ratio versus the percentage of live bacteria (Fig. 4). A fitted
linear regression method will provide with an equation to
deduce the percentage of live bacteria from the F 530 /F 630
value of any tested samples (see Note 23).
3.4.2 Survival Test of a
Bacterial Suspension
1. Harvest the bacteria (treated and non-treated control samples
obtained in Subheading 3.1) by centrifugation for 15 min at
2600 Â g at room temperature in conical polypropylene tubes
(see Note 20).
2. Wash the cells twice with 0.5% NaCl and resuspend them in
0.5% NaCl (see Note 24).
3. Adjust all the samples to the same concentration (as assessed by
absorbance measurement at 420 nm or enumeration with a
Petroff-Hausser chamber under a dark-field microscope) (see
Note 25).
4. In a 96-well flat bottom plate suitable for fluorescence measurement (see Note 22), mix 100 μl of bacterial suspension
washed in 0.5% NaCl with 100 μl of Syto9/PI mixture. Mix
thoroughly by pipetting up and down.
5. Incubate for 15 min at room temperature in the dark by
enveloping the plate in aluminum foil.
6. Measure the fluorescence of each well with a plate reader as
described in step 11 in Subheading 3.4.1.
7. Calculate the ratio of Syto9 fluorescence (F 530 ) to that of the PI
(F 630 ) for each sample.
Fig. 4 Linear relationship between the percentage of live Leptospira and Syto9/PI
fluorescence. Live and heat-killed Leptospira were obtained and treated with the
Syto9/PI mixture in a flat bottom 96-well plates (μClear Black 96-well plate) as
described in Subheading 3.4.1. Green Syto9 fluorescence (F 530 ; λ exc ¼ 485 nm,
λ em ¼ 530 nm) and red PI fluorescence (F 630 ; λ exc ¼ 485 nm, λ em ¼ 630 nm)
were measured. The F 530 /F 630 values (green Syto9-to-red PI fluorescence ratio)
were plotted versus the percentage of live bacteria
224
Cle ´ mence Mouville and Nadia Benaroudj
linear regression method will provide with an equation to
deduce the percentage of live bacteria from the F 530 /F 630
value of any tested samples (see Note 23).
3.4.2 Survival Test of a
Bacterial Suspension
1. Harvest the bacteria (treated and non-treated control samples
obtained in Subheading 3.1) by centrifugation for 15 min at
2600 Â g at room temperature in conical polypropylene tubes
(see Note 20).
2. Wash the cells twice with 0.5% NaCl and resuspend them in
0.5% NaCl (see Note 24).
3. Adjust all the samples to the same concentration (as assessed by
absorbance measurement at 420 nm or enumeration with a
Petroff-Hausser chamber under a dark-field microscope) (see
Note 25).
4. In a 96-well flat bottom plate suitable for fluorescence measurement (see Note 22), mix 100 μl of bacterial suspension
washed in 0.5% NaCl with 100 μl of Syto9/PI mixture. Mix
thoroughly by pipetting up and down.
5. Incubate for 15 min at room temperature in the dark by
enveloping the plate in aluminum foil.
6. Measure the fluorescence of each well with a plate reader as
described in step 11 in Subheading 3.4.1.
7. Calculate the ratio of Syto9 fluorescence (F 530 ) to that of the PI
(F 630 ) for each sample.
Fig. 4 Linear relationship between the percentage of live Leptospira and Syto9/PI
fluorescence. Live and heat-killed Leptospira were obtained and treated with the
Syto9/PI mixture in a flat bottom 96-well plates (μClear Black 96-well plate) as
described in Subheading 3.4.1. Green Syto9 fluorescence (F 530 ; λ exc ¼ 485 nm,
λ em ¼ 530 nm) and red PI fluorescence (F 630 ; λ exc ¼ 485 nm, λ em ¼ 630 nm)
were measured. The F 530 /F 630 values (green Syto9-to-red PI fluorescence ratio)
were plotted versus the percentage of live bacteria
224
Cle ´ mence Mouville and Nadia Benaroudj