100) colonies on plate. Generally, sample dilution will also
dilute the killing chemical agent used for the survival test to
sublethal concentration. However, in some cases, it might be
necessary to wash the bacteria with EMJH before plating them
on solid media.
8. We generally use cleaned and autoclaved 4 mm glass beads
from Sigma. You can also use a glass spreader or inoculation
loops to spread bacteria, but this will make handling multiple
plates difficult.
9. Glass beads are reusable. Wash them thoroughly with a decontaminating solution, rinse them extensively with distilled water,
and autoclave them for further use.
10. It is important to ensure that the colonies will remain isolated.
It might be necessary to check regularly the plates during their
incubation at 30
C as Leptospira spp. are motile and can
produce spread large and confluent colonies when overgrown
on agar plate.
11. The use of a black paper sheet facilitates the visualization of
Leptospira colonies.
12. We generally use the Alamar Blue
® Assay provided by Thermo
Fisher Scientific Invitrogen but a resazurin solution from
another manufacturer might give satisfactory results as well.
13. You have to anticipate which method will be used to quantify
resazurin reduction. If fluorescence will be used, the reaction
has to be performed in plates that are compatible with fluorescence measurement. We recommend using clear-bottom blackwalled 96-well plates. We have obtained satisfactory results
with the μClear Black 96-well plate provided by Greiner. But,
these plates are not suitable to take a picture for recording the
change in color upon resazurin reduction.
14. The optimum pH for resazurin reduction is around 7.0 [9]. In
fact, resazurin and resorufin will be respectively pink and colorless under acidic pH. You have to ensure that the reaction mix
for resazurin reduction is buffered.
15. It is crucial to verify that the chemical used as killing agent in
the survival test does not interfere with resazurin reduction.
This can be done by pipetting the chemical at the desired
concentration into a well of the 96-well plate and completing
with EMJH medium to have a final volume of 180 μl. Add
20 μl of the 10Â resazurin solution and mix by pipetting.
16. The optimal temperature for resazurin reduction is 37
C, but
we prefer to perform the resazurin reduction reaction at 30
C
to reduce evaporation. 200 μl of sterile deionized water can
also be added to all outer-perimeter wells of the 96-well plate.
226
Cle ´ mence Mouville and Nadia Benaroudj
dilute the killing chemical agent used for the survival test to
sublethal concentration. However, in some cases, it might be
necessary to wash the bacteria with EMJH before plating them
on solid media.
8. We generally use cleaned and autoclaved 4 mm glass beads
from Sigma. You can also use a glass spreader or inoculation
loops to spread bacteria, but this will make handling multiple
plates difficult.
9. Glass beads are reusable. Wash them thoroughly with a decontaminating solution, rinse them extensively with distilled water,
and autoclave them for further use.
10. It is important to ensure that the colonies will remain isolated.
It might be necessary to check regularly the plates during their
incubation at 30
C as Leptospira spp. are motile and can
produce spread large and confluent colonies when overgrown
on agar plate.
11. The use of a black paper sheet facilitates the visualization of
Leptospira colonies.
12. We generally use the Alamar Blue
® Assay provided by Thermo
Fisher Scientific Invitrogen but a resazurin solution from
another manufacturer might give satisfactory results as well.
13. You have to anticipate which method will be used to quantify
resazurin reduction. If fluorescence will be used, the reaction
has to be performed in plates that are compatible with fluorescence measurement. We recommend using clear-bottom blackwalled 96-well plates. We have obtained satisfactory results
with the μClear Black 96-well plate provided by Greiner. But,
these plates are not suitable to take a picture for recording the
change in color upon resazurin reduction.
14. The optimum pH for resazurin reduction is around 7.0 [9]. In
fact, resazurin and resorufin will be respectively pink and colorless under acidic pH. You have to ensure that the reaction mix
for resazurin reduction is buffered.
15. It is crucial to verify that the chemical used as killing agent in
the survival test does not interfere with resazurin reduction.
This can be done by pipetting the chemical at the desired
concentration into a well of the 96-well plate and completing
with EMJH medium to have a final volume of 180 μl. Add
20 μl of the 10Â resazurin solution and mix by pipetting.
16. The optimal temperature for resazurin reduction is 37
C, but
we prefer to perform the resazurin reduction reaction at 30
C
to reduce evaporation. 200 μl of sterile deionized water can
also be added to all outer-perimeter wells of the 96-well plate.
226
Cle ´ mence Mouville and Nadia Benaroudj