Oral Biofilm Sampling for Microbiome Analysis in Healthy Children
December 31st, 2017
Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.
The overall goal of this procedure is to present a standardized method for oral biofilm sampling for microbiome analysis in healthy children. This is accomplished by first preparing and sterilizing instruments and material. Next, the child is prepared for biofilm sampling.
Then biofilm sampling is performed from three sites, the subgingival sulcus, the mucosa from the vestibular fold, and saliva. Finally, samples are systematically stored. Ultimately, the oral microbiome analyses will be performed to answer clinical and scientific questions.
Hello, I'm Elizabeth, and I'm an orthodontist. My research focuses on the oral microbiome changes that come along with orthodontic treatment. The sampling of oral biofilm can be tricky.
The space in the subgingival sulcus is limited, especially in children. Comparing our microbiome data to worldwide research, we found a lack of standardized sampling protocols. With this video, we take the first step to fill this gap.
Calibrated and sterile paper points that are usually used in endodontic therapy are cut at the first ring mark in order to standardize their length. After cutting, paper points have to be UV-irradiated in order to avoid DNA and RNA contamination. The tubes for storage will be autoclaved and UV-irradiated as well.
Before biofilm sampling, teeth need to be cleaned completely, starting with the application of cocoa butter. Then teeth are stained with plaque disclosure. After staining, the mouth is rinsed with water.
Teeth are brushed properly with an electric toothbrush and water. No toothpaste is applied, as this would alter the oral biofilm. A cheek and tongue retractor is used to keep the mouth open and dry.
The placing of the retractor requires skilled and steady hands, so as to minimize discomfort to the child and to keep the procedure as short as possible. Sterile dental tweezers are used and the sampling site remains unaffected throughout the procedure. Teeth are cleaned and dried with sterile cotton swabs to avoid absorption of supragingival fluid during paper point sampling.
Different methods can be used for biofilm sampling in the oral cavity. Due to limited space in the children's subgingival sulcus, paper point sampling is the method of choice. Paper points are inserted tangentially up to a defined length of four millimeters for 20 seconds.
Special care has to be taken not to traumatize the junctional epithelium. Paper points are then cut at the third mark to a standardized length of four millimeters. Paper point can also be inserted simultaneously into the same site or samples can be pooled from several sites.
The dry field apparatus is removed for mucosal and saliva sampling. Paper points can be applied in the same way to sample mucosal biofilm in the vestibular fold. Salivary biofilm can be collected by spitting unstimulated saliva.
Depending on the study design, paper points will be stored as single, pooled, or parallel samples. A color-coded storage system will ease further sample management. Finally, the samples are stored for microbiome analyses at minus 80 degrees Celsius.
Several commercial kits have been developed for the quick and accurate identification of periodontopathogenic bacteria. Paper points are sent to specialized laboratories. DNA extraction, preparation, and analysis are then performed within three hours.
The DNA can also be used in research for microbiome studies. Next generation sequencing for example results in complete microbiome datasets. The overall relative abundance of certain bacterial species can be analyzed, as can shifts in the microbiota due to changing conditions.
This bar chart and heat map display the relative abundance in the subgingival microbiota on the phylum and on the order level. Differences in the microbial composition over time, comparisons between or within individuals or different sampling methods are presented in histograms. This figure shows the results of a comparison of two sampling methods, Mode A and Mode B, on different taxonomic levels.
Statistical analysis of the values created with next generation sequencing can be presented in tables. Different time points as shown in this table can be analyzed on different taxonomic levels. P-values are calculated to prove significant findings.
Finally, the oral microbiome on the OTU level can be visualized with principal coordinate analyses. This figure displays intra-and inter-individual differences in five subjects. A clustering of certain groups can show shifts in the microbiome composition.
This 3D animation shows the results of a case control study. Red dots are the cases. Blue dots are the controls.
A clear clustering of the cases after orthodontic intervention at T3, the big red dots, visualize a shift in the microbiome. Blue dots showing the control group are evenly distributed. This method can provide insight into clinical biofilm sampling in the subgingival sulcus of healthy children.
We chose paper points as most suitable for this vulnerable compartment. In this video, we applied the method to only permanent condition. It also can applied to mixed or mature condition.
With some of the patients, you can also sample periodontally diseased sites according to the same protocol. Hi, my name is Barbara, and I'm a molecular microbiologist. When I analyze biofilm in the lab, I need to rely on a library of unbiased clinical samples.
Each step in the interdisciplinary field of oral microbial research has to be fully standardized. And there is one more important fact. You can only be successful in a team.
So always treat your colleagues nicely.
