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Cryopreservation of stool samples altered the microbial viability quantitively and compositionally.
Aolei Chen1, Yingxin Hu1, Yajie Zhang1
1State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Archives of Microbiology
|August 16, 2022
Summary
Cryopreservation of stool at -80°C reduces viable bacteria, altering gut microbiota composition. Room temperature storage is worse, decreasing diversity and increasing pathogens.
Area of Science:
- Microbiology
- Gut Microbiome Research
- Molecular Biology
Background:
- Stool analysis is crucial for understanding gut microbiota in humans and animals.
- Cryopreservation at -80°C is a common method for storing stool samples for research.
- Quantitative and compositional effects of cryopreservation on viable gut bacteria remain unclear.
Purpose of the Study:
- To quantitatively and compositionally assess the viable gut microbiota in stool samples after cryopreservation at -80°C.
- To compare the effects of -80°C cryopreservation with ambient temperature storage on viable gut bacteria.
- To identify specific bacterial groups affected by freezing and ambient storage.
Main Methods:
- Stool samples from three healthy adults were analyzed.
- Propidium monoazide (PMA) treatment was used to distinguish viable from non-viable bacteria.
- Quantitative PCR (qPCR) and 16S rRNA gene sequencing were employed to analyze viable microbiota.
Main Results:
- Cryopreservation at -80°C led to a decrease in the overall number of viable bacteria, with inter-individual variations.
- Alpha diversity of viable microbiota remained largely unchanged after freezing, but composition was altered.
- Freezing reduced Gram-negative bacteria (Bacteroidetes, Firmicutes) while proportionally increasing Gram-positive bacteria (Actinobacteria, Firmicutes), including Bifidobacterium.
- Ambient temperature storage significantly decreased viable bacteria, reduced alpha diversity, and enriched opportunistic pathogens like Escherichia-Shigella.
Conclusions:
- Cryopreservation at -80°C alters the composition of viable gut microbiota, with Gram-positive bacteria showing increased relative abundance.
- Bacterial cell envelope structure may influence sensitivity to freezing.
- Ambient temperature storage poses a greater risk to sample integrity, reducing viable bacteria and diversity.
- Researchers must consider the impact of cryopreservation on viable microbiota for accurate downstream analysis.
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