Assessment of stool collection and storage conditions for in vitro human gut model studies

The role of the gut microbiota in health and disease is becoming increasingly apparent. Faeces is the most accessible sample to collect from human volunteers for studying the gut microbiota. However, the impact of stool collection and storage conditions on microbial and metabolic profiles have not been fully evaluated. By understanding the effect of different stool collection and storage conditions on microbial and metabolic composition, we can consider these parameters in the design of in vitro fermentation studies.Stool samples from 3 volunteers were stored under 5 different conditions to mimic methods that researchers may use to collect and store stool samples for study of the gut microbiota, including: fresh sample used within 10 min; stored on wet ice (4 °C) for 60 min; stored in an anaerobic chamber in a temperature-controlled bag (4 °C) for 60 min; freezing at -20 °C for 60 min and freezing at -20 °C for 60 min and then at -80 °C for 2 weeks. The stored samples were added to basal medium in batch culture fermenters alone (negative control) or with 5 g 2'-Fucosyllactose (2'FL) Human Milk Oligosaccharide (HMO) (as a positive fermentation control). Samples were collected at 3 timepoints (0, 12 and 24 h) for analysis by Flow Cytometry-Fluorescent In Situ Hybridisation (FC-FISH) and 1H-Nuclear Magnetic Resonance (NMR) spectroscopy to assess the impact on microbial and metabolic profiles, respectively.Freezing stool significantly impacted microbial numbers and activity during in vitro fermentations, whereas storing the stool on wet ice (4 °C) or in an anaerobic chamber at 4 °C for 60 min had minimal effects on microbial and metabolic profiles throughout the 24 h batch culture fermentation experiments.For in vitro batch culture fermentation studies where it may not be practical or possible to use fresh stool, either storing the stool on wet ice (4 °C) or in an anaerobic chamber at 4 °C for 60 min could be plausible alternatives to maintain microbial and metabolic profiles for analysis.