The maintenance of microbial community in human fecal samples by a cost effective preservation buffer

Abstract

In the burgeoning microbiome field, powerful sequencing approaches and accompanied bioanalytical methods have made tremendous contributions to the discoveries of breakthroughs, which favor to unravel the intimate interplay between gut microbiota and human health. The proper preservation of samples before being processed is essential to guarantee the authenticity and reliability of microbiome studies. Hence, the development of preservation methods is extremely important to hold samples eligible for the consequent analysis, especially population cohort-based investigations or those spanning species or geography, which frequently facing difficulties in suppling freezing conditions. Although there are several commercial products available, the exploration of cost-efficient and ready-to-use preservation methods are still in a large demand. Here, we performed shotgun metagenomic sequencing and demonstrated that microbial consortia in human fecal samples were substantially preserved within a temporary storage of 4 h, independent of the storage temperature. We also verified a previous reported self-made preservation buffer (PB buffer) could not only preserve fecal microbiota at room temperature up to 4 weeks but also enable samples to endure a high temperature condition which mimics temperature variations in summer logistics. Moreover, PB buffer exhibited suitability for human saliva as well. Collectively, PB buffer may be a valuable choice to stabilize samples if neither freezing facilities nor liquid nitrogen is available.

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

“…No statistical differences were observed for Bacteroides , Prevotellaceae , and Bifidobacterium ( 9 ). Storage temperature had little effect on the microbial community during 4 h of temporary storage ( 33 ). Eight literatures studied the differences between different preservation methods and the gold standard for 24-h storage ( 8 , 9 , 16 , 21 , 24 – 26 , 28 ).…”

“…Furthermore, the impacts of preservation conditions are smaller than the difference between individuals. Seven studies showed that the bacterial composition was relatively stable after preservation for 3 h, 4 h, 24 h, and 48 h ( 9 , 16 , 21 , 25 , 28 , 31 – 33 ). In brief, the composition of the microbial community was relatively stable when the feces were stored at room temperature for up to 24 h ( 9 ).…”

“…The alpha diversity of most of the samples preserved for 3 days, 4 days, or 5 days was similar to that of immediately frozen (−80°C) samples ( 8 – 10 , 13 – 18 , 21 , 25 , 28 , 31 , 35 ). Similarly, principal-coordinate analysis (PCoA) or nonmetric multidimensional scaling (NMDS) analysis showed that clustering of samples from the same individual is more evident than preservation conditions or preservation duration ( 7 – 18 , 20 , 25 , 26 , 29 , 31 , 33 – 35 ), suggesting that individual differences are more remarkable than differences caused by different preservation methods. The study found that the stability of 95% ethanol preservation is poor and the intraclass correlation coefficient (ICC) is lower and more diverse ( 34 ).…”

Standards for Collection, Preservation, and Transportation of Fecal Samples in TCM Clinical Trials

“…The preservation conditions of the fecal samples can significantly affect the integrity of the extracted DNA and RNA as well as the composition of the microbial community ( Wu et al., 2021 ). A reasonable protocol on the preservation conditions is of great significance to ensure the quality of the samples.…”

Colonic drug delivery: Formulating the next generation of colon-targeted therapeutics

“…Though not truly representative of colonic microbiome composition, human faecal material is easily obtained and represents an effective method for predicting formulation behaviour in vivo, provided that faecal samples are processed correctly and repeatedly [212,[214][215][216]. There is also the advantage that patient samples can be obtained to allow analysis of formulation behaviour in a target disease state [217].…”