Autoclaving and long-term storage deplete glutamine sources in complex media and affect bacterial phenotypes

Autoclave sterilization is the most common method for sterilizing reagents and media in biology. However, the effects of heat-induced loss or modification of complex medium components on bacterial growth and phenotypes remain poorly understood. Here, we investigated the impact of autoclaving on glutamine sources in bacterial complex media using an Escherichia coli {Delta}glnA mutant, which requires exogenous glutamine for growth. {Delta}glnA exhibited impaired growth in LB medium after autoclaving compared with non-autoclaved LB, whereas its residual growth indicated the presence of heat-stable glutamine sources. Growth assays and HPLC quantification revealed that free glutamine in LB decreased from 58 {micro}M to 12 {micro}M upon autoclaving, while heat-stable glutamine sources remained at 117 {micro}M. Similar growth defects were observed for {Delta}glnA in autoclaved BHI, TSB, and M17 media compared with their non-autoclaved counterparts. Long-term storage of LB at room temperature for 24 weeks also reduced {Delta}glnA growth regardless of autoclaving, compared with freshly prepared LB. Furthermore, supplementation of glutamine sources into glutamine-deficient MHB medium enhanced biofilm formation by Pseudomonas aeruginosa. Collectively, these results demonstrate that autoclaving and storage reduce glutamine sources in complex media, thereby influencing bacterial growth and phenotypes.