Effects of Stool Sample Preservation Methods on Gut Microbiota Biodiversity: New Original Data and Systematic Review with Meta-Analysis

Abstract

Here, we aimed to compare the effects of different preservation methods on outcomes of fecal microbiota. We evaluated the effects of different preservation methods using stool sample preservation experiments for up to 1 year. The stool samples from feces of healthy volunteers were grouped based on whether absolute ethanol was added and whether they were hypothermically preserved. Besides, we performed a systematic review to combine current fecal microbiota preservation evidence. We found that Proteobacteria changed significantly and Veillonellaceae decreased significantly in the 12th month in the room temperature + absolute ethanol group. The four cryopreservation groups have more similarities with fresh sample in the 12 months; however, different cryopreservation methods have different effects on several phyla, families, and genera. A systematic review showed that the Shannon diversity and Simpson index of samples stored in RNAlater for 1 month were not statistically significant compared with those stored immediately at -80°C (P = 0.220 and P = 0.123, respectively). The -80°C refrigerator and liquid nitrogen cryopreservation with 10% glycerine can both maintain stable microbiota of stool samples for long-term preservation. The addition of absolute ethanol to cryopreserved samples had no significant difference in the effect of preserving fecal microbial characteristics. Our study provides empirical insights into preservation details for future studies of the long-term preservation of fecal microbiota. Systematic review and meta-analysis found that the gut microbiota structure, composition, and diversity of samples preserved by storage methods, such as preservation solution, are relatively stable, which were suitable for short-term storage at room temperature. IMPORTANCE The study of gut bacteria has become increasingly popular, and fecal sample preservation methods and times need to be standardized. Here, we detail a 12-month study of fecal sample preservation, and our study provides an empirical reference about experimental details for long-term high-quality storage of fecal samples in the field of gut microbiology research. The results showed that the combination of -80°C/liquid nitrogen deep cryopreservation and 10% glycerol was the most effective method for the preservation of stool samples, which is suitable for long-term storage for at least 12 months. The addition of anhydrous ethanol to the deep cryopreserved samples did not make a significant difference in the preservation of fecal microbiological characteristics. Combined with the results of systematic reviews and meta-analyses, we believe that, when researchers preserve fecal specimens, it is essential to select the proper preservation method and time period in accordance with the goal of the study.

Keywords: biological sample; gut microbiome; preservation; stool.

FIG 1

Relative abundance of bacterial phylum according to different storage conditions. (A) Room temperature + absolute ethanol group. (B) The −80°C refrigerator group. (C) The −80°C refrigerator + absolute ethanol group. (D) Liquid nitrogen tank group. (E) Liquid nitrogen + absolute ethanol group.

FIG 2

Relative abundance of bacterial families according to different storage conditions. (A) Room temperature + absolute ethanol group. (B) The −80°C refrigerator group. (C) The −80°C refrigerator + absolute ethanol group. (D) Liquid nitrogen tank group. (E) Liquid nitrogen + absolute ethanol group.

FIG 3

Diversity curves of sample microbial communities under different preservation methods. (A) Species richness index. (B) Shannon diversity index. (C) Simpson index. (D) Rank abundance curve.

FIG 4

PCoA plot based on unweighted and weighted UniFrac distance matrices of samples in in different storage methods.

FIG 5

Flow chart for retrieving articles.

FIG 6

Alpha diversity of samples preserved for 1 month in RNAlater. (A) Shannon diversity index. (B) Simpson index.