Supplementary Information:
Direct optical monitoring of flow generated by bacterial
flagellar rotation
Silke R. Kirchner1, Spas Nedev1, Sol Carretero-Palacios1, Andreas Mader2, Madeleine Opitz2,
Theobald Lohmüller1,a), and Jochen Feldmann1,a)
1
Photonics and Optoelectronics Group, Physics Department and CeNS, Ludwig-Maximilians-Universität München,
Germany
2
Chair for Experimental Physics: Soft Matter Physics and Biophysics, Physics Department and CeNS, LudwigMaximilians-Universität München, Germany
MATERIALS AND METHODS
Bacterial growth: Two different strains of Bacillus subtilis are used in this study: BM 77, a
flagellated bacterium1 and BD3458, a non-flagellated bacterium2. The bacteria were grown
overnight in LB-medium. On the next day the bacterial solution was diluted 1:50 with mineralsalt-solution (50 mM potassium-phosphate-buffer pH 7.0, 0.12 mM MgCl2, 1 mM (NH4)2SO4,
0.14 mM CaCl2, 0.01 mM MnCl2, 20 mM D-sorbitol, 0.3 mM methionine, 0.3 mM leucine and
0.3 mM histidine) and incubated at 37 °C, 300 rpm until reaching an optical density OD600 0.3.
After adding 5 mM potassium-phosphate-buffer and 0.05% glycerine the bacteria were again
incubated at 37 °C, 300 rpm for 15 min. In the next step, the bacteria were washed with the
Chemotaxis medium3, 4 (10mM potassium-phosphate-buffer (pH 7.0), 0.14 mM CaCl2, 0.3 mM
(NH4)2SO4, 0.1 mM EDTA (pH ca. 5.0), 5 mM sodium lactate, 0.05% glycerol and ddH2O) and
subsequently separated into aliquots. The aliquots were frozen in liquid nitrogen with 10%
glycerine and stored at -80°C. For each measurement, one aliquot was defrosted at room
temperature and the bacterial solution was diluted with Chemotaxis medium in that way, that just
one or two bacteria could be seen simultaneously in the field of view of the dark field
microscope. 120 µl of the end solution was drop casted on a common glass substrate and placed
under the microscope.
Optical Tweezers Setup: The setup was based on an up-right microscope (Zeiss Axio Scope.A1)
with dark-field illumination (NA=1.2, Zeiss).
A 1064 nm CW laser (2W Cobolt Rumba,
λ=1064nm, TEM00) was expanded by a beam-expander (Edmund Optics) to slightly overfill the
back aperture of a 100x/NA=1 water dipping objective (Zeiss). The expanded laser beam was
split into two beams using a polarizing beam-splitter with a lambda-half plate, defining the
power ratio 1 to 1 between them. Both beams were recombined using a second polarizing beamsplitter and two mirrors for steering of one of the beams with respect to the other. Finally the
beams were focused in the objective focal plane, forming two optical traps separated at a
predefined distance. For all experiments the distance was set to be 2µm with a trapping power
per beam Pbeam varying between 25mW and 40mW. Image sequences were recorded with a Highspeed camera (PCO, Dimax HD) at a frame rate of 500 fps. A Notch Filter (NF) (StopLine
532/1064, Semrock) was used to prevent the laser from entering the camera. The trapped
bacterium and the bead coordinates for each frame were evaluated using a particle tracking
software (Video Spot Tracker).
Analyzing bacterial movement in an optical trap: The body rotation of a flagellated bacterium
measured in an optical trap compared to the motion analysis of the non-flagellated strain can be
seen in Supplementary Figure S2a. The non-flagellated strain did not show any body rotation
(Supplementary Figure S2b).
Supplementary Movie V1: Video file of a rotating flagellated bacterium recorded with 1500 fps.
Numerical Simulations: Numerical simulations of the velocity field generated by a flagellated
bacterium were performed using “COMSOL Multiphysics”.
References:
1. M. Leisner, K. Stingl, J. O. Rädler, B. Maier, Molecular Microbiology 63, 1806 (2007).
2. Hoa, T.T., Tortosa, P., Albano, M., Dubnau, D. Rok, Molecular Microbiology 43, 15 (2002).
3. G. W. Ordal, D. J. Goldman, Science 189, 802 (1975).
4. G. W. Ordal, D. J. Goldman, J Bacteriol., 129, 151 (1977).
Supplementary Figure S1
Figure S1. The frequency spectrum of two 1.76 µm silica beads trapped next to each other did
not show any signal. Only white noise was observed in the Fourier space, similar to
measurements of a non-flagellated bacteria strain.
Supplementary Figure S2
Figure S2. Comparison between the movement of a (a) flagellated (BM77) and a (b) nonflagellated (BD3458) B. subtilis strain in an optical trap (red: movement in y-direction, black:
movement in x-direction). (a) A periodic body rotation around the long axis of the bacterial cell
at ~ 4Hz was only observed for the flagellated cell. (b) No periodic movement or cell body
rotation was observed for a non-flagellated bacterium.
Supplementary Figure S3
Figure S3. (a) Amplitude of the FFT spectra of the detector bead trapped next to a flagellated
bacterium in x- and y-direction. The sum of both amplitudes (SQRT[(FFT X)2 + (FFT Y)2])
gives the maximum signal intensity at 62Hz. (b) Measurements of a non-flagellated bacterium
revealed no signal in the FFT spectra.
Supplementary Figure S4
Figure S4. Static representation of the attached multimedia file. The movie shows the periodic
cell body rotation of an active bacterium trapped in an optical tweezer. The body rotation
frequency is 4Hz.
Supplementary Figure S5
Figure S5. Time dependent analysis of the cell body rotation (red: movement in y-direction,
black: movement in x-direction) and the corresponding frequency spectrum measured for a
single flagellated bacterial cell. A secondary 17Hz peak is observed between 12s and 16s. This
peak, however, was not reliably observed in the majority of our measurements and did not occur
in the control experiments (see also Figure S6, S7).
Supplementary Figure S6
Figure S6. (a) Time dependent analysis of the cell body rotation (black: movement in x-direction,
red: fitted curve to guide the eye) and the corresponding frequency spectrum for 2s time intervals
(b-e).
Supplementary Figure S7
Figure S7. (a) Time dependent analysis of the cell body rotation (black: movement in x-direction,
red: fitted curve to guide the eye) and the corresponding frequency spectrum for 2s time intervals
(b-e). The bacterium stopped to move after 9s. At this time point, the 62Hz peak also vanished
completely.
Supplementary Figure S8
Figure S8. Control measurement: Time dependent analysis performed with a non-flagellated
strain for comparison. The cell body did not show any periodic movement over time and no peak
was observed in the analysis of the detector bead.
Supplementary Figure S9
Figure S9. Success rates for measurements on single bacterial cells as a function of the distance
between the source and the detector bead. The success rate was found to be decreasing when the
particle was further away from the bacterium.