Phosphate buffer

The impact of GCTA on the morphology and structure of S. Enteritidis was investigated using TEM (Hitachi H7700, Tokyo, Japan). S. Enteritidis cultures (1 × 10⁸ CFU/mL) were treated with 1 MIC of GCTA for 2 h. Then the culture was centrifuged, and the resulting precipitates were immersed in fixative (2.5% glutaraldehyde in 0.1 M phosphate buffer, Servicebio, China) at 4 °C overnight. Precipitates underwent triple rinsing with 0.1 M phosphoric acid, followed by pre-embedded in agarose and fixation with 1% OsO4 in 0.1 M phosphate buffer (PB, pH 7.4) for 2 h. After dehydration, the samples were double-stained with 2% uranium acetate and 2.6% lead citrate for TEM analysis. Control samples were prepared without GCTA.

The malondialdehyde (MDA) expression levels in each group of samples were detected using an MDA assay kit (A003-1-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions; and the intracellular reactive oxygen species (ROS) levels were determined with dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma, USA). Specifically, 50 μM DCFH-DA was applied to incubate the samples for 30 min at 37°C in the dark. Then, the samples were washed twice with cold phosphate buffer (Servicebio, Wuhan, China). Immediately thereafter, a fluorescence microscope (Olympus IX51, Japan) was conducted to capture intracellular ROS fluorescence images.