Centro Universitario de los Lagos, Universidad de Guadalajara.
Centro Universitario de los Lagos, Universidad de Guadalajara;
J Vis Exp. 2023 Jun 30(196). doi: 10.3791/64823.
Electron microscopy is one of the tools required to characterize cellular structures. However, the procedure is complicated and expensive due to the sample preparation for observation. Atomic force microscopy (AFM) is a very useful characterization technique due to its high resolution in three dimensions and because of the absence of any requirement for vacuum and sample conductivity. AFM can image a wide variety of samples with different topographies and different types of materials. AFM provides high-resolution 3D topography information from the angstrom level to the micron scale. Unlike traditional microscopy, AFM uses a probe to generate an image of the surface topography of a sample. In this protocol, the use of this type of microscopy is suggested for the morphological and cell damage characterization of bacteria fixed on a support. Strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Pseudomonas hunanensis (isolated from garlic bulb samples) were used. In this work, bacterial cells were grown in specific culture media. To observe cell damage, Staphylococcus aureus and Escherichia coli were incubated with different concentrations of nanoparticles (NPs). A drop of bacterial suspension was fixed on a glass support, and images were taken with AFM at different scales. The images obtained showed the morphological characteristics of the bacteria. Further, employing AFM, it was possible to observe the damage to the cellular structure caused by the effect of NPs. Based on the images obtained, contact AFM can be used to characterize the morphology of bacterial cells fixed on a support. AFM is also a suitable tool for the investigation of the effects of NPs on bacteria. Compared to electron microscopy, AFM is an inexpensive and easy-to-use technique.
电子显微镜是用于表征细胞结构的工具之一。然而，由于观察前的样品制备过程复杂且昂贵，因此该技术的应用受到限制。原子力显微镜（AFM）是一种非常有用的特性分析技术，因为它具有在三维空间中实现高分辨率的能力，并且不需要真空和样品导电性。AFM 可以对具有不同形貌和不同类型材料的各种样品进行成像。AFM 可提供从埃级到微米级的高分辨率 3D 形貌信息。与传统显微镜不同，AFM 使用探针生成样品表面形貌的图像。在本方案中，建议将这种类型的显微镜用于固定在支持物上的细菌的形态学和细胞损伤特征分析。使用金黄色葡萄球菌（ATCC 25923）、大肠杆菌（ATCC 25922）和湖南假单胞菌（从大蒜鳞茎样本中分离得到）作为实验菌株。在这项工作中，细菌细胞在特定的培养基中生长。为了观察细胞损伤，将金黄色葡萄球菌和大肠杆菌分别与不同浓度的纳米颗粒（NPs）孵育。将一滴细菌悬浮液固定在玻璃支持物上，并在不同尺度下用 AFM 拍摄图像。获得的图像显示了细菌的形态特征。此外，通过 AFM 还可以观察到 NPs 对细胞结构造成的损伤。基于获得的图像，可以使用接触 AFM 来表征固定在支持物上的细菌细胞的形态。AFM 也是研究 NPs 对细菌影响的合适工具。与电子显微镜相比，AFM 是一种经济且易于使用的技术。
