4. Measure motility after incubation using a Petroff-Hausser
chamber, approximately 60% of cells should be translated, and
the remaining cells should display gyrating ends.
5. Prepare chemotaxis chambers using two storage plates of
96 wells with 1.2 ml rounded bottom facing each other. Take
one of the plates, and pierce holes at the bottom of wells
through the plastic using a heated tweezer. The holes need to
be large enough to easily allow capillaries to enter the chamber.
Fill the other plate with 200 μl suspension of 2 Â 10
7 bacteria
in motility buffer. Invert the perforated plate and face it to the
plate filled with bacterial suspension. Tape the two plates
together.
6. Prepare four capillary tubes for each condition, and fill capillaries by plunging them in control or hypothetical chemoattractant solution (see Note 1). Once filled seal the end of the
capillary with wax seal. Wipe carefully with low lint wipes all
excess fluid remaining outside of the capillary’s end immersed
in attractant solution. Plunge capillaries carefully in chemotaxis
chamber immersed in bacterial suspension.
7. Incubate the chemotaxis chamber from 30 m to 1 h at 30
C
(Fig. 1). Maintain the chemotaxis chamber with capillaries in
horizontal position while in the incubator and after.
8. After incubation, remove capillaries and wipe out any excess
fluid of bacteria suspension on the outside of capillary. Over a
15 ml conical tube, break the seal wax extremity and dispose of
it (see Note 2). Centrifuge capillaries with liquid in conical tube
for 1 m at 3000 Â g. Approximately 50 μl of suspension should
remain at the end of the procedure.
96 well plate inverted with
holes for capillary tubes in
bottom of wells
96 well plate : cell suspension
in bottom of wells
Capillary tubes : filled with
putative attractant plunged
into cell suspension
Plates are tape attached
together
Fig. 1 Chemotaxis chamber setup (picture annotated)
Leptospira spp. Toolbox for Chemotaxis Assay
127
chamber, approximately 60% of cells should be translated, and
the remaining cells should display gyrating ends.
5. Prepare chemotaxis chambers using two storage plates of
96 wells with 1.2 ml rounded bottom facing each other. Take
one of the plates, and pierce holes at the bottom of wells
through the plastic using a heated tweezer. The holes need to
be large enough to easily allow capillaries to enter the chamber.
Fill the other plate with 200 μl suspension of 2 Â 10
7 bacteria
in motility buffer. Invert the perforated plate and face it to the
plate filled with bacterial suspension. Tape the two plates
together.
6. Prepare four capillary tubes for each condition, and fill capillaries by plunging them in control or hypothetical chemoattractant solution (see Note 1). Once filled seal the end of the
capillary with wax seal. Wipe carefully with low lint wipes all
excess fluid remaining outside of the capillary’s end immersed
in attractant solution. Plunge capillaries carefully in chemotaxis
chamber immersed in bacterial suspension.
7. Incubate the chemotaxis chamber from 30 m to 1 h at 30
C
(Fig. 1). Maintain the chemotaxis chamber with capillaries in
horizontal position while in the incubator and after.
8. After incubation, remove capillaries and wipe out any excess
fluid of bacteria suspension on the outside of capillary. Over a
15 ml conical tube, break the seal wax extremity and dispose of
it (see Note 2). Centrifuge capillaries with liquid in conical tube
for 1 m at 3000 Â g. Approximately 50 μl of suspension should
remain at the end of the procedure.
96 well plate inverted with
holes for capillary tubes in
bottom of wells
96 well plate : cell suspension
in bottom of wells
Capillary tubes : filled with
putative attractant plunged
into cell suspension
Plates are tape attached
together
Fig. 1 Chemotaxis chamber setup (picture annotated)
Leptospira spp. Toolbox for Chemotaxis Assay
127