摘要：SummaryFixation 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, someEscherichia colicells were able to grow directly after fixation. Moreover, methanol fixation caused lysis in a subpopulation of cells, with virtually allBacillus subtiliscells lysing after one day. These findings highlight tradeoffs between maintenance of fluorescence and membrane integrity for future applications of fixation.Graphical abstractDisplay OmittedHighlights•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 integrityBiological Sciences; Microbiology; Cell Biology
