3.2.4 Cesium Chloride
Gradient
Ultracentrifugation
A purification step by cesium chloride gradient ultracentrifugation
is recommended for particle characterization (TEM or mass spectrometry) but should be avoided prior to infection of bacteria
because cesium chloride is toxic to leptospires.
1. Add 45% final concentration cesium chloride to sample.
2. Ultracentrifuge between 110,000 and 260,000 Â g for 24 h at
10
C in an ultracentrifuge tube.
3. Recover formed bands with a syringe (see Note 3).
4. Dialyze the sample with TN buffer with a 3.5 kDa cassette.
3.3 Double Agar
Overlay Plaque
Lysis Assay
3.3.1 Phage Isolation and
Counting
1. Prepare Petri dishes containing 20 mL EMJH with 1.2% agar
and wait for complete solidification.
2. Prepare an equivalent number of Falcon tubes containing 5 mL
EMJH with 0.6% agar, stored in 43
C water bath to prevent
solidification before plating.
3. Incubate 500 μL of exponential phase Leptospira sp. culture
with 10 μL of different dilutions of an extract of leptophages, at
30
C for 15 min.
4. Mix these co-cultures with 5 mL EMJH with 0.6% agar, and
pour immediately over 1.2% agar EMJH plates. Work efficiently, as the overlays harden quickly.
5. Incubate at 30
C and check every 2 days for the appearance of
lysis plaques (see Fig. 2). The number of plaques formed is
proportional to the bacteriophage concentration in the extract,
considering the dilution used.
3.3.2 Fast Dropping
1. Prepare Petri dishes containing 20 mL EMJH with 1.2% agar
and allow for complete solidification.
Fig. 2 LE4 leptophage plaque lysis on Leptospira biflexa overlay
Leptophage Isolation and Characterization
71
Gradient
Ultracentrifugation
A purification step by cesium chloride gradient ultracentrifugation
is recommended for particle characterization (TEM or mass spectrometry) but should be avoided prior to infection of bacteria
because cesium chloride is toxic to leptospires.
1. Add 45% final concentration cesium chloride to sample.
2. Ultracentrifuge between 110,000 and 260,000 Â g for 24 h at
10
C in an ultracentrifuge tube.
3. Recover formed bands with a syringe (see Note 3).
4. Dialyze the sample with TN buffer with a 3.5 kDa cassette.
3.3 Double Agar
Overlay Plaque
Lysis Assay
3.3.1 Phage Isolation and
Counting
1. Prepare Petri dishes containing 20 mL EMJH with 1.2% agar
and wait for complete solidification.
2. Prepare an equivalent number of Falcon tubes containing 5 mL
EMJH with 0.6% agar, stored in 43
C water bath to prevent
solidification before plating.
3. Incubate 500 μL of exponential phase Leptospira sp. culture
with 10 μL of different dilutions of an extract of leptophages, at
30
C for 15 min.
4. Mix these co-cultures with 5 mL EMJH with 0.6% agar, and
pour immediately over 1.2% agar EMJH plates. Work efficiently, as the overlays harden quickly.
5. Incubate at 30
C and check every 2 days for the appearance of
lysis plaques (see Fig. 2). The number of plaques formed is
proportional to the bacteriophage concentration in the extract,
considering the dilution used.
3.3.2 Fast Dropping
1. Prepare Petri dishes containing 20 mL EMJH with 1.2% agar
and allow for complete solidification.
Fig. 2 LE4 leptophage plaque lysis on Leptospira biflexa overlay
Leptophage Isolation and Characterization
71