2.2 Determination
of Colony-Forming Unit
1. Safety cabinet level 2.
2. P1000, P200, P20 micropipettes and sterile tips.
3. Sterile 1.5 ml polypropylene tubes.
4. EMJH medium (see Subheading 2.1).
5. Concentrated EMJH medium: dissolve 2.3 g of Difco Leptospira medium base EMJH (Becton Dickenson) in 560 ml sterile
WFI. Add 100 ml of albumin supplement. Adjust the pH to 7.5
and filter sterilize the solution.
6. Agar solution: Dissolve 12 g of agar noble in 340 ml of sterile
WFI. Autoclave the solution.
7. Solid EMJH agar medium: melt 340 ml of agar noble solution
and keep it at 50–55
C in a water bath. Pre-warm 660 ml of
concentrated EMJH medium at 50–55
C. Mix the agar noble
solution with the concentrated EMJH medium. Pour
25–30 ml of the solution into petri plates (see Note 2).
8. 90 mm  14.2 mm petri dishes.
9. Sterile inoculation loop or glass spreader or glass beads.
10. Microbiological incubator at 30
C.
11. Aluminum foil.
12. Parafilm.
2.3 Measurement
of Resazurin Reduction
by the Alamar
Blue
® Assay
1. Safety cabinet level 2.
2. P1000, P200, P20 micropipettes and sterile tips.
3. 10Â resazurin solution (Alamar Blue
® Assay).
4. EMJH medium (see Subheading 2.1).
5. Sterile 96-well flat bottom microplates for absorbance or fluorescence reading.
6. Microplate reader for absorbance or fluorescence reading.
7. Plastic storage box (to accommodate 96-well microplates).
8. Microbiological incubator at 30
C.
2.4 DNA Staining
with Syto9 and PI
1. Safety cabinet level 2.
2. P1000, P200, P20 micropipettes and sterile tips.
3. Sterile 15 ml conical polypropylene tubes.
4. Sterile 1.5 ml polypropylene tubes.
5. 10 ml disposable serological sterile plastic pipettes.
6. Sterile ultrapure H 2 O.
7. 0.5% NaCl solution: 0.5% NaCl in ultrapure sterile H 2 O.
8. Water bath at 55
C.
9. Centrifuge with rotor for 2600 Â g.
220
Cle ´ mence Mouville and Nadia Benaroudj