5. PHEM buffer (2Â): 60 mM PIPES, 25 mM HEPES, pH 7.0,
10 mM EGTA, 4 mM MgSO 4 .
6. Filter paper.
2.5 Leptophage
Kinetics
1. Incubator.
2. EMJH medium.
3. 50 mL flasks.
4. Fresh Leptospira cultures.
5. Petroff-Hausser counting chamber.
6. TN buffer.
7. Centrifuge.
8. 0.22 μm dead-end filter.
3 Methods
3.1 Prophage
Inductions in
Leptospira spp.
1. Grow the Leptospira sp. bacterial host in EMJH flasks at 30
C
(we suggest 400 mL).
2. Measure the optical density of the culture daily at a wavelength
of 420 nm.
3. When the optical density of the culture reaches 0.1 (3–7 days
depending on the species), divide the culture in two identical
volumes. Induce one culture with 0.05 μg/mL final concentration of mitomycin C.
4. Measure the optical density of the culture daily at a wavelength
of 420 nm. If a significant difference (>50%) in optical density
is observed between the control and the induced culture, this is
potentially due to prophage expression.
5. Bacteriophages or induced particles are stored at 4
C.
3.2 Bacteriophage
Extraction and
Purification
Two protocols exist for leptophage extraction. Tangential flow
filtration is available to extract viruses from huge volumes of environmental water (see Note 1), but, due to high density of EMJH, it
is not practical for the extraction of viruses from leptospiral lysates.
In this case, phage recovery is facilitated by polyethylene glycol
precipitation (see Note 2). If working with bacterial lysate, the
supernatant should be recovered after pelleting cells and other
debris at 6000 Â g for 45 min.
3.2.1 Tangential Flow
Filtration
1. Filter up to 2 L of environmental water with a 0.22 μm deadend filter, and recover the filtrate.
2. Concentrate this sample up to 400 times with a 100 kDa
tangential flow filtration cassette (see Fig. 1 for assembly).
Leptophage Isolation and Characterization
69
10 mM EGTA, 4 mM MgSO 4 .
6. Filter paper.
2.5 Leptophage
Kinetics
1. Incubator.
2. EMJH medium.
3. 50 mL flasks.
4. Fresh Leptospira cultures.
5. Petroff-Hausser counting chamber.
6. TN buffer.
7. Centrifuge.
8. 0.22 μm dead-end filter.
3 Methods
3.1 Prophage
Inductions in
Leptospira spp.
1. Grow the Leptospira sp. bacterial host in EMJH flasks at 30
C
(we suggest 400 mL).
2. Measure the optical density of the culture daily at a wavelength
of 420 nm.
3. When the optical density of the culture reaches 0.1 (3–7 days
depending on the species), divide the culture in two identical
volumes. Induce one culture with 0.05 μg/mL final concentration of mitomycin C.
4. Measure the optical density of the culture daily at a wavelength
of 420 nm. If a significant difference (>50%) in optical density
is observed between the control and the induced culture, this is
potentially due to prophage expression.
5. Bacteriophages or induced particles are stored at 4
C.
3.2 Bacteriophage
Extraction and
Purification
Two protocols exist for leptophage extraction. Tangential flow
filtration is available to extract viruses from huge volumes of environmental water (see Note 1), but, due to high density of EMJH, it
is not practical for the extraction of viruses from leptospiral lysates.
In this case, phage recovery is facilitated by polyethylene glycol
precipitation (see Note 2). If working with bacterial lysate, the
supernatant should be recovered after pelleting cells and other
debris at 6000 Â g for 45 min.
3.2.1 Tangential Flow
Filtration
1. Filter up to 2 L of environmental water with a 0.22 μm deadend filter, and recover the filtrate.
2. Concentrate this sample up to 400 times with a 100 kDa
tangential flow filtration cassette (see Fig. 1 for assembly).
Leptophage Isolation and Characterization
69