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[Objective] To establish a method of semi-thin section preparation and scanning electron microscopy (SEM) observation with a simple operation, a short cycle, and a low technical threshold, which can be used as a rapid pre-screening strategy for the complex process of transmission electron microscopy (TEM) ultra-thin sections, in response to the requirements of rapid identification and dynamic observation of typical subcellular structures (such as cell wall, cytoplasm, spore, and vacuole) of microbial samples in the process of fermentation production. [Methods] Bacillus subtilis, Escherichia coli, and Pichia pastoris, taken as the research objects, were embedded in Embed 812 epoxy resin to prepare semi-thin sections with different thicknesses (200, 500, and 1 000 nm) and resin embedding blocks (> 1 000 nm) with samples. After platinum coating by ion sputtering, the subcellular structure was observed by SEM. Ultra-thin sections (70 nm) of the above microbial samples were also prepared, stained with lead and uranium, and imaged by TEM. The imaging effect and operation efficiency of the above two methods were compared. [Results] The 200 nm semi-thin section can clearly and completely display various kinds of microbial subcellular structures under a scanning electron microscope. The image quality was significantly better than that of 500 and 1 000 nm sections and resin block samples. Its resolution was close to the TEM observation level, and it can save about 6.5 h. [Conclusion] The method of SEM combined with 200 nm semi-thin sections was successfully applied to the high-resolution imaging of microbial subcellular structure for the first time, which can clearly identify the typical ultrastructural morphology, and has the advantages of a simple operation, a short cycle, a low cost, and high safety. It has strong versatility and promotion value, and provides a new and effective solution for the biological electron microscopy technology system.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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