2. Add 5 mL EMJH with 0.6% agar as an overlay and wait for
complete solidification. Store dishes at 4
C up to 1 month.
3. Spread 1 mL of exponential phase Leptospira sp. culture on a
room temperature overlay medium.
4. Incubate at 30
C for 2 h.
5. Remove any remaining liquid with a micropipette.
6. Leave the dish open under a sterile biosafety cabinet up to
15 min to allow the small volume of remnant liquid to dry.
7. (a) For screening: Drop 10 μL of each purified phage sample to
test for clearing.
(b) For raw counting: Drop 10 μL of serial dilutions of the
bacteriophage extract to titer.
8. Incubate at 30
C and check every 2 days for the appearance of
lysis plaques. The number of plaques is proportional to the
concentration of bacteriophages in the extract, considering
the dilution used (see Note 5).
3.4 Leptophage
Transmission Electron
Microscopy
3.4.1 Induced Particle
Morphological
Characterization
1. Deposit a carbon-coated copper grid on a 15 μL drop of phage
sample for 15 min.
2. Deposit the grid on a 30 μL drop of 4% uranyl acetate solution
for 3 s for washing and staining.
3. Deposit the grid on another 30 μL drop of 4% uranyl acetate
solution for 8 s for staining.
4. Dry the grid with a filter paper; take care to remove any
remnant liquid.
5. Observe the grid with a transmission electron microscope at a
magnification of 23,000 for screening and 49,000 for characterization (see Fig. 3a).
3.4.2 Infected Bacteria
Observation
1. Deposit a carbon-coated copper grid on a 15 μL drop of phageinfected Leptospira culture (we suggest a MOI of 10) at the
desired infection time, for 15 min.
2. Deposit the grid on a 15 μL drop of 2% glutaraldehyde in
PHEM buffer for 10 min.
3. Deposit the grid successively on four 30 μL drops of distilled
water for washing.
4. Deposit the grid on a 30 μL drop of 4% uranyl acetate solution
for 3 s for washing and staining.
5. Deposit the grid on another 30 μL drop of 4% uranyl acetate
solution for 8 s for staining.
6. Dry the grid with a filter paper; take care to remove any
remnant liquid.
72
Olivier Schiettekatte and Pascale Bourhy
complete solidification. Store dishes at 4
C up to 1 month.
3. Spread 1 mL of exponential phase Leptospira sp. culture on a
room temperature overlay medium.
4. Incubate at 30
C for 2 h.
5. Remove any remaining liquid with a micropipette.
6. Leave the dish open under a sterile biosafety cabinet up to
15 min to allow the small volume of remnant liquid to dry.
7. (a) For screening: Drop 10 μL of each purified phage sample to
test for clearing.
(b) For raw counting: Drop 10 μL of serial dilutions of the
bacteriophage extract to titer.
8. Incubate at 30
C and check every 2 days for the appearance of
lysis plaques. The number of plaques is proportional to the
concentration of bacteriophages in the extract, considering
the dilution used (see Note 5).
3.4 Leptophage
Transmission Electron
Microscopy
3.4.1 Induced Particle
Morphological
Characterization
1. Deposit a carbon-coated copper grid on a 15 μL drop of phage
sample for 15 min.
2. Deposit the grid on a 30 μL drop of 4% uranyl acetate solution
for 3 s for washing and staining.
3. Deposit the grid on another 30 μL drop of 4% uranyl acetate
solution for 8 s for staining.
4. Dry the grid with a filter paper; take care to remove any
remnant liquid.
5. Observe the grid with a transmission electron microscope at a
magnification of 23,000 for screening and 49,000 for characterization (see Fig. 3a).
3.4.2 Infected Bacteria
Observation
1. Deposit a carbon-coated copper grid on a 15 μL drop of phageinfected Leptospira culture (we suggest a MOI of 10) at the
desired infection time, for 15 min.
2. Deposit the grid on a 15 μL drop of 2% glutaraldehyde in
PHEM buffer for 10 min.
3. Deposit the grid successively on four 30 μL drops of distilled
water for washing.
4. Deposit the grid on a 30 μL drop of 4% uranyl acetate solution
for 3 s for washing and staining.
5. Deposit the grid on another 30 μL drop of 4% uranyl acetate
solution for 8 s for staining.
6. Dry the grid with a filter paper; take care to remove any
remnant liquid.
72
Olivier Schiettekatte and Pascale Bourhy