Physical fixation by plunge-freezing offers minimum sample manipulation and preserves bacterial samples in a near native state. In conjunction with cryo-EM imaging, we gained insights into a range of research projects, including the studies on the Porphyromonas gingivalis protein secretion system and surface layer, biogenesis of outer membrane vesicles (OMVs), and bactericidal mechanisms of anti-microbial agents. Cryo-EM analysis of various secretion mutants led to the discovery of an electron dense surface layer (EDSL) in P. gingivalis and its linkage to secreted protein substrates that are post-translationally modified and attached to the surface of the bacterium. OMVs generated by Gram-negative bacteria play an important role in bacterial pathogenicity. Cryo-EM revealed the ultrastructure of purified OMVs from P. gingivalis, Treponema denticola and Tannerella forsythia, in addition to their size and shape variations. Changes in P. gingivalis OMV biogenesis were observed when limiting the supply of hemin in a chemostat setting. Various isogenic mutants, lacking proteins that are selectively sorted into P. gingivalis OMVs, are being studied to elucidate their roles in OMV biogenesis. Cryo-EM imaging allowed the visualisation of differential actions by anti-microbial peptides, caerin and melittin, on Streptococcus mutans. The mechanism employed by star polymers in the killing of E. coli was proposed based on the cryo-EM images acquired. Cryo-EM not only shed light on the original research questions but also often provided additional structural insights into bacterial samples, leading to new research directions and focuses.