Impact of Autoclaving and Long‐Term Storage on Bacterially Available Glutamine Sources in Complex Media

Free glutamine is unstable in aqueous solution and rapidly degrades at elevated temperatures. In complex microbiological media, glutamine may exist in free and peptide-bound forms; however, how autoclaving and prolonged storage affect bacterially available glutamine sources remains unclear. Here, we used an Escherichia coli glutamine synthetase-deficient mutant (ΔglnA) as a biological indicator to assess glutamine availability in complex media. The ΔglnA mutant exhibited reduced but detectable growth in autoclaved LB compared with non-autoclaved LB, indicating the presence of both heat-labile and heat-stable glutamine sources. Prolonged storage of autoclaved LB at room temperature further reduced ΔglnA growth. Brain heart infusion, tryptic soy broth, and M17 contained both heat-labile and heat-stable glutamine sources, whereas Mueller-Hinton broth (MHB) and nutrient broth contained neither. Furthermore, supplementation of MHB, which appeared to lack bacterially available glutamine sources, with glutamine resulted in a greater reduction in viable Staphylococcus aureus cells in the presence of gentamicin than gentamicin treatment alone. These findings demonstrate that medium processing, storage, and composition influence the availability of glutamine sources and can affect bacterial growth and antibiotic susceptibility.