Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis
Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis
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DOI：
10.1111/j.1365-2818.2009.03299.x
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发表时间：
2010-01-01
影响因子：
2
通讯作者：
Griffiths, G.
中科院分区：
文献类型：
--
作者：
Bleck, C. K. E.;Merz, A.;Griffiths, G.
细菌和分枝杆菌通常很难制备常规树脂切片电子显微镜的标本，它们的厚而复杂的细胞被膜层特别容易产生伪影。本文对不同的耻垢分枝杆菌电镜薄片制备方法进行了系统的比较。这些方法是：（1）常温下用固定剂和环氧树脂常规制备。(2)Tokuyasu化学固定细菌的冷冻切片。(3)快速冷冻，然后冷冻置换并在室温下包埋在环氧树脂中，或（4）结合Lowicryl HM 20包埋和低温下紫外（UV）聚合，和（5）CEMOVIS，或玻璃体切片的冷冻电子显微镜。如预期的那样，用玻璃体切片的冷冻电子显微镜方法获得了细菌的最佳保存，特别是在细胞被膜和脂质体的保存方面。通过与玻璃体切片的冷冻电子显微镜比较，常规方法和Tokuyasu方法产生了不同的不期望的伪影。两种不同类型的冷冻置换方案显示细胞包膜的可变保存，但细胞质的保存可接受，但脂质体和细菌DNA的保存不可接受。总之，尽管玻璃体切片的冷冻电子显微镜必须被认为是电子显微镜切片方法中的“金标准”，因为它避免了溶剂和染色剂，使用最佳制备的冷冻替代物也提供了一些优势，用于细菌的超微结构分析。
P>Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.