Overview
This article presents a standardized method for oral biofilm sampling in healthy children, focusing on the challenges of sampling in the limited space of the subgingival sulcus. The method aims to improve the reliability of microbiome analyses by addressing the lack of standardized protocols.
Key Study Components
Area of Science
- Oral microbiome research
- Pediatric dentistry
- Microbial sampling techniques
Background
- Changes in the oral microbiome during childhood are increasingly studied.
- Current microbiome studies lack standardized sampling protocols.
- Sampling in the subgingival sulcus of children presents unique challenges.
- Paper point sampling is proposed as a suitable method for this area.
Purpose of Study
- To establish a reliable method for oral biofilm sampling in children.
- To standardize procedures for microbiome analysis.
- To facilitate comparisons between microbiome data across studies.
Methods Used
- Preparation and sterilization of instruments and materials.
- Cleaning and preparation of the child's teeth before sampling.
- Sampling from the subgingival sulcus, mucosa, and saliva using paper points.
- Systematic storage of samples for microbiome analysis.
Main Results
- Standardized paper point sampling effectively collects biofilm samples.
- Samples can be pooled or stored individually for analysis.
- Next generation sequencing provides detailed microbiome datasets.
- Statistical analyses reveal significant differences in microbial composition.
Conclusions
- The proposed method enhances the reliability of oral microbiome studies in children.
- Standardization is crucial for interdisciplinary research in oral microbiology.
- Collaboration among researchers is essential for successful outcomes.
Frequently Asked Questions
Executive Industry Relevance
Standardized oral biofilm sampling in children addresses a critical gap in pediatric microbiome research, enabling reliable data generation for target validation in oral health interventions. The method supports mechanistic de-risking by providing reproducible biological samples that reflect true microbial composition, reducing variability in preclinical assessments. This enhances predictive confidence in early discovery stages where oral microbiota shifts may indicate disease susceptibility or treatment response.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of oral microbiome composition as a biomarker for cariogenic and periopathogenic mechanisms in children.
- Operational Value: Provides a standardized sampling protocol that reduces technical variability in microbiome data generation.
- Scientific Value: Supports hypothesis testing regarding microbial shifts during childhood development and intervention periods.
Screening & Assay Development
- Scientific Value: Prepares validated biological samples for downstream NGS and bioinformatic analysis, ensuring assay readiness.
- Operational Value: Facilitates sample pooling or parallel processing, improving throughput and standardization across study sites.
- Scientific Value: Generates quantitative microbial abundance data suitable for assay optimization and screening campaign design.
Translational & Preclinical Research
- Scientific Value: Delivers disease-relevant samples from healthy pediatric oral sites, establishing baseline for comparative disease modeling.
- Operational Value: Enables longitudinal tracking of microbiome shifts, supporting risk-adjusted advancement decisions in preclinical pipelines.
- Scientific Value: Provides mechanistic insights into host-microbe interactions that inform target selection and validation strategies.
Pipeline & Workflow Integration
The method integrates into early discovery workflows by supplying standardized biological inputs for microbiome profiling, which informs target identification and validation efforts prior to lead generation.
- Discovery Biology: Supports hypothesis testing of microbial community shifts linked to oral health status and developmental stages.
- Screening: Delivers reproducible samples enabling consistent NGS-based microbial profiling for comparative compound or intervention evaluation.
- Analytics: Generates relative abundance and diversity metrics that allow cross-condition comparison and statistical assessment of microbial changes.
- Translational Research: Connects discovery-phase microbiome data to preclinical continuity by establishing healthy baselines for disease model validation.
- Enterprise Reuse: Establishes a reusable sampling protocol applicable across pediatric oral health studies, reducing protocol redevelopment costs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in microbiome-associated target validation by minimizing sampling-induced noise.
- Operational Value: Ensures reproducibility and standardization across sites and time points, critical for multi-center studies.
- Strategic Value: Improves go/no-go decision reliability in oral health programs by reducing false positives from methodological artifacts.
- Portfolio Impact: Enables risk-adjusted prioritization of oral microbiome targets based on robust, comparable longitudinal data.
Implementation Considerations
- Requires training in pediatric dental techniques and sterile handling to ensure sample integrity and child cooperation.
- Dependent on access to UV irradiation, autoclaving, and cold storage (-80°C) infrastructure for contamination control and sample preservation.
- Necessitates cross-disciplinary coordination between dental clinicians and molecular biologists for standardized execution.
- Requires adaptation considerations when applying to mixed dentition or periodontally diseased sites, as noted in the protocol.
- Limited by the physical constraints of subgingival access in very young children, which may affect sampling consistency.
