Chapter 4
Cell Enumeration of Leptospira by Flow Cytometry
Ce ´ lia Fontana and Jean Reyes
Abstract
Rapid and reliable enumeration of Leptospira spp., the causative agent of leptospirosis, represents a technical
challenge because leptospires are thin, highly motile, and slow-growing bacteria. The current gold standard
for cell enumeration is the use of a Petroff-Hausser counting chamber and a dark-field microscope, but this
method remains time-consuming and lacks reproducibility. New alternative techniques are then of great
interest. Here we describe the protocol for counting leptospires by flow cytometry. This method is rapid,
reproducible, sensitive, and hence suitable to become a new standard to enumerate Leptospira spp.
Key words Leptospira, Enumeration, Flow cytometry, Fluorescent dyes, Cell viability
1 Introduction
Tools to manipulate Leptospira spp. are still in their infancy. Even
today, rapid and reliable enumeration of these atypical bacteria
represents a technical challenge. Classical methods like colonyforming unit (CFU) counts are not suitable for routine testing
considering the slow growth rate of leptospires—more than
4 weeks are necessary to obtain colonies of pathogenic strains on
agar plates [1]. The current standard method for enumerating
Leptospira spp. is the count of motile bacteria on a Petroff-Hausser
chamber under dark-field microscopy. Due to high motility of
Leptospira spp. within or between microscope fields, this technique
remains time-consuming, requires a well-trained operator, and
lacks reproducibility [2]. Other approaches like bioluminescence
have been described using an ATP assay, but the protocol was laborintensive and valid only in the limited range of 4 Â 10
8 –8 Â 10
9 leptospires/mL [3]. More recently, the modification of L. interrogans
and L. biflexa strains by inserting the luxCDABE cassette in their
genomes resulted in an efficient counting method of bioluminescent strains but restricted to these genetically modified strains
[2]. Alternatively, a viability PCR using propidium monoazide
was implemented for efficient quantification of living Leptospira
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_4, © Springer Science+Business Media, LLC, part of Springer Nature 2020
31
Cell Enumeration of Leptospira by Flow Cytometry
Ce ´ lia Fontana and Jean Reyes
Abstract
Rapid and reliable enumeration of Leptospira spp., the causative agent of leptospirosis, represents a technical
challenge because leptospires are thin, highly motile, and slow-growing bacteria. The current gold standard
for cell enumeration is the use of a Petroff-Hausser counting chamber and a dark-field microscope, but this
method remains time-consuming and lacks reproducibility. New alternative techniques are then of great
interest. Here we describe the protocol for counting leptospires by flow cytometry. This method is rapid,
reproducible, sensitive, and hence suitable to become a new standard to enumerate Leptospira spp.
Key words Leptospira, Enumeration, Flow cytometry, Fluorescent dyes, Cell viability
1 Introduction
Tools to manipulate Leptospira spp. are still in their infancy. Even
today, rapid and reliable enumeration of these atypical bacteria
represents a technical challenge. Classical methods like colonyforming unit (CFU) counts are not suitable for routine testing
considering the slow growth rate of leptospires—more than
4 weeks are necessary to obtain colonies of pathogenic strains on
agar plates [1]. The current standard method for enumerating
Leptospira spp. is the count of motile bacteria on a Petroff-Hausser
chamber under dark-field microscopy. Due to high motility of
Leptospira spp. within or between microscope fields, this technique
remains time-consuming, requires a well-trained operator, and
lacks reproducibility [2]. Other approaches like bioluminescence
have been described using an ATP assay, but the protocol was laborintensive and valid only in the limited range of 4 Â 10
8 –8 Â 10
9 leptospires/mL [3]. More recently, the modification of L. interrogans
and L. biflexa strains by inserting the luxCDABE cassette in their
genomes resulted in an efficient counting method of bioluminescent strains but restricted to these genetically modified strains
[2]. Alternatively, a viability PCR using propidium monoazide
was implemented for efficient quantification of living Leptospira
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_4, © Springer Science+Business Media, LLC, part of Springer Nature 2020
31