 1 h after exposure to glucose. Observed pH drops differed considerably between the different species and also between replicate biofilms of the same species. Candida albicans biofilms showed the highest acidogenicity, with pH drops occurring much faster than in planktonic culture. pH ratiometry with C-SNARF-4 is a valuable tool to get insight into fungal biofilm metabolism and may shed new light on both disease-related and industrially relevant processes in fungal biofilms.