Establishment of a Standard Tongue Coating Collection Method for Microbiome Studies

Abstract

Objective: Recently, researchers have been focusing on characterizing the tongue coating microbiome from patients with digestive tract disease. However, to the best of our knowledge, the tongue coating collection methods have not been standardized until now. This article focuses on bridging this gap by exploring and validating the conditions suitable for the collection of tongue coating samples. Methods: One hundred forty-one healthy subjects were involved in the standardization of the tongue coating collection method. We conducted our standardization experiment by comparing different sampling tools, different preservation solutions, different scraping times, and different storage days with preservation at room temperature. The tongue coating samples from 59 normal individuals were analyzed using 16S ribosomal RNA (rRNA) gene-sequencing technology. The assessment of the quality of extracted DNA was used to verify our established method. We separated the 59 subjects into two groups (aged and younger), and the sequencing results were used to explore the age-related changes in microbiome. Results: Sterile oral swab B is suitable for the collection of tongue coating samples. To obtain a sufficient amount of DNA from a tongue coating sample, we recommend 30 times of tongue coating scraping. Normal saline, phosphate-buffered saline, and commercial preservation solution are all suitable for short-term sample storage (p < 0.05), and Neisseria, Streptococcus, and Porphyromonas are significantly higher in younger individuals (all p < 0.05). Conclusion: A participant-friendly tongue coating collection method for microbiome analyses can be established with good reliability and reproducibility. By taking advantage of our established method and 16S rRNA gene sequencing, significant differences were found in diversity and composition of tongue coating microbiota between aged and younger individuals, which contributes to a better understanding of the age-related composition of tongue coating microbiota.