absorbance (Fig. 2b) or fluorescence measurement (Fig. 2c). This
method is easy to perform and not labor-intensive, as it does not
require a high number of steps, and is therefore compatible with
processing multiple samples. Reduction of resazurin can be visible
after a few hours, and the signal of reduced resazurin is stable for
several days. In addition, it allows flexibility in the measurement
method used (qualitative or quantitative). This method, commercially known as the Alamar Blue
® Assay, has been extensively used
with bacteria including the slow-growing Mycobacterium tuberculosis [10]. Since the EMJH medium used to cultivate Leptospira does
not interfere with the reduction of resazurin, Alamar Blue
® Assay
was also successfully used to measure Leptospira viability [11–14].
Another criterion that can be used to rapidly assess bacterial
viability is the membrane integrity. In this method, bacterial viability is correlated with the level of membrane damage and is assessed
using a mixture of two fluorescent nucleic acid stains, the Syto9 and
the propidium iodide (PI). These two dyes differ in their spectroscopic properties and in their capacities to penetrate bacteria. The
green fluorescent Syto9 penetrates bacteria regardless their membrane integrity and stains DNA of all bacteria in a population. On
the contrary, the red fluorescent PI penetrates only permeable
bacteria and stains DNA of only bacteria with damaged membranes. Importantly, PI has a higher affinity for nucleic acids than
Syto9 [15]. When a bacterial population containing intact and
membrane-damaged bacteria is exposed to a mixture of Syto9 and
PI, intact bacteria will be stained by green fluorescence, whereas
membrane-damaged bacteria will be stained by red fluorescence.
Because the two dyes have different fluorescent properties, respective nucleic acid staining can be quantified by measuring fluorescence of the bacterial suspension at different wavelengths. The
green/red fluorescence ratio is proportional to the relative number
of live bacteria, and a standard curve will determine the proportionality factor. This method is known as the LIVE/DEAD BacLight
stain. Since Boulos et al. [16] evaluated this procedure in bacteria
and demonstrated its ease 20 years ago, this method is widely used
to assess bacterial viability. This method allows staining of viable
and dead cells in a single step, and the acquisition of results is rapid.
In addition to quantify Leptospira viability by fluorescent measurement, staining of Leptospira by Syto9 and PI can be measured by
flow cytometry [17] (see Chapter 4, Fontana et al.) and fluorescence
microscopy [18]. It should be pointed out however that several
studies have raised some concerns about the Syto9/PI staining; in
fact, under certain conditions, this method has led to under- or
overestimation of the number of viable cells [19–21].
In conclusion, when Leptospira viability needs to be estimated,
multiple approaches are available. The traditional gold standard
colony-forming unit method is labor-intensive but the most sensitive and linear method. The Alamar Blue
® and LIVE/DEAD
Survival Tests for Leptospira spp.
217
method is easy to perform and not labor-intensive, as it does not
require a high number of steps, and is therefore compatible with
processing multiple samples. Reduction of resazurin can be visible
after a few hours, and the signal of reduced resazurin is stable for
several days. In addition, it allows flexibility in the measurement
method used (qualitative or quantitative). This method, commercially known as the Alamar Blue
® Assay, has been extensively used
with bacteria including the slow-growing Mycobacterium tuberculosis [10]. Since the EMJH medium used to cultivate Leptospira does
not interfere with the reduction of resazurin, Alamar Blue
® Assay
was also successfully used to measure Leptospira viability [11–14].
Another criterion that can be used to rapidly assess bacterial
viability is the membrane integrity. In this method, bacterial viability is correlated with the level of membrane damage and is assessed
using a mixture of two fluorescent nucleic acid stains, the Syto9 and
the propidium iodide (PI). These two dyes differ in their spectroscopic properties and in their capacities to penetrate bacteria. The
green fluorescent Syto9 penetrates bacteria regardless their membrane integrity and stains DNA of all bacteria in a population. On
the contrary, the red fluorescent PI penetrates only permeable
bacteria and stains DNA of only bacteria with damaged membranes. Importantly, PI has a higher affinity for nucleic acids than
Syto9 [15]. When a bacterial population containing intact and
membrane-damaged bacteria is exposed to a mixture of Syto9 and
PI, intact bacteria will be stained by green fluorescence, whereas
membrane-damaged bacteria will be stained by red fluorescence.
Because the two dyes have different fluorescent properties, respective nucleic acid staining can be quantified by measuring fluorescence of the bacterial suspension at different wavelengths. The
green/red fluorescence ratio is proportional to the relative number
of live bacteria, and a standard curve will determine the proportionality factor. This method is known as the LIVE/DEAD BacLight
stain. Since Boulos et al. [16] evaluated this procedure in bacteria
and demonstrated its ease 20 years ago, this method is widely used
to assess bacterial viability. This method allows staining of viable
and dead cells in a single step, and the acquisition of results is rapid.
In addition to quantify Leptospira viability by fluorescent measurement, staining of Leptospira by Syto9 and PI can be measured by
flow cytometry [17] (see Chapter 4, Fontana et al.) and fluorescence
microscopy [18]. It should be pointed out however that several
studies have raised some concerns about the Syto9/PI staining; in
fact, under certain conditions, this method has led to under- or
overestimation of the number of viable cells [19–21].
In conclusion, when Leptospira viability needs to be estimated,
multiple approaches are available. The traditional gold standard
colony-forming unit method is labor-intensive but the most sensitive and linear method. The Alamar Blue
® and LIVE/DEAD
Survival Tests for Leptospira spp.
217