Establishing Methods for In Vitro Static Culture of Gut Microbiomes for Nutritional Intervention Studies

Author

Jeremy Mathew Case, Utah State University

Date of Award

5-2025

Degree Type

Thesis

Degree Name

Departmental Honors

Department

Animal, Dairy, and Veterinary Sciences

Abstract

Lipid oxidation products (LOPs), which form during high-heat processing of oils common in Western diets, are known to cause inflammation and cellular damage. While LOPs have been implicated in exacerbating colitis and promoting colon tumorigenesis in mouse models, their direct impact on the gut microbiome remains poorly understood. This capstone project aimed to develop a low-cost in vitro protocol to culture the mouse gut microbiome under anaerobic conditions, enabling future investigations into the direct effects of bioactive dietary compounds such as LOPs. The study also assessed whether viable microbial communities could be recovered from fecal samples stored at -80°C. Fecal inocula from fresh and frozen samples were cultured in an anaerobic Atmos bag system for 48 hours. Microbial growth was monitored by optical density, and 16S rRNA sequencing was used to assess community composition. Results indicated a significant loss in microbial diversity and a shift toward dominance by facultative anaerobes, particularly Enterobacteriaceae, within 24 hours of culture. These changes were consistent across replicates and timepoints and were likely driven by trace oxygen exposure and limitations of batch culture conditions. While the system enabled microbial growth, limitations in maintaining strict anaerobic conditions likely contributed to reduced community diversity over time. In contrast, microbial communities from fresh and frozen samples exhibited similar profiles, indicating that short-term freezing does not compromise microbiome recovery. Although the protocol did not fully preserve the complexity of a healthy gut microbiome, this study provides a valuable foundation for refining in vitro culture methods and confirms the feasibility of using archived fecal material in future experiments.

Recommended Citation

Case, Jeremy Mathew, "Establishing Methods for In Vitro Static Culture of Gut Microbiomes for Nutritional Intervention Studies" (2025). Undergraduate Honors Capstone Projects. 1014.
https://digitalcommons.usu.edu/honors/1014