3.5 Enumeration
of Leptospira by Flow
Cytometry
1. As determined in Subheading 3.3, in the “Collect” panel of BD
Accuri™ C6 software, set the threshold on FL1-H (green
fluorescence) at 1000 to remove background noise.
2. Shortly vortex the tube containing the stained bacteria prior to
analysis.
3. Place the tube under the SIP.
4. Click on the “Run” button to launch analysis. The recorded
events will automatically be displayed on FSC/SSC and
FL1/FL3 dot plots (Fig. 5a) (see Notes 11 and 12).
5. To assess culture viability, look at the calculated percentage of
events contained in the “Live” gate (see Note 13).
6. To obtain sample enumeration titer, go to the “Statistics”
software panel, and choose to display “Events/μL” contained
in the “Live” gate (Fig. 5b). Calculate the enumeration titer as
follows: number of leptospires/mL ¼ number of events in the
“Live” gate per microliter  dilution factor  1000 (Fig. 5c)
(see Note 14).
Fig. 5 Enumeration of two Leptospira cultures, a viable and a dying one. (a) FCM fluorescence (FL1/FL3)
profiles. (b) Recorded number of events in the “Live” gate for each culture automatically calculated by flow
cytometer software. (c) Obtention of enumeration titer for each culture diluted at 1:1000 prior to FCM analysis
Enumeration of Leptospira Cells
37
of Leptospira by Flow
Cytometry
1. As determined in Subheading 3.3, in the “Collect” panel of BD
Accuri™ C6 software, set the threshold on FL1-H (green
fluorescence) at 1000 to remove background noise.
2. Shortly vortex the tube containing the stained bacteria prior to
analysis.
3. Place the tube under the SIP.
4. Click on the “Run” button to launch analysis. The recorded
events will automatically be displayed on FSC/SSC and
FL1/FL3 dot plots (Fig. 5a) (see Notes 11 and 12).
5. To assess culture viability, look at the calculated percentage of
events contained in the “Live” gate (see Note 13).
6. To obtain sample enumeration titer, go to the “Statistics”
software panel, and choose to display “Events/μL” contained
in the “Live” gate (Fig. 5b). Calculate the enumeration titer as
follows: number of leptospires/mL ¼ number of events in the
“Live” gate per microliter  dilution factor  1000 (Fig. 5c)
(see Note 14).
Fig. 5 Enumeration of two Leptospira cultures, a viable and a dying one. (a) FCM fluorescence (FL1/FL3)
profiles. (b) Recorded number of events in the “Live” gate for each culture automatically calculated by flow
cytometer software. (c) Obtention of enumeration titer for each culture diluted at 1:1000 prior to FCM analysis
Enumeration of Leptospira Cells
37