A simple, rapid method for demonstrating bacterial flagella

We developed a simple, rapid method for demonstrating flagellation of bacte ria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugen e, 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 c ell bodies, as well as flagella and other appendages, which could be direct ly observed by epifluorescence microscopy, Detection of flagella was furthe r improved by using a charge-coupled device camera for image capture and pr ocessing. The reliability of the method was tested by using 37 pure culture s of marine bacteria, Detection of flagella on the isolates by NanoOrange s taining was compared to detection by transmission electron microscopy (TEM) . For 36 of 37 cultures, the two methods yielded the same results, In one c ase, flagella were detected by TEM but not by NanoOrange, although the diff erence 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 gener al protein stain and works directly in seawater, it may also prove to be us eful for staining other proteinaceous material that is of interest to aquat ic microbial ecologists.