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Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy

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Fixation ability of five common fixation solutions, including 2.5% glutaraldehyde, 10% formalin, 4% paraformaldehyde, methanol/acetone (1:1), and ethanol/acetic acid (3:1) were evaluated by using atomic force microscopy in the present study. Three model bacteria, i.e., Escherichia coli, Pseudomonas putida, and Bacillus subtilis were applied to observe the above fixation methods for the morphology preservation of bacterial cells and surface ultrastructures. All the fixation methods could effectively preserve cell morphology. However, for preserving bacterial surface ultrastructures, the methods applying aldehyde fixations performed much better than those using alcohols, since the alcohols could detach the surface filaments (i.e., flagella and pili) significantly. Based on the quantitative and qualitative assessments, the 2.5% glutaraldehyde was proposed as a promising fixation solution both for observing morphology of both bacterial cell and surface ultrastructures, while the methonal/acetone mixture was the worst fixation solution which may obtain unreliable results.

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The authors wish to thank the Hong Kong UGC One-off Special Equipment Grant Scheme (SEG HKU10) for the financial support on this study, and Yuanqing Chao wishes to thank the University of Hong Kong for the postgraduate studentship. The technical assistance of Ms. Vicky Fung is greatly appreciated.

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Correspondence to Tong Zhang.

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Chao, Y., Zhang, T. Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy. Appl Microbiol Biotechnol 92, 381–392 (2011).

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  • Fixation methods
  • Cell morphology
  • Ultrastructure
  • Atomic force microscopy