Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

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.