4 Notes
1. The protocol assumes that insertion mutants were generated
with a transposon carrying a Kan
R marker. Transposon mutants
can be generated from saprophytic [10] or pathogenic strains
[2]. The creation of the library of mutants is described in
Chapter 8 of the book. The Tn-Seq protocol can be applied
to both strains.
2. To determine the density of Leptospira using a Petroff-Hausser
chamber, dilute the culture 1:100 in EMJH. Place coverslip
over the slide, and put 10 μl of diluted bacterial suspension
under the coverslip. Count bacterial cells located within the big
center square (corresponding to 25 small squares) under a
dark-field microscope at 400Â magnification. Calculate the
density of Leptospira using the following equation: cells/
ml ¼ number of cells  dilution factor  50,000.
3. The bacterial cell density of the inoculum or input pool
depends on the goals of the experiment. For animal experiments, the challenge dose, the strain’s ED 50 , the route of
inoculation, the animal model, and the length of the experiment need to be considered to determine the appropriate input
pool cell density. The kinetics of dissemination of Leptospira is
affected by all of these characteristics. For example, after intraperitoneal challenge with the Fiocruz L1-130 strain of
L. interrogans, bacteria can be detected in blood after an hour
when the inoculum is 10
8 leptospires, but when the dose is only
250 leptospires, bacteria are not detected until the fifth day of
infection. The route of challenge also influences the kinetics of
dissemination. With the same dose of L. interrogans, bacteria
are detected in the kidney 1 day after intraperitoneal challenge
but 4 days after conjunctival challenge [11]. The number of
Leptospira cells in the inoculum required to generate a lethal
infection also depends on the animal model.
The starting density and the length of the in vitro experiment depend on the conditions being examined. For in vitro
testing, the main criterion to consider is the properties of the
strain itself. Saprophytic and pathogenic strains have very different in vitro doubling times, 4.5 and 14–18 h, respectively
[12]. Because of these differences, the density of the input pool
and the length of the experiment need to be determined for
each strain. To detect subtle effects on fitness, a longer period
of incubation of the culture and a lower starting density may be
necessary so that a larger number of cell doublings occur.
4. When possible, transposon insertion mutants unaffected by the
condition to be examined should be included. Mutants whose
fitness is known to be affected by the condition under study
104
Kristel Lourdault and James Matsunaga
1. The protocol assumes that insertion mutants were generated
with a transposon carrying a Kan
R marker. Transposon mutants
can be generated from saprophytic [10] or pathogenic strains
[2]. The creation of the library of mutants is described in
Chapter 8 of the book. The Tn-Seq protocol can be applied
to both strains.
2. To determine the density of Leptospira using a Petroff-Hausser
chamber, dilute the culture 1:100 in EMJH. Place coverslip
over the slide, and put 10 μl of diluted bacterial suspension
under the coverslip. Count bacterial cells located within the big
center square (corresponding to 25 small squares) under a
dark-field microscope at 400Â magnification. Calculate the
density of Leptospira using the following equation: cells/
ml ¼ number of cells  dilution factor  50,000.
3. The bacterial cell density of the inoculum or input pool
depends on the goals of the experiment. For animal experiments, the challenge dose, the strain’s ED 50 , the route of
inoculation, the animal model, and the length of the experiment need to be considered to determine the appropriate input
pool cell density. The kinetics of dissemination of Leptospira is
affected by all of these characteristics. For example, after intraperitoneal challenge with the Fiocruz L1-130 strain of
L. interrogans, bacteria can be detected in blood after an hour
when the inoculum is 10
8 leptospires, but when the dose is only
250 leptospires, bacteria are not detected until the fifth day of
infection. The route of challenge also influences the kinetics of
dissemination. With the same dose of L. interrogans, bacteria
are detected in the kidney 1 day after intraperitoneal challenge
but 4 days after conjunctival challenge [11]. The number of
Leptospira cells in the inoculum required to generate a lethal
infection also depends on the animal model.
The starting density and the length of the in vitro experiment depend on the conditions being examined. For in vitro
testing, the main criterion to consider is the properties of the
strain itself. Saprophytic and pathogenic strains have very different in vitro doubling times, 4.5 and 14–18 h, respectively
[12]. Because of these differences, the density of the input pool
and the length of the experiment need to be determined for
each strain. To detect subtle effects on fitness, a longer period
of incubation of the culture and a lower starting density may be
necessary so that a larger number of cell doublings occur.
4. When possible, transposon insertion mutants unaffected by the
condition to be examined should be included. Mutants whose
fitness is known to be affected by the condition under study
104
Kristel Lourdault and James Matsunaga