You can also seal the 96-well plate with Parafilm for a longer
incubation or storage.
17. The time for the completion of the resazurin reduction reaction depends on the number of viable cells used in the assay.
The change of blue to pink color can be seen after few hours,
but it can also require longer incubation time (up to 3 days).
Extended incubation times are not recommended to record the
results. Indeed, the resazurin reduction into resorufin is irreversible but resorufin can be further reduced into hydroresorufin, a colorless and non-fluorescent chemical [8].
18. Resazurin reduction into resorufin is detectable only if Leptospira concentration is above 10
6
/ml (Fig. 2). A good linearity is
observed between resazurin reduction and Leptospira concentration only between 10
6 and 10
8 bacteria/ml, as assessed by
absorbance (Fig. 2b) or by fluorescence (Fig. 2c).
19. We have verified that 2 h incubation at 55
C results in more
than 99.99% of Leptospira killing as measured by colonyforming unit on EMJH agar plate.
20. Do not centrifuge Leptospira at a speed above 2600 Â g as it is
generally believed that this might result in damage to
Leptospira.
21. Complete removal of EMJH medium is important as we have
observed that this medium interferes with fluorescence measurement of Syto9 and PI. We have also verified that incubating
Leptospira with 0.5% NaCl did not affect their viability. Noteworthily, Leptospira resuspended in 0.5% NaCl cannot be used
in the Alamar Blue
® Assay. Therefore, if different methods are
simultaneously used to assess Leptospira viability, bacteria will
have to be treated accordingly to each assay.
22. We generally use clear-bottom black-walled 96-well plates, and
we have obtained satisfactory results with the μClear Black
96-well plate provided by Greiner.
23. The F 530 /F 630 value exhibits a linear relationship with the
percentage of live bacteria. The linear regression by the leastsquares method gave a R square coefficient of 0.9907, indicative of a very good linearity.
24. You should expect to lose some bacteria upon washing and a
decreased bacteria concentration at the end of the washing
steps.
25. We have observed that a bacterial concentration below 10
7 Leptospira/ml is not detected by this method. We generally perform this assay with a bacterial suspension at 10
8 Leptospira/
ml.
Survival Tests for Leptospira spp.
227
incubation or storage.
17. The time for the completion of the resazurin reduction reaction depends on the number of viable cells used in the assay.
The change of blue to pink color can be seen after few hours,
but it can also require longer incubation time (up to 3 days).
Extended incubation times are not recommended to record the
results. Indeed, the resazurin reduction into resorufin is irreversible but resorufin can be further reduced into hydroresorufin, a colorless and non-fluorescent chemical [8].
18. Resazurin reduction into resorufin is detectable only if Leptospira concentration is above 10
6
/ml (Fig. 2). A good linearity is
observed between resazurin reduction and Leptospira concentration only between 10
6 and 10
8 bacteria/ml, as assessed by
absorbance (Fig. 2b) or by fluorescence (Fig. 2c).
19. We have verified that 2 h incubation at 55
C results in more
than 99.99% of Leptospira killing as measured by colonyforming unit on EMJH agar plate.
20. Do not centrifuge Leptospira at a speed above 2600 Â g as it is
generally believed that this might result in damage to
Leptospira.
21. Complete removal of EMJH medium is important as we have
observed that this medium interferes with fluorescence measurement of Syto9 and PI. We have also verified that incubating
Leptospira with 0.5% NaCl did not affect their viability. Noteworthily, Leptospira resuspended in 0.5% NaCl cannot be used
in the Alamar Blue
® Assay. Therefore, if different methods are
simultaneously used to assess Leptospira viability, bacteria will
have to be treated accordingly to each assay.
22. We generally use clear-bottom black-walled 96-well plates, and
we have obtained satisfactory results with the μClear Black
96-well plate provided by Greiner.
23. The F 530 /F 630 value exhibits a linear relationship with the
percentage of live bacteria. The linear regression by the leastsquares method gave a R square coefficient of 0.9907, indicative of a very good linearity.
24. You should expect to lose some bacteria upon washing and a
decreased bacteria concentration at the end of the washing
steps.
25. We have observed that a bacterial concentration below 10
7 Leptospira/ml is not detected by this method. We generally perform this assay with a bacterial suspension at 10
8 Leptospira/
ml.
Survival Tests for Leptospira spp.
227