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Effects of storage temperature, storage time, and Cary-Blair transport medium on the stability of the gut microbiota.

Author(s): Nagata N, Tohya M, Takeuchi F, Suda W, Nishijima S, Ohsugi M, Ueki K, Tsujimoto T, Nakamura T, Kawai T, Miyoshi-Akiyama T, Uemura N, Hattori M

Publication: Drug Discov Ther, 2019, Vol. 13, Page 256-260

PubMed ID: 31611489 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of storage temperature and duration and the use of Cary-Blair transport medium on bacteria counts and microbial composition of fecal specimens. The effect of preservation with Cary-Blair transport medium or RNAlater on colony forming units was also investigated. Fecal specimens from 11 healthy adults were transferred to the laboratory within 15 min of defecation, homogenized, and split into 231 aliquots. Aliquots either underwent immediate DNA extraction (fresh) or were stored in containers with or without Cary-Blair transport medium at -80°C (unspecified duration), 4°C (1, 3, or 7 days), or 25°C (1, 3, or 7 days). Fecal bacterial DNA was extracted using an enzymatic lysis method with lysozyme and achromopeptidase. Bacterial 16S rRNA was amplified by PCR, resolved by gel electrophoresis, and bands were cut out and then quantified by TapeStation. Amplicons were subjected to next-generation sequencing on a MiSeq and sequences were grouped into operational taxonomic units.

   Summary of Findings:

   Significant differences in the bacterial composition between fresh and stored fecal specimens were found when stored without Cary-Blair transport medium at 4°C for ≥3 days or at 25°C for ≥1 day (P<0.05, all), but there were no significant differences at either temperature even after 7 days when stored with Cary-Blair transport medium. Further, the differences in bacterial composition at both the genus and species level from fresh specimens were significant when stored without Cary-Blair transport medium at 4°C for ≥3 days or at 25°C for ≥1 day (P<0.05, all). Examination at the phyla level revealed that effects of storage temperature, duration, and use of Cary-Blair transport medium depended on the phyla in question, with significant declines in Firmicates with any storage at 25°C or 4°C for ≥3 days, regardless of the addition of Cary-Blair transport medium, and significant increases in Actinobacteria when stored without Cary-Blair transport media for 3 days or more at either temperature. In contrast, Bacteroidetes and proteobacteria were unaffected by storage at 4°C, regardless of use of Cary-Blair transport medium, but Bacteroidetes decreased when stored at 25°C only in the absence of Cary-Blair transport medium and Proteobacteria increased with storage at 25°C with or without Cary-Blair transport medium. Finally, bacterial survival counts were much lower in RNAlater-preserved specimens than the unpreserved or Cary-Blair transport media specimens under aerobic and anaerobic culture conditions (P<0.05).

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • None (Fresh)
   • RNAlater
   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Microbiological assay
   Cell count/volume	Next generation sequencing
   Cell count/volume	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Cary-Blair transport medium Refrigeration None (fresh) Frozen RNAlater
   Storage	Storage duration	1 day 3 days 7 days
   Storage	Storage temperature	4°C 25°C -80°C

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