How Proper Fecal Sample Collection Matters in Outcomes

Scientist holding experiment sample in laboratory medical freezer

Having robust and reliable fecal samples is the cornerstone of any gut microbiome study. However, fecal material is not homogeneous and requires careful handling to obtain accurate and replicable results. A recent study published in Scientific Reports systematically compared different fecal sample collection and storage methods to provide recommendations for reducing variability in microbiome and metabolite profiling.

The researchers found that subsampling small aliquots from unhomogenized stool resulted in sporadic detection of some microbial taxa and inconsistent metabolite levels between samples. This heterogeneity was detected both within a single bowel movement and between consecutive stools collected less than a day apart. To minimize sampling bias, the study recommends homogenizing the entire stool sample prior to aliquotting for different analyses.

Additionally, immediate freezing of samples was found to best preserve the native bacterial and fungal composition compared to collection tubes with stabilizing liquid, which significantly altered the abundance of certain taxa. The study also detected differences in microbial diversity, metabolite levels, and community structure between two consecutive stools from the same individual, highlighting the importance of consistent collection timing.

Based on these results, the researchers advise participants in microbiome studies to collect their first bowel movement of the day, homogenize the entire sample, and freeze it immediately. For situations where immediate freezing is not possible, commercial stabilization tubes may be used but should be kept consistent across the study. Precise collection protocols can maximize sample quality and study reproducibility in the dynamic landscape of gut microbiome research.

Here are some interesting viewpoints from the study:

- The surface of stool samples showed higher variability in microbial richness and diversity compared to homogenized samples, indicating the mucus layer on the exterior of stool contributes to patchy microbial distribution.
- Fungal communities were more variable and seemed to have distinct composition patterns dominated by 1-2 species, unlike the distinct bacterial profiles per individual.
- Short chain fatty acid levels were linked to transit time and stool consistency, with higher SCFAs in looser stools and shorter transit times.
- Consecutive stools showed differences including higher acetic acid, lower diversity, and enrichment of certain bacterial taxa, suggesting normal microbiome volatility between bowel movements.
- Immediate freezing was superior to stabilization tubes for preserving microbial composition, though tubes altered abundances of certain taxa like decreased Actinobacteria.
- Stabilization tubes significantly increased Ruminococcaceae, so caution is needed when interpreting Firmicutes abundance if this collection method is used.
- The study provides direct evidence that methods like homogenization and optimized storage reduce variability from intrinsic stool heterogeneity and are critical for multi-omics microbiome studies.

With growing interest in the links between gut microbes to health, high-quality fecal samples are invaluable. By adopting evidence-based protocols for proper fecal sample collection, researchers can optimize their microbiome study outcomes.

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