Effective treatment of bacterial infections is becoming increasingly difficult with the emergence of resistance to multiple classes of antimicrobial drugs. In particular, sepsis due to the multidrug-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and E. coli/Enterobacter species) causes significant morbidity and mortality worldwide. During an investigation of drug interactions, multi-drug resistant E. coli proved difficult to embed for transmission electron microscopy (TEM), using conventional resin embedding. In addition to infiltration problems, traditional resin embedding destroyed ultrastructure and created artefacts. In this study we compared different methods for preparing the Gram-negative bacteria, E. coli for TEM analysis of morphology. Aldehyde-fixed bacteria were prepared using conventional embedding in Epon-Araldite or LR White resin at ambient temperature, and compared with ultrathin Tokuyasu cryo-sections. Results of this study indicate the superiority of ultrathin cryo-sections, with a clear delineation of the outer and inner membrane as well as the peptidoglycan layer. This technique opens opportunities for visualising drug interaction mechanisms on the bacterial cell membrane.