mucin is a gel-like, viscous component of mucosa, 3% of commercial mucin solution (9.5 mPa  s) or 0.5% methylcellulose
solution (7.5 mPa  s) is useful for examining Leptospira
motility under conditions resembling environments near tissue
surfaces [15]. Ficoll is better to investigate the effect of viscous
drag on the bacterial movements, because mucin and methylcellulose change not only viscosity but also elasticity of polymer
solutions.
2. According to the data provided by the company, the efficiency
of antibody labeling depends on the amount of antibody and
the bead size. It is necessary to examine appropriate conditions
for each experiment.
3. Leptospira cells are too thin to be visualized by transmitted light
through conventional optical microscopes, because the spatial
resolution is about a half of wavelength of illumination light:
When observed using 550 nm (green), the observation limit is
about 280 nm. Largely refracted illumination through a high
numerical aperture (NA) condenser of a dark-field microscope
visualizes such thin cells as scattered objects in a dark field.
High-intensity illumination system, such as combination of
oil-immersion condenser and mercury lamp or laser, is required
for high-speed recording.
4. Although either digital charge-coupled device (CCD) or complementary metal-oxide semiconductor (CMOS) video camera
is available, a frame rate of recording enough to acquire target
parameters should be set. Swimming speeds of Leptospira cells
can be determined with normal video rate (30 frames per
second, fps), whereas measurements of cell-body rotation
rates (~100 revolutions per second) require more than
200 fps. We use IDP-Express R2000 (Photron, CMOS) for
motility assays.
5. In general, bacterial suspension is placed between two glasses
with different sizes, e.g., a glass slide and a coverslip. A possible
event is unexpected flow of media, disturbing accurate measurements of motility. Especially, the effect of the flow on slow
motile mutant is problematic, because the flow of media is
usually faster than the actual movements of such mutant. To
prevent the flow of media, use a flow chamber made by sticking
a glass slide with a coverslip with double-sided tape (5–10 μm
in thickness) (Fig. 3). Bacterial suspension placed on one side
of the chamber spontaneously enters the space between two
glasses by capillarity. Careful removal of bacterial suspension
overflown from the sides stabilizes suspension within the chamber for several minutes. Sealing the opened sides of the flow
chamber with silicone grease enables longer time observation.
6. Beads attached to the cell surface via anti-lipopolysaccharide
(LPS) antibody move along the cell body in the bead-sizeMotility assays for Leptospira
147
solution (7.5 mPa  s) is useful for examining Leptospira
motility under conditions resembling environments near tissue
surfaces [15]. Ficoll is better to investigate the effect of viscous
drag on the bacterial movements, because mucin and methylcellulose change not only viscosity but also elasticity of polymer
solutions.
2. According to the data provided by the company, the efficiency
of antibody labeling depends on the amount of antibody and
the bead size. It is necessary to examine appropriate conditions
for each experiment.
3. Leptospira cells are too thin to be visualized by transmitted light
through conventional optical microscopes, because the spatial
resolution is about a half of wavelength of illumination light:
When observed using 550 nm (green), the observation limit is
about 280 nm. Largely refracted illumination through a high
numerical aperture (NA) condenser of a dark-field microscope
visualizes such thin cells as scattered objects in a dark field.
High-intensity illumination system, such as combination of
oil-immersion condenser and mercury lamp or laser, is required
for high-speed recording.
4. Although either digital charge-coupled device (CCD) or complementary metal-oxide semiconductor (CMOS) video camera
is available, a frame rate of recording enough to acquire target
parameters should be set. Swimming speeds of Leptospira cells
can be determined with normal video rate (30 frames per
second, fps), whereas measurements of cell-body rotation
rates (~100 revolutions per second) require more than
200 fps. We use IDP-Express R2000 (Photron, CMOS) for
motility assays.
5. In general, bacterial suspension is placed between two glasses
with different sizes, e.g., a glass slide and a coverslip. A possible
event is unexpected flow of media, disturbing accurate measurements of motility. Especially, the effect of the flow on slow
motile mutant is problematic, because the flow of media is
usually faster than the actual movements of such mutant. To
prevent the flow of media, use a flow chamber made by sticking
a glass slide with a coverslip with double-sided tape (5–10 μm
in thickness) (Fig. 3). Bacterial suspension placed on one side
of the chamber spontaneously enters the space between two
glasses by capillarity. Careful removal of bacterial suspension
overflown from the sides stabilizes suspension within the chamber for several minutes. Sealing the opened sides of the flow
chamber with silicone grease enables longer time observation.
6. Beads attached to the cell surface via anti-lipopolysaccharide
(LPS) antibody move along the cell body in the bead-sizeMotility assays for Leptospira
147