of the dyes, it is important to keep the stock solution in the dark
to retain maximum efficiency.
8. Slow speed of analysis usually results in more accurate acquisitions. Changing speed leads to a population shift. Thus, always
use the same speed of analysis along the experiments.
9. It can be necessary to wash the flow cytometer with decontamination, cleaning, and deionized water solution between analyses of stained and unstained samples, since fluorescent dyes
tend to adhere to the pipes.
10. Contrarily to eukaryotic cells or bacteria such as Escherichia coli
[10], Leptospira cannot be differentiated from background
noise on FSC or SSC parameters because they are too thin to
be distinguished based on morphological parameters.
11. When performing the first stained bacteria analysis of the day,
FCM profile can be diffuse at first. It can be necessary to delete
and restart the run after 15–30 s to obtain a more defined and
accurate profile.
12. As an indication, the timescale for Leptospira enumeration is
approximately 6 min: 2 min of sample preparation, 3 min of
incubation with the dyes, 1 min of analysis by FCM.
13. Be aware that percentage of events contained in the “Live
Leptospires” gate strictly depends on the fixed threshold and
cannot be translated to an absolute percentage of bacterial
viability. Indeed, as dying and dead cells’ fluorescence is low,
a part—large or small depending on FL1 threshold value—of
the corresponding events are under the threshold and therefore not considered for percentage calculation. However, this
percentage still provides valuable indications. High percentages (>70%) usually correspond to viable bacteria, whereas
lower percentages imply dying ones. Besides, it is relevant to
compare viability of different suspensions based on this percentage when analyzed with the same threshold.
14. FCM enumeration technique is highly reproducible, with no
observed operator effect, and a repeatability variance,
corresponding to the variability of the technique itself, is very
low. Sensitivity of Leptospira enumeration by FCM is statistically determined to be 10
4 leptospires/mL. A bias in the
measures is observed for titers as low as 10
3 leptospires/mL,
leading to a slight overestimation of the titers,but this overestimation can be acceptable for most applications [9].
References
1. Picardeau M (2015) Genomics, proteomics,
and genetics of leptospira. Curr Top Microbiol
Immunol 387:43–63. https://doi.org/10.
1007/978-3-662-45059-8_4
2. Murray GL, King AM, Srikram A, Sermswan
RW, Adler B (2010) Use of luminescent Leptospira interrogans for enumeration in
biological
assays.
J
Clin
Microbiol
Enumeration of Leptospira Cells
39
to retain maximum efficiency.
8. Slow speed of analysis usually results in more accurate acquisitions. Changing speed leads to a population shift. Thus, always
use the same speed of analysis along the experiments.
9. It can be necessary to wash the flow cytometer with decontamination, cleaning, and deionized water solution between analyses of stained and unstained samples, since fluorescent dyes
tend to adhere to the pipes.
10. Contrarily to eukaryotic cells or bacteria such as Escherichia coli
[10], Leptospira cannot be differentiated from background
noise on FSC or SSC parameters because they are too thin to
be distinguished based on morphological parameters.
11. When performing the first stained bacteria analysis of the day,
FCM profile can be diffuse at first. It can be necessary to delete
and restart the run after 15–30 s to obtain a more defined and
accurate profile.
12. As an indication, the timescale for Leptospira enumeration is
approximately 6 min: 2 min of sample preparation, 3 min of
incubation with the dyes, 1 min of analysis by FCM.
13. Be aware that percentage of events contained in the “Live
Leptospires” gate strictly depends on the fixed threshold and
cannot be translated to an absolute percentage of bacterial
viability. Indeed, as dying and dead cells’ fluorescence is low,
a part—large or small depending on FL1 threshold value—of
the corresponding events are under the threshold and therefore not considered for percentage calculation. However, this
percentage still provides valuable indications. High percentages (>70%) usually correspond to viable bacteria, whereas
lower percentages imply dying ones. Besides, it is relevant to
compare viability of different suspensions based on this percentage when analyzed with the same threshold.
14. FCM enumeration technique is highly reproducible, with no
observed operator effect, and a repeatability variance,
corresponding to the variability of the technique itself, is very
low. Sensitivity of Leptospira enumeration by FCM is statistically determined to be 10
4 leptospires/mL. A bias in the
measures is observed for titers as low as 10
3 leptospires/mL,
leading to a slight overestimation of the titers,but this overestimation can be acceptable for most applications [9].
References
1. Picardeau M (2015) Genomics, proteomics,
and genetics of leptospira. Curr Top Microbiol
Immunol 387:43–63. https://doi.org/10.
1007/978-3-662-45059-8_4
2. Murray GL, King AM, Srikram A, Sermswan
RW, Adler B (2010) Use of luminescent Leptospira interrogans for enumeration in
biological
assays.
J
Clin
Microbiol
Enumeration of Leptospira Cells
39