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Labeling Bacterial Flagella with Fluorescent Dyes

  • Linda Turner
  • Howard C. Berg
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1729)

Abstract

We describe labeling of bacteria with amino-specific or sulfhydryl-specific Alexa Fluor dyes, methods that allow visualization of flagellar filaments, even in swimming cells. Bacterial flagellar filaments are long (~10 μm), but of small diameter (~20 nm), and their rotation rates are high (>100 Hz), so visualization is difficult. Dark-field microscopy works well with isolated filaments, but visualization in situ is hampered by light scattered from cell bodies, which obscures short filaments or the proximal ends of long filaments. Differential interference contrast microscopy also works, but is technically difficult and suffers from a narrow depth of field and low image contrast; background subtraction and contrast enhancement are necessary. If filaments are fluorescent, they can be imaged in their entirety using standard fluorescence microscopes. For imaging in vivo, blurring can be prevented by strobing the light source or by using a camera with a fast shutter. The former method is preferred, since it minimizes bleaching.

Keywords

Bacteria Flagellar staining Motility 

Notes

Acknowledgment

This work was supported by grant AI016478 from the US National Institutes of Health, by a grant from the Physics of Living Systems program of the US National Science Foundation, and by the Rowland Institute at Harvard University.

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Departments of Molecular and Cellular Biology and of PhysicsHarvard UniversityCambridgeUSA
  2. 2.The Rowland InstituteCambridgeUSA
  3. 3.The Rowland Institute at HarvardCambridgeUSA

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