(f) Remove free antibodies and EDAC by centrifugation at
17,000 Â g for 15 min at 23
C, and suspend the pellet
into 10 mM Tris–HCl buffer (pH 8.0).
2. Centrifuge 300 μL of Leptospira culture at 1000 Â g for
10 min, and suspend the pellet into 500 μL of motility
medium.
3. Mix 5 μL of the cell suspension with 15 μL of the antibodycoated bead.
4. Infuse the cell-bead mixture into a flow chamber, and observe
motility of cells attached with beads by dark-field microscope.
5. Record the cell movements as described in Subheading 3.2.
6. Example data are shown in Fig. 5: The bead rotation rate is
measured by the time trace of the bead position; information of
the focal plane is required for determining the rotational direction (see the figure legend and Note 6 for more details).
Fig. 4 Measurements of spiral-end and hook-end gyrations. (a) A still of a Leptospira biflexa cell swimming to
the right, displaying spiral shape at the right (anterior side) (b) and hook shape at the left (posterior side) (c).
The montages in b and c show about a half of one revolution of spiral-end and hook-end, respectively. The
gyration speeds are determined from the periodic changes in the angles of the lines fitted to the cell ends (right
panels in b and c). The angles of the fitted lines (red dashed lines in b and c) were given by Fit ellipse in
ImageJ (see text). In this example data, the spiral-end speed and the hook-end speed are 30 Hz and 22 Hz,
respectively
Motility assays for Leptospira
145
17,000 Â g for 15 min at 23
C, and suspend the pellet
into 10 mM Tris–HCl buffer (pH 8.0).
2. Centrifuge 300 μL of Leptospira culture at 1000 Â g for
10 min, and suspend the pellet into 500 μL of motility
medium.
3. Mix 5 μL of the cell suspension with 15 μL of the antibodycoated bead.
4. Infuse the cell-bead mixture into a flow chamber, and observe
motility of cells attached with beads by dark-field microscope.
5. Record the cell movements as described in Subheading 3.2.
6. Example data are shown in Fig. 5: The bead rotation rate is
measured by the time trace of the bead position; information of
the focal plane is required for determining the rotational direction (see the figure legend and Note 6 for more details).
Fig. 4 Measurements of spiral-end and hook-end gyrations. (a) A still of a Leptospira biflexa cell swimming to
the right, displaying spiral shape at the right (anterior side) (b) and hook shape at the left (posterior side) (c).
The montages in b and c show about a half of one revolution of spiral-end and hook-end, respectively. The
gyration speeds are determined from the periodic changes in the angles of the lines fitted to the cell ends (right
panels in b and c). The angles of the fitted lines (red dashed lines in b and c) were given by Fit ellipse in
ImageJ (see text). In this example data, the spiral-end speed and the hook-end speed are 30 Hz and 22 Hz,
respectively
Motility assays for Leptospira
145