undifferentiated from background noise, contrarily to stained
ones (Fig. 3a, b). In addition, application of a fluorescence
threshold leads to significant enhancement of bacterial population detection (Fig. 3c) (see Note 10).
9. Repeat steps 1–7 with tubes of stained (1) fresh viable Leptospira culture, (2) the same culture heat-killed at 70
C for
15 min, and (3) a mix of 50% fresh and 50% heat-killed cultures. Live bacteria should emit high green fluorescence and
low red fluorescence on FL1/FL3 dot plot, contrarily to dead
cells, emitting high red fluorescence and low green fluorescence (Fig. 4). Based on these analyses, draw a polygonal gate
on FL1/FL3 dot plot containing only live leptospires. Name
this gate “Live.” Dead bacteria should not appear in the drawn
“Live” gate but be in the upper and/or left part of the
FL1/FL3, as shown in Fig. 4. Reuse this gate for all future
analyses.
Fig. 2 Determination of proper thresholds for background noise removal based on saline solution analyses.
FCM morphology (FSC/SSC) and fluorescence (FL1/FL3) profiles of (a) unstained saline with a threshold at
1000 on FSC, (b) stained saline with a threshold at 1000 on FSC, and (c) stained saline with a threshold at
1000 on FL1. Analysis of EMJH gives identical results (not shown). The red lines represent the thresholds
Enumeration of Leptospira Cells
35
ones (Fig. 3a, b). In addition, application of a fluorescence
threshold leads to significant enhancement of bacterial population detection (Fig. 3c) (see Note 10).
9. Repeat steps 1–7 with tubes of stained (1) fresh viable Leptospira culture, (2) the same culture heat-killed at 70
C for
15 min, and (3) a mix of 50% fresh and 50% heat-killed cultures. Live bacteria should emit high green fluorescence and
low red fluorescence on FL1/FL3 dot plot, contrarily to dead
cells, emitting high red fluorescence and low green fluorescence (Fig. 4). Based on these analyses, draw a polygonal gate
on FL1/FL3 dot plot containing only live leptospires. Name
this gate “Live.” Dead bacteria should not appear in the drawn
“Live” gate but be in the upper and/or left part of the
FL1/FL3, as shown in Fig. 4. Reuse this gate for all future
analyses.
Fig. 2 Determination of proper thresholds for background noise removal based on saline solution analyses.
FCM morphology (FSC/SSC) and fluorescence (FL1/FL3) profiles of (a) unstained saline with a threshold at
1000 on FSC, (b) stained saline with a threshold at 1000 on FSC, and (c) stained saline with a threshold at
1000 on FL1. Analysis of EMJH gives identical results (not shown). The red lines represent the thresholds
Enumeration of Leptospira Cells
35