20240820_TEM

Transmission electron microscopy of cells were grown overnight and then sub-cultured into fresh LB, grown at 37°C shaking for 3 hours before being rinsed and then resuspended in TEM buffer and delivered for preparation.Wild-type MG1655 (MJG0001) and <i>ppk relA spoT </i>(MJG1282) were grown in LB at 37°C until exponential phase growth (approximately OD600 0.3–0.5). Cells were then spun down and collected. Remove media from pellet and fixin 1% Osmium tetroxide (148) in 0.1M Sodium Cacodylate BufferpH 7.4 at room temperature in the dark for 1 hour, then 3 times 0.1M Sodium Cacodylate BufferpH 7.4 rinse for 15 minutes each. 1% Low molecular weight tannic acid (Ted Pella Inc) for 20 minutes, 3 times 0.1M Sodium Cacodylate BufferpH 7.4 rinse for 15 minutes each.

将细胞过夜培养后，转接至新鲜LB培养基中，于37℃振荡培养3小时，随后经冲洗并重悬于透射电镜（Transmission Electron Microscopy, TEM）缓冲液，送至样本制备流程。野生型MG1655（MJG0001）及*ppk relA spoT*菌株（MJG1282）于LB培养基中37℃培养至指数生长期（OD600约为0.3~0.5），随后离心收集细胞。弃去上清培养基，将细胞沉淀置于1%四氧化锇（货号148）与0.1M二甲胂酸钠缓冲液（pH 7.4）的混合固定液中，室温避光固定1小时；之后用0.1M二甲胂酸钠缓冲液（pH 7.4）冲洗3次，每次15分钟。再用1%低分子量单宁酸（Ted Pella Inc）处理20分钟，随后再次用0.1M二甲胂酸钠缓冲液（pH 7.4）冲洗3次，每次15分钟。