aluminum foil to protect them from light and lower risk of
contamination.
4. Incubate the plates at 30
C for 3–4 weeks (see Note 10).
5. Count the number of colonies on each plate and calculate the
mean of the replicates (see Note 11). Multiplicate this number
by the dilution factor and by 10 to obtain the number of
cfu/ml. Calculate the ratio of cfu of the treated sample to the
cfu of the non-treated sample. The percentage of survival after
treatment is determined by multiplying this ratio by 100.
3.3 Assessing
Survival by Measuring
Cellular Reducing
Environment
We describe the protocol to measure Leptospira spp. viability with
the Alamar Blue
® Assay (see Note 12). You can perform this assay
with the bacterial suspension as prepared in Subheading 3.1 or
directly perform the cell treatment in 96-well plates, depending
on the nature of the treatment or whether cell viability will be
simultaneously assessed by different techniques.
1. Transfer 100 μl of treated cells or non-treated control cells into
each well of a 96-well plate (see Note 13), and add 80 μl of
EMJH medium to each well. Proceed directly to step 3.
2. If the treatment is being performed directly in a 96-well plate,
pipette 100 μl of Leptospira suspension obtained at step 3 of
Subheading 3.1 into each well of the plate. Add the desired
amount of the chemical used in the survival test, and complete
with EMJH medium to a final volume of 180 μl. Mix by
pipetting and incubate the plate at 30
C for a given time.
3. Add 20 μl of the 10Â resazurin solution beforehand warmed at
room temperature. Mix by pipetting (see Note 14).
4. Prepare a negative control sample that does not contain Leptospira: pipette 180 μl of EMJH medium and add 20 μl of the
10Â resazurin solution. Mix by pipetting (see Note 15).
5. Place the 96-well plate into a plastic box and incubate the plate
at 30
C (see Note 16) until the reaction is completed, as seen
by the appearance of a pink solution (see Note 17).
6. You can record the results simply by taking a picture (Fig. 2a).
7. The quantification can be done by absorbance measurement at
570 (absorbance maximum of resorufin) and 600 (absorbance
maximum of resazurin) nm (Fig. 2b). The optical zero is made
using the negative control well that does not contain any cell.
Percentage of resazurin reduction is calculated using the equation indicated in Fig. 3 (as recommended by the manufacturer). The quantification can be also done by fluorescence
measurement with an excitation and emission wavelengths of
560 nm and 590 nm, respectively (Fig. 2c) (see Note 18).
222
Cle ´ mence Mouville and Nadia Benaroudj
contamination.
4. Incubate the plates at 30
C for 3–4 weeks (see Note 10).
5. Count the number of colonies on each plate and calculate the
mean of the replicates (see Note 11). Multiplicate this number
by the dilution factor and by 10 to obtain the number of
cfu/ml. Calculate the ratio of cfu of the treated sample to the
cfu of the non-treated sample. The percentage of survival after
treatment is determined by multiplying this ratio by 100.
3.3 Assessing
Survival by Measuring
Cellular Reducing
Environment
We describe the protocol to measure Leptospira spp. viability with
the Alamar Blue
® Assay (see Note 12). You can perform this assay
with the bacterial suspension as prepared in Subheading 3.1 or
directly perform the cell treatment in 96-well plates, depending
on the nature of the treatment or whether cell viability will be
simultaneously assessed by different techniques.
1. Transfer 100 μl of treated cells or non-treated control cells into
each well of a 96-well plate (see Note 13), and add 80 μl of
EMJH medium to each well. Proceed directly to step 3.
2. If the treatment is being performed directly in a 96-well plate,
pipette 100 μl of Leptospira suspension obtained at step 3 of
Subheading 3.1 into each well of the plate. Add the desired
amount of the chemical used in the survival test, and complete
with EMJH medium to a final volume of 180 μl. Mix by
pipetting and incubate the plate at 30
C for a given time.
3. Add 20 μl of the 10Â resazurin solution beforehand warmed at
room temperature. Mix by pipetting (see Note 14).
4. Prepare a negative control sample that does not contain Leptospira: pipette 180 μl of EMJH medium and add 20 μl of the
10Â resazurin solution. Mix by pipetting (see Note 15).
5. Place the 96-well plate into a plastic box and incubate the plate
at 30
C (see Note 16) until the reaction is completed, as seen
by the appearance of a pink solution (see Note 17).
6. You can record the results simply by taking a picture (Fig. 2a).
7. The quantification can be done by absorbance measurement at
570 (absorbance maximum of resorufin) and 600 (absorbance
maximum of resazurin) nm (Fig. 2b). The optical zero is made
using the negative control well that does not contain any cell.
Percentage of resazurin reduction is calculated using the equation indicated in Fig. 3 (as recommended by the manufacturer). The quantification can be also done by fluorescence
measurement with an excitation and emission wavelengths of
560 nm and 590 nm, respectively (Fig. 2c) (see Note 18).
222
Cle ´ mence Mouville and Nadia Benaroudj