Immediate Processing of Fresh Fecal Maximizes Microbial Stability and Functional Fidelity in Fecal Incubation System
Fecal incubation systems are widely employed as in vitro platforms to investigate gut microbial responses to dietary and therapeutic interventions. However, pre-incubation handling of fecal material may substantially influence microbial composition and downstream functional readouts. In this study, we systematically compared commonly used fecal preservation strategies—ethanol fixation (EtOH), freeze–thaw at -80 °C, and glycerol cryopreservation—using samples from ten healthy adult donors. All samples were subjected to standardized 24 h anaerobic incubation. Freshly processed samples and EtOH-treated samples retained microbial richness, community structure, and core microbiome overlap comparable to baseline controls (0 h). In contrast, freeze–thaw and glycerol preservation resulted in reduced alpha diversity, increased intra-group variability, and significant compositional divergence. Core microbiome analysis revealed substantial loss of shared taxa under freeze–thaw and glycerol conditions, whereas EtOH-treated samples maintained high core overlap with fresh controls. Predicted functional profiling further indicated preservation-dependent differences, with glycerol-treated samples exhibiting reduced abundance of key metabolic pathways. Collectively, these findings demonstrate that fecal preservation method critically shapes microbial stability during in vitro incubation. Immediate processing of fresh fecal samples should be prioritized to preserve ecological fidelity, while ethanol treatment represents the most suitable alternative when logistical constraints preclude fresh processing.
Fecal incubation systems are widely employed as in vitro platforms to investigate gut microbial responses to dietary and therapeutic interventions. However, pre-incubation handling of fecal material may substantially influence microbial composition and downstream functional readouts. In this study, we systematically compared commonly used fecal preservation strategies—ethanol fixation (EtOH), freeze–thaw at -80 °C, and glycerol cryopreservation—using samples from ten healthy adult donors. All samples were subjected to standardized 24 h anaerobic incubation. Freshly processed samples and EtOH-treated samples retained microbial richness, community structure, and core microbiome overlap comparable to baseline controls (0 h). In contrast, freeze–thaw and glycerol preservation resulted in reduced alpha diversity, increased intra-group variability, and significant compositional divergence. Core microbiome analysis revealed substantial loss of shared taxa under freeze–thaw and glycerol conditions, whereas EtOH-treated samples maintained high core overlap with fresh controls. Predicted functional profiling further indicated preservation-dependent differences, with glycerol-treated samples exhibiting reduced abundance of key metabolic pathways. Collectively, these findings demonstrate that fecal preservation method critically shapes microbial stability during in vitro incubation. Immediate processing of fresh fecal samples should be prioritized to preserve ecological fidelity, while ethanol treatment represents the most suitable alternative when logistical constraints preclude fresh processing.
University of California San Diego Microbiome Initiative