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Comparative evaluation of microbial profiles of oral samples obtained at different collection time points and using different methods



Recently, the oral microbiome has been found to be associated with oral and general health status. Although various oral sample collection protocols are available, the potential differences between the results yielded by these protocols remain unclear. In this study, we aimed to determine the effects of different time points and methods of oral sample collection on the outcomes of microbiome analysis.

Materials and methods

Oral samples were collected from eight healthy individuals at four different time points: 2 h after eating, immediately after teeth brushing, immediately after waking up, and 2 h after eating on the subsequent day. Four methods of saliva collection were evaluated: spitting, gum chewing, cotton swab, and oral rinse. Oral microbiomes of these samples were compared by analyzing the bacterial 16S rRNA gene sequence data.


The oral microbial composition at the genus level was similar among all sample collection time points and methods. Alpha diversity was not significantly different among the groups, whereas beta diversity was different between the spitting and cotton swab methods. Compared with the between-subject variations, the weighted UniFrac distances between the groups were not minor.


Although the oral microbiome profiles obtained at different collection time points and using different methods were similar, some differences were detected.

Clinical relevance

The results of the present study suggest that although all the described protocols are useful, comparisons among microbiomes of samples collected by different methods are not appropriate. Researchers must be aware of the issues regarding the impact of saliva collection methods.

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The work was supported and conducted at the Department of Dentistry and Oral Surgery, Osaka Medical College, Osaka, Japan.

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Authors and Affiliations



All authors contributed to the study conception and design. Material preparation was conducted by Michi Omori, Nozomu Fukui, Kayoko Yamamoto, Yoichiro Nakajima, Kazuya Inoue, and Hiroyuki Nakano. The data collection and analysis were conducted by Nahoko Kato-Kogoe, Shoichi Sakaguchi, Daisuke Motooka, and Shota Nakamura. The first draft of the manuscript was written by Nahoko Kato-Kogoe and review and editing by Takashi Nakano, Shota Nakamura, and Takaaki Ueno, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Nahoko Kato-Kogoe.

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The authors declare that they have no conflict of interest.

Ethical approval

All procedures conducted in studies involving human participants were according to the ethical standards of the Ethics Committee of Osaka Medical College, approval no. 2145, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

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Informed consent was obtained from all individual participants included in the present study.

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Omori, M., Kato-Kogoe, N., Sakaguchi, S. et al. Comparative evaluation of microbial profiles of oral samples obtained at different collection time points and using different methods. Clin Oral Invest 25, 2779–2789 (2021).

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  • Human
  • Metagenome
  • Methods
  • Microbiota
  • Saliva