Effects of fixation on bacterial cellular dimensions and integrity

• Main Authors: Lillian Zhu, Manohary Rajendram, Kerwyn Casey Huang
• Format: Article
• Language: English
• Published: Elsevier 2021-04-01
• Series: iScience
• Subjects: Biological Sciences; Microbiology; Cell Biology
• Online Access: http://www.sciencedirect.com/science/article/pii/S2589004221003163

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.