8. You can deduce the percentage of live bacteria from the F 530 /
F 630 value using the standard curve and its proportionality
factor (Fig. 4; see Note 23). Alternatively, you can normalize
the fluorescence ratio of the treated sample with that of the
non-treated control one and calculate a percentage of live cells.
4 Notes
1. We use autoclaved glassware dedicated only to EMJH medium
preparation. To avoid contaminating the glassware with components that could prevent growth of Leptospira, we rinse
beforehand the glassware with sterile water for injection
(WFI), and all the chemical stock solutions are prepared with
sterile WFI.
2. We recommend pouring rather thick EMJH agar plates to
avoid drying during the long storage at 30
C.
3. This Syto9/PI combination has given satisfactory results; however it might be necessary to adapt dye concentrations to the
condition the assay will be performed. We have used the LIVE/
DEAD™ BacLight™ Bacterial Viability Kit from Thermo
Fisher Scientific. This kit provides with 3.34 mM Syto9 and
20 mM PI solutions. Separated Syto9 and PI solutions allow
finding the optimal dye combination for your specific assay
condition.
4. We usually perform survival tests with exponentially growing
L. interrogans serovar Manilae, which are at a concentration of
about 10
8 bacteria/ml (OD 420 % 0.1–0.3). These values might
vary depending of laboratory conditions including the oxygenation level, the composition of EMJH medium, and the size
of leptospiral strain used. We advise the experimenter to perform a growth curve with the strain used in survival test
beforehand in order to have a knowledge of its growth feature
in EMJH medium in his/her laboratory condition.
5. Rigorous viability comparison implies that all bacterial samples
are taken at the same growth phase.
6. If a killing kinetic is performed, the non-treated control sample
can be a bacterial suspension incubated in the same conditions
and for the same duration without the killing agent. A sample
of the bacterial suspension before the addition of the killing
agent can also serve as non-treated control sample.
7. Finding the suitable dilution for plating is crucial for the accuracy of bacterial enumeration on agar plates. Indeed, to accurately count Leptospira on plates, it is important to obtain
isolated colonies. Also, discrepancies in counting might occur
when having too few (less than 10) or too many (more than
Survival Tests for Leptospira spp.
225
F 630 value using the standard curve and its proportionality
factor (Fig. 4; see Note 23). Alternatively, you can normalize
the fluorescence ratio of the treated sample with that of the
non-treated control one and calculate a percentage of live cells.
4 Notes
1. We use autoclaved glassware dedicated only to EMJH medium
preparation. To avoid contaminating the glassware with components that could prevent growth of Leptospira, we rinse
beforehand the glassware with sterile water for injection
(WFI), and all the chemical stock solutions are prepared with
sterile WFI.
2. We recommend pouring rather thick EMJH agar plates to
avoid drying during the long storage at 30
C.
3. This Syto9/PI combination has given satisfactory results; however it might be necessary to adapt dye concentrations to the
condition the assay will be performed. We have used the LIVE/
DEAD™ BacLight™ Bacterial Viability Kit from Thermo
Fisher Scientific. This kit provides with 3.34 mM Syto9 and
20 mM PI solutions. Separated Syto9 and PI solutions allow
finding the optimal dye combination for your specific assay
condition.
4. We usually perform survival tests with exponentially growing
L. interrogans serovar Manilae, which are at a concentration of
about 10
8 bacteria/ml (OD 420 % 0.1–0.3). These values might
vary depending of laboratory conditions including the oxygenation level, the composition of EMJH medium, and the size
of leptospiral strain used. We advise the experimenter to perform a growth curve with the strain used in survival test
beforehand in order to have a knowledge of its growth feature
in EMJH medium in his/her laboratory condition.
5. Rigorous viability comparison implies that all bacterial samples
are taken at the same growth phase.
6. If a killing kinetic is performed, the non-treated control sample
can be a bacterial suspension incubated in the same conditions
and for the same duration without the killing agent. A sample
of the bacterial suspension before the addition of the killing
agent can also serve as non-treated control sample.
7. Finding the suitable dilution for plating is crucial for the accuracy of bacterial enumeration on agar plates. Indeed, to accurately count Leptospira on plates, it is important to obtain
isolated colonies. Also, discrepancies in counting might occur
when having too few (less than 10) or too many (more than
Survival Tests for Leptospira spp.
225