10. Syto9 and PI nucleic acid dye mix: Prepare a mixture of 10 μM
of Syto9 and 60 μM of PI in ultrapure sterile H 2 O (see Note 3).
Keep the stock and the mixed solutions at À20
C and protected them from light.
11. 96-well flat bottom microplates for fluorescence reading.
12. Microplate reader for fluorescence reading.
3 Methods
3.1 Preparation
and Treatment of a
Bacterial Suspension
1. Inoculate EMJH medium-containing flasks or tubes with Leptospira cultures to achieve a final concentration of %10
7 bacteria/ml as assessed by absorbance measurement at 420 nm
(OD 420 % 0.01–0.02) or enumerating the bacteria under a
dark-field microscope with a Petroff-Hausser chamber.
2. Cultivate Leptospira at 30
C with shaking at 100 rpm until
they reach the suitable and desirable growth phase (see Note 4).
3. If comparing different Leptospira strains, adjust all samples to
the same bacterial concentration with EMJH medium (see
Note 5).
4. Divide the bacterial suspension in different samples according
to the number of treatments you want to apply to the bacterial
suspension.
5. Apply the treatment to the samples, e.g., temperature shift and
addition of chemicals or antibiotics, for a given time. You
should have the proper control of the treatment, i.e., a sample
that is not subjected to the treatment (see Note 6).
3.2 Assessing
Survival by
Determination
of Colony-Forming
Unit (cfu)
The colony-forming unit method is considered as the most quantitative and linear method if the appropriate dilution is applied to the
bacterial suspension.
1. Treated and non-treated Leptospira samples are tenfold diluted
serially. For instance, 15 μl of bacterial suspension are added to
135 μl of EMJH medium in 1.5 ml polypropylene sterile tubes.
A wide range of tenfold dilutions is necessary to determine the
appropriate dilution allowing obtaining readily countable
isolated colonies on agar plate (see Note 7).
2. Place 5–6 cleaned and sterile glass beads at the surface of the
EMJH agar plates. Add 100 μl of the diluted treated or
non-treated leptospires samples onto EMJH agar plates in
duplicate, and spread the cells by agitating the plates (see
Note 8).
3. Allow some time for the plated bacterial suspension to penetrate the agar and remove the glass beads (see Note 9). Wrap
the plates in Parafilm to avoid drying off the medium and in
Survival Tests for Leptospira spp.
221
of Syto9 and 60 μM of PI in ultrapure sterile H 2 O (see Note 3).
Keep the stock and the mixed solutions at À20
C and protected them from light.
11. 96-well flat bottom microplates for fluorescence reading.
12. Microplate reader for fluorescence reading.
3 Methods
3.1 Preparation
and Treatment of a
Bacterial Suspension
1. Inoculate EMJH medium-containing flasks or tubes with Leptospira cultures to achieve a final concentration of %10
7 bacteria/ml as assessed by absorbance measurement at 420 nm
(OD 420 % 0.01–0.02) or enumerating the bacteria under a
dark-field microscope with a Petroff-Hausser chamber.
2. Cultivate Leptospira at 30
C with shaking at 100 rpm until
they reach the suitable and desirable growth phase (see Note 4).
3. If comparing different Leptospira strains, adjust all samples to
the same bacterial concentration with EMJH medium (see
Note 5).
4. Divide the bacterial suspension in different samples according
to the number of treatments you want to apply to the bacterial
suspension.
5. Apply the treatment to the samples, e.g., temperature shift and
addition of chemicals or antibiotics, for a given time. You
should have the proper control of the treatment, i.e., a sample
that is not subjected to the treatment (see Note 6).
3.2 Assessing
Survival by
Determination
of Colony-Forming
Unit (cfu)
The colony-forming unit method is considered as the most quantitative and linear method if the appropriate dilution is applied to the
bacterial suspension.
1. Treated and non-treated Leptospira samples are tenfold diluted
serially. For instance, 15 μl of bacterial suspension are added to
135 μl of EMJH medium in 1.5 ml polypropylene sterile tubes.
A wide range of tenfold dilutions is necessary to determine the
appropriate dilution allowing obtaining readily countable
isolated colonies on agar plate (see Note 7).
2. Place 5–6 cleaned and sterile glass beads at the surface of the
EMJH agar plates. Add 100 μl of the diluted treated or
non-treated leptospires samples onto EMJH agar plates in
duplicate, and spread the cells by agitating the plates (see
Note 8).
3. Allow some time for the plated bacterial suspension to penetrate the agar and remove the glass beads (see Note 9). Wrap
the plates in Parafilm to avoid drying off the medium and in
Survival Tests for Leptospira spp.
221