Panessa-Warren Barbara J, Tortora George T, Warren John B
Department of Condensed Matter Physics and Materials Science, Building 480, Brookhaven National Laboratory, Upton, NY 11973, USA.
Microsc Microanal. 2007 Aug;13(4):251-66. doi: 10.1017/S1431927607070651.
Transmission electron microscopy (TEM) studies in the 1960s and early 1970s using conventional thin section and freeze fracture methodologies revealed ultrastructural bacterial spore appendages. However, the limited technology at that time necessitated the time-consuming process of imaging serial sections and reconstructing each structure. Consequently, the distribution and function of these appendages and their possible role in colonization or pathogenesis remained unknown. By combining high resolution field emission electron microscopy with TEM images of identical bacterial spore preparations, we have been able to obtain images of intact and sectioned Bacillus and Clostridial spores to clearly visualize the appearance, distribution, resistance (to trypsin, chloramphenicol, and heat), and participation of these structures to facilitate attachment of the spores to glass, agar, and human cell substrates. Current user-friendly commercial field emission scanning electron microscopes (FESEMs), permit high resolution imaging, with high brightness guns at lower accelerating voltages for beam sensitive intact biological samples, providing surface images at TEM magnifications for making direct comparisons. For the first time, attachment structures used by pathogenic, environmental, and thermophile bacterial spores could be readily visualized on intact spores to reveal how specific appendages and outer spore coats participated in spore attachment, colonization, and invasion.
20世纪60年代和70年代初，利用传统的薄切片和冷冻断裂方法进行的透射电子显微镜（TEM）研究揭示了细菌孢子的超微结构附属物。然而，当时有限的技术使得对连续切片进行成像并重建每个结构的过程非常耗时。因此，这些附属物的分布和功能以及它们在定殖或发病机制中可能发挥的作用仍然未知。通过将高分辨率场发射电子显微镜与相同细菌孢子制剂的TEM图像相结合，我们能够获得完整的和切片的芽孢杆菌和梭菌孢子的图像，以清楚地观察这些结构的外观、分布、抗性（对胰蛋白酶、氯霉素和热），以及这些结构在促进孢子附着于玻璃、琼脂和人类细胞底物中的作用。当前用户友好型的商用场发射扫描电子显微镜（FESEM），允许进行高分辨率成像，在较低加速电压下使用高亮度电子枪对束敏感的完整生物样品进行成像，以TEM放大倍数提供表面图像以便进行直接比较。首次能够在完整的孢子上轻松观察到致病性、环境性和嗜热细菌孢子所使用的附着结构，以揭示特定的附属物和孢子外层如何参与孢子的附着、定殖和侵袭。
