Chapter 20
Survival Tests for Leptospira spp.
Cle ´ mence Mouville and Nadia Benaroudj
Abstract
Measuring viability is an important and necessary assessment in studying microorganisms. Several methods
can be applied to Leptospira spp., each with advantages and inconveniencies. Here, we describe the
traditional colony-forming unit method, together with two other methods based, respectively, on the
reducing capacity of live cells (Alamar Blue
® Assay) and differential staining of live and dead cells (LIVE/
DEAD BacLight
® ). The Alamar Blue
® Assay uses the blue reagent resazurin, which can be reduced into the
pink reagent resorufin by live cell oxidoreductases. Production of resorufin can be quantified by absorbance
or fluorescence reading. The LIVE/DEAD BacLight
® assay uses a mixture of two nucleic acid dyes (Syto9
and propidium iodide) that differentially penetrate and stain nucleic acid of cells with decreased membrane
integrity. The colony-forming unit method is labor-intensive but the most sensitive and linear method. The
two other methods are not laborious and well-adapted to high-throughput studies, but the range of
detection and linearity are limited.
Key words Spirochetes, Leptospira, Survival, Plating, Colony-forming unit, Resazurin, Resorufin,
Syto9, Propidium iodide, Fluorescence, Absorbance
1 Introduction
Assessing and quantifying the viability of a microorganism is a basic
but fundamental procedure in microbiology. This is particularly
important when the efficiency of an antibiotic or other antimicrobial compound needs to be determined or to characterize bacterial
species and their ability to survive under different environments.
Evaluating whether a bacterium is alive or dead is rather complex as finding the best criteria to define bacterial viability is still
matter of debate. Several different qualitative and quantitative
techniques are available to measure bacterial survival, each having
their advantages and limitations. Here, we will describe three methods that can be applied to Leptospira spp.
The gold standard is the colony-forming unit (CFU) method
based on the ability of a bacterium to form visible colonies on agar
plates [1]. In this method, bacterial viability is defined by their
cultivability and ability to divide on solid medium. This technique
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_20, © Springer Science+Business Media, LLC, part of Springer Nature 2020
215
Survival Tests for Leptospira spp.
Cle ´ mence Mouville and Nadia Benaroudj
Abstract
Measuring viability is an important and necessary assessment in studying microorganisms. Several methods
can be applied to Leptospira spp., each with advantages and inconveniencies. Here, we describe the
traditional colony-forming unit method, together with two other methods based, respectively, on the
reducing capacity of live cells (Alamar Blue
® Assay) and differential staining of live and dead cells (LIVE/
DEAD BacLight
® ). The Alamar Blue
® Assay uses the blue reagent resazurin, which can be reduced into the
pink reagent resorufin by live cell oxidoreductases. Production of resorufin can be quantified by absorbance
or fluorescence reading. The LIVE/DEAD BacLight
® assay uses a mixture of two nucleic acid dyes (Syto9
and propidium iodide) that differentially penetrate and stain nucleic acid of cells with decreased membrane
integrity. The colony-forming unit method is labor-intensive but the most sensitive and linear method. The
two other methods are not laborious and well-adapted to high-throughput studies, but the range of
detection and linearity are limited.
Key words Spirochetes, Leptospira, Survival, Plating, Colony-forming unit, Resazurin, Resorufin,
Syto9, Propidium iodide, Fluorescence, Absorbance
1 Introduction
Assessing and quantifying the viability of a microorganism is a basic
but fundamental procedure in microbiology. This is particularly
important when the efficiency of an antibiotic or other antimicrobial compound needs to be determined or to characterize bacterial
species and their ability to survive under different environments.
Evaluating whether a bacterium is alive or dead is rather complex as finding the best criteria to define bacterial viability is still
matter of debate. Several different qualitative and quantitative
techniques are available to measure bacterial survival, each having
their advantages and limitations. Here, we will describe three methods that can be applied to Leptospira spp.
The gold standard is the colony-forming unit (CFU) method
based on the ability of a bacterium to form visible colonies on agar
plates [1]. In this method, bacterial viability is defined by their
cultivability and ability to divide on solid medium. This technique
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_20, © Springer Science+Business Media, LLC, part of Springer Nature 2020
215