Bacterial pore-forming toxins(PFTs) are essential virulence factors of many human pathogens. Knowledge of their structure within the membrane is critical for an understanding of their function in pathogenesis and for the development of useful therapy. Atomic force microscopy(AFM) has often been employed to structurally interrogate many membrane proteins, including PFTs, owing to its ability to produce sub-nanometer resolution images of samples under aqueous solution. However, an absolute prerequisite for AFM studies is that the samples are single-layered and closely-packed, which is frequently challenging with PFTs. Here, using the prototypical member of the cholesterol-dependent cytolysin family of PFTs, perfringolysin O(PFO), as a test sample, we have developed a simple, highly robust method that routinely produces clean, closely-packed samples across the entire specimen surface. In this approach, we first use a small Teflon well to prepare the supported lipid bilayer, remove the sample from the well, and then directly apply the proteins to the bilayer. For reasons that are not clear,bilayer preparation in the Teflon well is essential. We anticipate that this simple method will prove widely useful for the preparation of similar samples, and thereby enable AFM imaging of the greatest range of bacterial PFTs to the highest possible resolution. Bacterial pore-forming toxins (PFTs) are essential virulence factors of many human pathogens. Knowledge of their structure within the membrane is critical for an understanding of their function in pathogenesis and for the development of useful therapy. Atomic force microscopy (AFM) has often been employed to structurally interrogate many membrane proteins, including PFTs, due to its ability to produce sub-nanometer resolution images of samples prepared under aqueous solution. However, an absolute prerequisite for AFM studies is that the samples are single-layered and closely- packed, which is frequently challenging with PFTs. Here, using the prototypical member of the cholesterol-dependent cytolysin family of PFTs, perfringolysin O (PFO), as a test sample, we have developed a simple, highly robust method that routinely produces clean, closely- packed samples across the entire specimen surface. In this approach, we first use a small Teflon well to prepare the supported lipid bilayer, remove the sam ple from the well, and then directly apply the proteins to the bilayer. For reasons that are not clear, bilayer preparation in the Teflon well is essential. We anticipate that this simple method will prove widely useful for the preparation of similar samples, and thus enable AFM imaging of the greatest range of bacterial PFTs to the highest possible resolution.