IRMS_c13_isotope_ratio

<i>S. aureus</i><i> </i>in the exponential growth phase was diluted 1:1 with fresh TSB. C13-labeled oleic acid or acetate was added to the medium to achieve a final concentration of 25 mg/mL for oleic acid and 250 mg/mL for acetate. After a 2-hour incubation along with specified treatments, <i>S. aureus</i><i> </i>was centrifuged and washed three times with deionized water to remove C13-labeled compounds. The supernatant was discarded by centrifugation, sonicated in an ice bath for 30 minutes. A 2 mL aliquot of chilled extraction solution (1:2 chloroform/methanol, v/v) was added and vortexed for 5 minutes. An additional 0.5 mL of water and chloroform were added to induce phase separation, followed by vortexing for 1 minute. The mixture was centrifuged at 2000 rpm for 10 minutes, after which the lower layer containing the lipids was collected for subsequent C13 quantification using Liquid Chromatography-Isotope Ratio Mass Spectrometry (LC-IRMS, MAT-253, Thermo).