Effects of fixation on bacterial cellular dimensions and integrity.
Effects of fixation on bacterial cellular dimensions and integrity.
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DOI：
10.1016/j.isci.2021.102348
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发表时间：
2021-04-23
期刊：
影响因子：
5.8
通讯作者：
Huang KC
中科院分区：
文献类型：
--
作者：
Zhu L;Rajendram M;Huang KC
固定有利于亚细胞定位和细胞形态的成像，但它仍然是未知的固定如何影响细胞的尺寸和细胞内的荧光模式，特别是在长期储存。在这里，我们描述了多种固定剂对几种细菌的影响。固定通常使细胞长度减少5-15%;微流体中的单细胞跟踪揭示，长度减少是固定方案中许多步骤的聚集效应，并且使用基于甲酰胺的固定剂，细胞质GFP而非膜结合的MreB-msfGFP的荧光迅速丧失。细胞尺寸在基于甲酰胺的固定剂中保存≥4天，但甲醇导致长度减少。虽然甲醇保存细胞质荧光比基于甲醛的固定剂，一些大肠杆菌细胞能够直接生长后固定。此外，甲醇固定引起细胞亚群中的裂解，几乎所有枯草芽孢杆菌细胞在一天后裂解。这些发现突出了荧光和膜完整性的维护之间的权衡，为未来的应用程序的固定。固定对细胞尺寸和荧光模式的影响被量化长度和细胞质GFP在甲醛固定过程中表现出快速的动力学甲醇保留了荧光，但没有完全抑制生长并导致裂解延长储存面临荧光维持和膜完整性的权衡生物科学;微生物学;细胞生物学
Fixation facilitates imaging of subcellular localization and cell morphology, yet it remains unknown how fixation affects cellular dimensions and intracellular fluorescence patterns, particularly during long-term storage. Here, we characterized the effects of multiple fixatives on several bacterial species. Fixation generally reduced cell length by 5–15%; single-cell tracking in microfluidics revealed that the length decrease was an aggregate effect of many steps in the fixation protocol and that fluorescence of cytoplasmic GFP but not membrane-bound MreB-msfGFP was rapidly lost with formaldehyde-based fixatives. Cellular dimensions were preserved in formaldehyde-based fixatives for ≥4 days, but methanol caused length to decrease. Although methanol preserved cytoplasmic fluorescence better than formaldehyde-based fixatives, some Escherichia coli cells were able to grow directly after fixation. Moreover, methanol fixation caused lysis in a subpopulation of cells, with virtually all Bacillus subtilis cells lysing after one day. These findings highlight tradeoffs between maintenance of fluorescence and membrane integrity for future applications of fixation. Effects of fixation on cellular dimensions and fluorescence patterns were quantified Length and cytoplasmic GFP exhibited rapid dynamics during formaldehyde fixation Methanol preserved fluorescence but did not fully inhibit growth and caused lysis Extended storage faces tradeoffs of fluorescence maintenance and membrane integrity Biological Sciences; Microbiology; Cell Biology
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DOI：
10.1038/ja.2015.116
发表时间：
2016-05
期刊：
The Journal of antibiotics
影响因子：
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作者：
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Bulgheresi S
影响因子：
9.9
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DOI：
10.1073/pnas.91.15.6808
发表时间：
1994-07-19
影响因子：
11.1
作者：
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通讯作者：
TRAVISANO, M
DOI：
10.1099/00221287-56-1-87
发表时间：
1969-01-01
期刊：
JOURNAL OF GENERAL MICROBIOLOGY
影响因子：
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作者：
BAZILL, GW;RETIEF, Y
通讯作者：
RETIEF, Y