4. Acquire 2 min video with ten frames per second. Use a darkfield microscope with 20Â magnification. For each strain
acquire at least ten independent 2 min videos from two independent culture batches. Make sure to dilute bacteria to a
density of ten bacteria per field of view.
5. Perform image analysis using ImageJ to binarize video,
decrease noise using background subtraction, and then export
as .tiff file for quantification and trajectory analysis. Then,
analyze movies using MATLAB scripts based from BACtrack
software, a boundary detection algorithm is applied, trajectories using the center of mass between consecutive frames
(http://www.rowland.harvard.edu/labs/bacteria/software/
index.php). This software allows to extract the length of trajectories, average speed, and number of reversal (any change of
direction of a total 180
Æ 45
).
4 Notes
1. When filling capillaries leave enough space at the other end of
capillaries to accommodate the wax that seals the capillary tube.
2. To extract capillary fluid, be su re to hold only hematocrit wax
to avoid breaking the capillary in the middle and losing fluid.
3. For DNA extraction, automated DNA extraction is preferred
but not mandatory for robust standardized results.
4. For DNA extraction from capillary fluid, avoid pipetting glass
after centrifuging capillary tubes, as the latter can result in
lower yield.
References
1. Costa F, Hagan JE, Calcagno J et al (2015)
Global morbidity and mortality of leptospirosis: a systematic review. PLoS Negl Trop Dis 9:
e0003898
2. McBride AJA, Athanazio DA, Reis MG, Ko AI
(2005) Leptospirosis. Curr Opin Infect Dis
18:376–386
3. Faine S, Adler B, Bolin C, Perolat P (1999)
Leptospira and leptospirosis. MediSci, Melbourne, Australia, p 259
4. Faine S, Vanderhoeden J (1964) Virulencelinked colonial and morphological variation in
Leptospira. J Bacteriol 88:1493–1496
5. Lambert A, Picardeau M, Haake DA et al
(2012) FlaA proteins in Leptospira interrogans
are essential for motility and virulence but are
not required for formation of the flagellum
sheath. Infect Immun 80:2019–2025
6. Lambert A, Takahashi N, Charon NW, Picardeau M (2012) Chemotactic behavior of pathogenic and nonpathogenic Leptospira species.
Appl Environ Microbiol 78:8467–8469
7. Eshghi A, Becam J, Lambert A et al (2014) A
putative regulatory genetic locus modulates
virulence in the pathogen Leptospira interrogans. Infect Immun 82:2542–2552
8. Lambert A, Wong JN, Picardeau M (2015)
Gene inactivation of a chemotaxis operon in
the pathogen Leptospira interrogans. FEMS
Microbiol Lett 362:1–8
9. Fontana C, Lambert A, Benaroudj N et al
(2016) Analysis of a spontaneous non-motile
and avirulent mutant shows that FliM is
required for full endoflagella assembly in Leptospira interrogans. PLoS One 11:e0152916
Leptospira spp. Toolbox for Chemotaxis Assay
129
acquire at least ten independent 2 min videos from two independent culture batches. Make sure to dilute bacteria to a
density of ten bacteria per field of view.
5. Perform image analysis using ImageJ to binarize video,
decrease noise using background subtraction, and then export
as .tiff file for quantification and trajectory analysis. Then,
analyze movies using MATLAB scripts based from BACtrack
software, a boundary detection algorithm is applied, trajectories using the center of mass between consecutive frames
(http://www.rowland.harvard.edu/labs/bacteria/software/
index.php). This software allows to extract the length of trajectories, average speed, and number of reversal (any change of
direction of a total 180
Æ 45
).
4 Notes
1. When filling capillaries leave enough space at the other end of
capillaries to accommodate the wax that seals the capillary tube.
2. To extract capillary fluid, be su re to hold only hematocrit wax
to avoid breaking the capillary in the middle and losing fluid.
3. For DNA extraction, automated DNA extraction is preferred
but not mandatory for robust standardized results.
4. For DNA extraction from capillary fluid, avoid pipetting glass
after centrifuging capillary tubes, as the latter can result in
lower yield.
References
1. Costa F, Hagan JE, Calcagno J et al (2015)
Global morbidity and mortality of leptospirosis: a systematic review. PLoS Negl Trop Dis 9:
e0003898
2. McBride AJA, Athanazio DA, Reis MG, Ko AI
(2005) Leptospirosis. Curr Opin Infect Dis
18:376–386
3. Faine S, Adler B, Bolin C, Perolat P (1999)
Leptospira and leptospirosis. MediSci, Melbourne, Australia, p 259
4. Faine S, Vanderhoeden J (1964) Virulencelinked colonial and morphological variation in
Leptospira. J Bacteriol 88:1493–1496
5. Lambert A, Picardeau M, Haake DA et al
(2012) FlaA proteins in Leptospira interrogans
are essential for motility and virulence but are
not required for formation of the flagellum
sheath. Infect Immun 80:2019–2025
6. Lambert A, Takahashi N, Charon NW, Picardeau M (2012) Chemotactic behavior of pathogenic and nonpathogenic Leptospira species.
Appl Environ Microbiol 78:8467–8469
7. Eshghi A, Becam J, Lambert A et al (2014) A
putative regulatory genetic locus modulates
virulence in the pathogen Leptospira interrogans. Infect Immun 82:2542–2552
8. Lambert A, Wong JN, Picardeau M (2015)
Gene inactivation of a chemotaxis operon in
the pathogen Leptospira interrogans. FEMS
Microbiol Lett 362:1–8
9. Fontana C, Lambert A, Benaroudj N et al
(2016) Analysis of a spontaneous non-motile
and avirulent mutant shows that FliM is
required for full endoflagella assembly in Leptospira interrogans. PLoS One 11:e0152916
Leptospira spp. Toolbox for Chemotaxis Assay
129