3. Add SYTO
® 9 and PI dyes to the bacteria at 1:1000 dilution
each, for example, 1 μL of each dye to 1 mL of bacterial
suspension. Shortly vortex after each addition (see Notes 5
and 6).
4. Incubate the stained bacteria for 3 min protected from light (see
Note 7).
3.3 Configuration
of the Flow Cytometer
Software
1. Open a new workspace on the BD Accuri™ C6 software.
2. In the “Collect” software panel, set the parameters as follows:
run limit at 20,000 events and speed of analysis at slow (14 μL/
min) (see Note 8).
3. In the “Data display” panel, add the following dot plots: SSC-A
versus FSC-A to visualize the shape of the population and
FL3-A (red fluorescence) versus FL1-A (green fluorescence)
to visualize fluorescent leptospires and enumerate live bacteria.
Set the plot axes in logarithmic scales.
3.4 Controls
to Perform Prior
to First Leptospira
Enumerations
Perform the steps described below before the first Leptospira enumerations to understand and fix proper flow cytometer parameters.
For routine analyses, go directly to Subheading 3.5.
1. Prepare 12 Â 75 mm or microcentrifuge tubes of at least
100 μL of (1) unstained saline solution, (2) unstained EMJH
medium, (3) stained saline solution, and (4) stained EMJH
medium.
2. Shortly vortex the tubes prior to analysis.
3. Place the first tube to analyze under the sample injection port
(SIP).
4. In the “Collect” panel of BD Accuri™ C6 software, fix a
permissive threshold at 1000 on FSC.
5. Click on the “Run” button. Events are automatically recorded
on FSC/SSC and FL1/FL3 dot plots. Zoom on the recorded
dots using the magnifying glass button to correctly visualize
the obtained profiles.
6. Analyze the other tubes following the same procedure. Events
of these four analyses all correspond to background noise and
should be removed for further analyses (Fig. 2a, b). This
explains the necessity of applying a higher threshold.
7. Fix a threshold at 1000 on FL1 and repeat analyses of the tubes
from step 1 (see Note 9). A minor quantity of events should be
recorded, meaning that background noise is correctly removed
from analysis (Fig. 2c).
8. Repeat steps 1–7 with tubes of (1) unstained fresh Leptospira
culture and (2) stained fresh Leptospira culture, diluted as
described in Subheading 3.2. Unstained Leptospira culture is
34
Ce ´ lia Fontana and Jean Reyes
® 9 and PI dyes to the bacteria at 1:1000 dilution
each, for example, 1 μL of each dye to 1 mL of bacterial
suspension. Shortly vortex after each addition (see Notes 5
and 6).
4. Incubate the stained bacteria for 3 min protected from light (see
Note 7).
3.3 Configuration
of the Flow Cytometer
Software
1. Open a new workspace on the BD Accuri™ C6 software.
2. In the “Collect” software panel, set the parameters as follows:
run limit at 20,000 events and speed of analysis at slow (14 μL/
min) (see Note 8).
3. In the “Data display” panel, add the following dot plots: SSC-A
versus FSC-A to visualize the shape of the population and
FL3-A (red fluorescence) versus FL1-A (green fluorescence)
to visualize fluorescent leptospires and enumerate live bacteria.
Set the plot axes in logarithmic scales.
3.4 Controls
to Perform Prior
to First Leptospira
Enumerations
Perform the steps described below before the first Leptospira enumerations to understand and fix proper flow cytometer parameters.
For routine analyses, go directly to Subheading 3.5.
1. Prepare 12 Â 75 mm or microcentrifuge tubes of at least
100 μL of (1) unstained saline solution, (2) unstained EMJH
medium, (3) stained saline solution, and (4) stained EMJH
medium.
2. Shortly vortex the tubes prior to analysis.
3. Place the first tube to analyze under the sample injection port
(SIP).
4. In the “Collect” panel of BD Accuri™ C6 software, fix a
permissive threshold at 1000 on FSC.
5. Click on the “Run” button. Events are automatically recorded
on FSC/SSC and FL1/FL3 dot plots. Zoom on the recorded
dots using the magnifying glass button to correctly visualize
the obtained profiles.
6. Analyze the other tubes following the same procedure. Events
of these four analyses all correspond to background noise and
should be removed for further analyses (Fig. 2a, b). This
explains the necessity of applying a higher threshold.
7. Fix a threshold at 1000 on FL1 and repeat analyses of the tubes
from step 1 (see Note 9). A minor quantity of events should be
recorded, meaning that background noise is correctly removed
from analysis (Fig. 2c).
8. Repeat steps 1–7 with tubes of (1) unstained fresh Leptospira
culture and (2) stained fresh Leptospira culture, diluted as
described in Subheading 3.2. Unstained Leptospira culture is
34
Ce ´ lia Fontana and Jean Reyes