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Applied and Environmental Microbiology, August 2000, p. 3632-3636, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Simple, Rapid Method for Demonstrating Bacterial Flagella

Hans-Peter Grossart,* Grieg F. Steward,dagger Josefina Martinez,Dagger and Farooq Azam

Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093-0202

Received 13 December 1999/Accepted 24 April 2000

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.

* Corresponding author. Present address: Institution for Chemistry and Biology of the Marine Environment, University of Oldenburg, 26111 Oldenburg, Germany. Phone: 49 441 7980. Fax: 49 441 798 3438. E-mail: hgrossart{at}icbm.uni-oldenburg.de.

dagger Present address: Monterey Bay Aquarium Research Institute, Moss Landing, CA 95039-0628.

Dagger Present address: Department of Microbiology, University of Barcelona, E-08028 Barcelona, Spain.

Applied and Environmental Microbiology, August 2000, p. 3632-3636, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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