Related Experiment Video
Updated: Apr 20, 2026
Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside
Published on: July 3, 2016
Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses
Leah Cuthbertson1, Geraint B Rogers2, Alan W Walker3
1NERC Centre for Ecology & Hydrology, Wallingford, OX10 8BB, UK; Institute of Pharmaceutical Science, Molecular Microbiology Research Laboratory, King's College London, London SE1 9NH, UK.
Background:
Best practice when performing culture-independent microbiological analysis of sputum samples involves their rapid freezing and storage at -80°C. However, accessing biobanked collections can mean that material has been passed through repeated freeze-thaw cycles. The aim of this study was to determine the impact of these cycles on microbial community profiles.
Methods:
Sputum was collected from eight adults with cystic fibrosis, and each sample was subjected to six freeze-thaw cycles. Following each cycle, an aliquot was removed and treated with propidium monoazide (PMA) prior to DNA extraction and 16S rRNA gene pyrosequencing.
Results:
The impact of freeze-thaw cycles was greatest on rare members of the microbiota, with variation beyond that detected with within-sample repeat analysis observed after three cycles.
Conclusion:
Four or more freeze thaw cycles result in a significant distortion of microbiota profiles from CF sputum.
