3.4 Quantitative
Survival Test Based
on Membrane Integrity
We describe the LIVE/DEAD BacLight staining method to assess
Leptospira spp. viability. In this method, the viability will be quantified by measurement of Syto9 and PI fluorescences. The ratio of
green (Syto9) to red (PI) fluorescence is proportional to the relative
number of live bacteria. A standard curve is necessary to verify the
linearity of the assay and determine the proportionality factor
between the fluorescence ratio and percentage of live bacteria.
3.4.1 Preparing
a Standard Curve
1. Cultivate Leptospira in 20 ml of EMJH medium at 30
C to
reach the exponential phase and a concentration of at least
10
8 bacteria/ml.
2. Transfer 10 ml of the culture in two different sterile conical
polypropylene tubes.
3. Obtain killed Leptospira by incubating one of the samples 2 h at
55
C in a water bath, while the other sample (live bacteria) is
maintained at 30
C (see Note 19).
4. Harvest the bacteria by centrifugation for 15 min at 2600 Â g
at room temperature (see Note 20).
5. Wash the bacteria twice with 10 ml of 0.5% NaCl (see Note 21).
6. Resuspend the bacteria in 10 ml of 0.5% NaCl.
7. Adjust the two samples (live and killed bacteria) to the same
concentration (as assessed by absorbance measurement at
420 nm or enumeration with a Petroff-Hausser chamber
under a dark-field microscope). We generally work with a bacterial suspension at about 10
8 Leptospira/ml.
8. Mix different proportions of killed and live Leptospira suspensions in sterile 1.5 ml polypropylene tubes in order to have, for
instance, the following live/killed bacteria ratio: 0:100, 10:90,
25:75, 50:50, 75:25, 90:10, and 100:0.
9. In a 96-well flat bottom plate suitable for fluorescence measurement (see Note 22), mix 100 μl of bacterial suspension
containing different proportions of killed and live Leptospira
with 100 μl of Syto9/PI mixture. Mix thoroughly by pipetting
up and down.
10. Incubate for 15 min at room temperature in the dark by
enveloping the plate in aluminum foil.
11. Measure the fluorescence of each well with a plate reader
(excitation at 485 nm and emission at 530 nm for the green
fluorescent Syto9 (F 530 ); excitation at 485 nm and emission at
630 nm for the red fluorescent PI (F 630 )).
12. Calculate the ratio of Syto9 fluorescence (F 530 ) to that of the
PI (F 630 ) for each sample.
13. The standard curve is obtained by plotting the F 530 /F 630 value
for each well corresponding to the different live/killed bacteria
Survival Tests for Leptospira spp.
223
Survival Test Based
on Membrane Integrity
We describe the LIVE/DEAD BacLight staining method to assess
Leptospira spp. viability. In this method, the viability will be quantified by measurement of Syto9 and PI fluorescences. The ratio of
green (Syto9) to red (PI) fluorescence is proportional to the relative
number of live bacteria. A standard curve is necessary to verify the
linearity of the assay and determine the proportionality factor
between the fluorescence ratio and percentage of live bacteria.
3.4.1 Preparing
a Standard Curve
1. Cultivate Leptospira in 20 ml of EMJH medium at 30
C to
reach the exponential phase and a concentration of at least
10
8 bacteria/ml.
2. Transfer 10 ml of the culture in two different sterile conical
polypropylene tubes.
3. Obtain killed Leptospira by incubating one of the samples 2 h at
55
C in a water bath, while the other sample (live bacteria) is
maintained at 30
C (see Note 19).
4. Harvest the bacteria by centrifugation for 15 min at 2600 Â g
at room temperature (see Note 20).
5. Wash the bacteria twice with 10 ml of 0.5% NaCl (see Note 21).
6. Resuspend the bacteria in 10 ml of 0.5% NaCl.
7. Adjust the two samples (live and killed bacteria) to the same
concentration (as assessed by absorbance measurement at
420 nm or enumeration with a Petroff-Hausser chamber
under a dark-field microscope). We generally work with a bacterial suspension at about 10
8 Leptospira/ml.
8. Mix different proportions of killed and live Leptospira suspensions in sterile 1.5 ml polypropylene tubes in order to have, for
instance, the following live/killed bacteria ratio: 0:100, 10:90,
25:75, 50:50, 75:25, 90:10, and 100:0.
9. In a 96-well flat bottom plate suitable for fluorescence measurement (see Note 22), mix 100 μl of bacterial suspension
containing different proportions of killed and live Leptospira
with 100 μl of Syto9/PI mixture. Mix thoroughly by pipetting
up and down.
10. Incubate for 15 min at room temperature in the dark by
enveloping the plate in aluminum foil.
11. Measure the fluorescence of each well with a plate reader
(excitation at 485 nm and emission at 530 nm for the green
fluorescent Syto9 (F 530 ); excitation at 485 nm and emission at
630 nm for the red fluorescent PI (F 630 )).
12. Calculate the ratio of Syto9 fluorescence (F 530 ) to that of the
PI (F 630 ) for each sample.
13. The standard curve is obtained by plotting the F 530 /F 630 value
for each well corresponding to the different live/killed bacteria
Survival Tests for Leptospira spp.
223