| Summary: | Summary: 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.
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