BacLight
® assays are not laborious and well-adapted to highthroughput studies, but their sensitivity and linearity are limited
to a short range of bacterial concentrations. We therefore would
like to emphasize that accurate and meaningful bacterial viability
assessment might require, under some circumstances, validation by
two different approaches.
2 Materials
2.1 Leptospira
Cultivation
1. Safety cabinet level 2.
2. EMJH albumin supplement: 10% (w/v) bovine serum albumin, 0.004% (w/v) zinc sulfate, 0.015% (w/v) magnesium
chloride, 0.015% (w/v) calcium chloride, 0.1% (w/v) sodium
pyruvate, 0.4% (w/v) glycerol, 1.25% (v/v) Tween
80, 0.0002% (w/v) vitamin B12, 0.05% (w/v) ferrous sulfate
(added at the last moment) in sterile water for injection (WFI)
(see Note 1).
3. EMJH base: dissolve 2.3 g of Difco Leptospira medium base
EMJH (Becton Dickenson) in 900 ml sterile WFI. Autoclave
the solution.
4. EMJH medium: add 100 ml of EMJH albumin supplement to
900 ml of EMJH base. Adjust the pH to 7.5 and filter sterilize
the solution.
5. Spectrophotometer.
6. Disposable serological plastic pipettes.
7. Culture flasks and/or tubes.
8. Incubator with orbital shaker at 30
C.
9. Dark-field microscope.
10. Petroff-Hausser counting chamber.
Fig. 3 Calculation of percentage of resazurin reduction. This formula is used to calculate the percentage of
resazurin reduction with E oxi 600, the molar extinction coefficient of oxidized Alamar Blue at 600 nm (117216);
E oxi 570, the molar extinction coefficient of oxidized Alamar Blue at 570 nm (80586); E red 600, the molar
extinction coefficient of reduced Alamar Blue at 600 nm (14652); E red 570, the molar extinction coefficient of
reduced Alamar Blue at 570 nm (155677); A 570 , absorbance of the tested well at 570 nm; A 600 , absorbance of
the tested well at 600 nm; C 570 , absorbance of the negative control well at 570 nm; and C 600 , absorbance of
the negative control well at 600 nm
Survival Tests for Leptospira spp.
219
® assays are not laborious and well-adapted to highthroughput studies, but their sensitivity and linearity are limited
to a short range of bacterial concentrations. We therefore would
like to emphasize that accurate and meaningful bacterial viability
assessment might require, under some circumstances, validation by
two different approaches.
2 Materials
2.1 Leptospira
Cultivation
1. Safety cabinet level 2.
2. EMJH albumin supplement: 10% (w/v) bovine serum albumin, 0.004% (w/v) zinc sulfate, 0.015% (w/v) magnesium
chloride, 0.015% (w/v) calcium chloride, 0.1% (w/v) sodium
pyruvate, 0.4% (w/v) glycerol, 1.25% (v/v) Tween
80, 0.0002% (w/v) vitamin B12, 0.05% (w/v) ferrous sulfate
(added at the last moment) in sterile water for injection (WFI)
(see Note 1).
3. EMJH base: dissolve 2.3 g of Difco Leptospira medium base
EMJH (Becton Dickenson) in 900 ml sterile WFI. Autoclave
the solution.
4. EMJH medium: add 100 ml of EMJH albumin supplement to
900 ml of EMJH base. Adjust the pH to 7.5 and filter sterilize
the solution.
5. Spectrophotometer.
6. Disposable serological plastic pipettes.
7. Culture flasks and/or tubes.
8. Incubator with orbital shaker at 30
C.
9. Dark-field microscope.
10. Petroff-Hausser counting chamber.
Fig. 3 Calculation of percentage of resazurin reduction. This formula is used to calculate the percentage of
resazurin reduction with E oxi 600, the molar extinction coefficient of oxidized Alamar Blue at 600 nm (117216);
E oxi 570, the molar extinction coefficient of oxidized Alamar Blue at 570 nm (80586); E red 600, the molar
extinction coefficient of reduced Alamar Blue at 600 nm (14652); E red 570, the molar extinction coefficient of
reduced Alamar Blue at 570 nm (155677); A 570 , absorbance of the tested well at 570 nm; A 600 , absorbance of
the tested well at 600 nm; C 570 , absorbance of the negative control well at 570 nm; and C 600 , absorbance of
the negative control well at 600 nm
Survival Tests for Leptospira spp.
219