1. Strains are diluted weekly from a 7-day culture, to 5 Â 10
6
leptospires/mL in a 25 cm
2 flask with ventilated cap, into a
20 mL culture volume.
2. Incubate for 7 days at 28
C.
3.2 Preparation of
Leptospira Inoculum
To ensure an optimal infection, take care to perform the following
steps carefully and without delay.
1. Prior to the animal infection, check the fitness of the strain by
measuring the light emitted with a known quantity of leptospires in the presence of D-luciferin, using a luminometer (see
Note 1).
2. Transfer the culture in a 50 mL tube and centrifuge 25 min at
3345 Â g, at room temperature (RT).
3. Remove the supernatant (in respect to regulations for disposal
of infectious waste), and resuspend the pellet with 5 mL of
sterile DPBS.
4. Take a 1 mL aliquot of the homogenized suspension and
prepare a 1:100 diluted solution in DPBS. Count the bacteria
Fig. 2 Five-port anesthesia manifold. This system including light baffle dividers
and transparent nose cones is inserted within the IVIS imaging chamber to
maintain mice under anesthesia during the imaging
152
Fre ´ de ´ rique Vernel-Pauillac and Catherine Werts