allows the determination of absolute number of bacteria, and the
range of detection is unlimited, although this method does not take
into consideration and cannot be applied to viable but
non-cultivable bacteria. After applying a treatment to a bacterial
population, the bacterial suspension is diluted to obtain isolated,
readily countable colonies on agar plate. This method is easy to
implement and does not require sophisticated equipment and is
thus feasible with Leptospira spp. However, optimization to find the
right bacterial suspension dilution for obtaining isolated colonies
on agar plates might be time-consuming [2]. Another limitation
lies in the generation time of Leptospira spp., and, as a consequence,
the results are obtained after 1 week or 3–4 weeks of incubation for
saprophytes and pathogenic species, respectively. An additional
potential issue is that Leptospira spp. grow below the surface of
the solid agar medium and appear as faint colonies, rendering their
macroscopic visualization difficult. Nevertheless, this method was
applied to Leptospira spp. and has allowed enumerating and quantifying their viability under different growth conditions [3–6].
Alternative methods using other viability criteria, such as metabolic activities, were developed and are now preferentially used
due to their adaptability to high-throughput screenings. One
method is based on the fact that live and actively respiring cells
maintain a reducing environment. This method uses resazurin
(7-hydroxy-10-oxidophenoxasin-10-ium-3-one), a chromogen
electron acceptor that is reduced into resorufin by cell oxidoreductases using NADH as cofactor (Fig. 1) [7]. Resazurin is permeable
through cell membranes, and its reduction is therefore an indicator
of the oxidoreduction state of the cells. Resazurin and resorufin
have different spectrophotometric and fluorometric properties
[8, 9]. Resazurin is a blue non-fluorescent reagent, and resorufin
is a pink highly fluorescent reagent. When mixed with live cells,
upon accepting electron, the blue-colored non-fluorescent resazurin is transformed into the pink fluorescent resorufin. Reduction
of resazurin can be qualitatively determined by observing the visible
change of the blue color into pink, indicative of the presence of live
cells (Fig. 2a). Resazurin reduction can be also quantified by
Fig. 1 Reaction of resazurin reduction into resorufin. Resazurin (a blue reagent) is an electron receptor that is
reduced into resorufin (a pink reagent) in the presence of cellular oxidoreductases and NADH
216
Cle ´ mence Mouville and Nadia Benaroudj
range of detection is unlimited, although this method does not take
into consideration and cannot be applied to viable but
non-cultivable bacteria. After applying a treatment to a bacterial
population, the bacterial suspension is diluted to obtain isolated,
readily countable colonies on agar plate. This method is easy to
implement and does not require sophisticated equipment and is
thus feasible with Leptospira spp. However, optimization to find the
right bacterial suspension dilution for obtaining isolated colonies
on agar plates might be time-consuming [2]. Another limitation
lies in the generation time of Leptospira spp., and, as a consequence,
the results are obtained after 1 week or 3–4 weeks of incubation for
saprophytes and pathogenic species, respectively. An additional
potential issue is that Leptospira spp. grow below the surface of
the solid agar medium and appear as faint colonies, rendering their
macroscopic visualization difficult. Nevertheless, this method was
applied to Leptospira spp. and has allowed enumerating and quantifying their viability under different growth conditions [3–6].
Alternative methods using other viability criteria, such as metabolic activities, were developed and are now preferentially used
due to their adaptability to high-throughput screenings. One
method is based on the fact that live and actively respiring cells
maintain a reducing environment. This method uses resazurin
(7-hydroxy-10-oxidophenoxasin-10-ium-3-one), a chromogen
electron acceptor that is reduced into resorufin by cell oxidoreductases using NADH as cofactor (Fig. 1) [7]. Resazurin is permeable
through cell membranes, and its reduction is therefore an indicator
of the oxidoreduction state of the cells. Resazurin and resorufin
have different spectrophotometric and fluorometric properties
[8, 9]. Resazurin is a blue non-fluorescent reagent, and resorufin
is a pink highly fluorescent reagent. When mixed with live cells,
upon accepting electron, the blue-colored non-fluorescent resazurin is transformed into the pink fluorescent resorufin. Reduction
of resazurin can be qualitatively determined by observing the visible
change of the blue color into pink, indicative of the presence of live
cells (Fig. 2a). Resazurin reduction can be also quantified by
Fig. 1 Reaction of resazurin reduction into resorufin. Resazurin (a blue reagent) is an electron receptor that is
reduced into resorufin (a pink reagent) in the presence of cellular oxidoreductases and NADH
216
Cle ´ mence Mouville and Nadia Benaroudj