Visualizing flagella of tracked bacteria
We began our work on motility of E. coli with construction of a microscope that can follow the motion of individual cells for ∼1 mm in any direction. This led to the description of the biased random walk: E. coli swims along a gently meandering track, called a run, counting molecules of interest as it goes along, e.g., sugars and amino acids, then abruptly changes course, picking a new direction via a maneuver called a tumble, more or less at random. If, during a run, the count of molecules that the cell likes, called attractants, goes up, the next tumble is postponed and the favorable run is extended. So the bias is positive, enabling the cell to move up spatial gradients of attractants. It has been found since, mainly by using tethered cells, that comparisons in concentrations are temporal, and that runs occur when all of the flagellar motors on a cell spin CCW (when observed from outside the cell), and that tumbles occur when one or more motors spin CW.
Now, we also know how to . So we have visualized flagella of tracked bacteria in order to study the dependence of behavior on the number of flagella on a cell and to learn more about the formation and disruption of flagellar bundles, events that characterize runs and tumbles. To that end, we rebuilt the tracker on an inverted microscope, first a Nikon Diaphot 200 with a 40x long-working-distance water-immersion objective and later a Nikon Eclipse Ti-U with a 25x water-immersion objective of higher numerical aperture. The original tracker used a student microscope (Nikon Ske) with a 20x objective (to provide adequate working distance).
That was in the days before lap-top computers and CCD cameras; the set-up included a 16-mm movie camera, but that was not very useful. Now, we also have better light sources. So we tracked cells in phase contrast at 660 nm (deep red), with light emitted by a high-power LED, visualized their cell bodies at 590 nm (amber), with light emitted by a second high-power LED, and excited filament fluorescence with a 532 nm (green) diode laser, chopped at video rates by an acousto-optic-modulator (AOM). The video data were recorded with a sensitive surveillance camera. We tracked E. coli of various lengths, B. subtilis, and a motile Streptococcus (now Enterococcus).
Our hope is that this system can be used to characterize the motion of other bacterial species about which less is known, and thus serve a broader community of workers interested in bacterial motility.
Berg, H.C. and Brown, D.A. Chemotaxis in Escherichia coli analysed by three-dimensional tracking. Nature 239, 500-504 (1972).
Berg, H.C. The tracking microscope. Adv. Opt. Elect. Microsc. 7, 1-15 (1978).
Segall, J.E., Block, S.M. and Berg, H.C. Temporal comparisons in bacterial chemotaxis. Proc. Natl. Acad. Sci. USA 83, 8987-8991 (1986).
Darnton, N.C., Turner, L., Rojevsky, S. and Berg, H.C. On torque and tumbling in swimming Escherichia coli. J. Bacteriol. 189, 1756-1764 (2007).
Turner, L., Ping, L., Neubauer, M., and Berg, H.C. Visualizing flagella while tracking bacteria. Biophys. J., (2016) in press.