3. Add 10% final volume of chloroform to eliminate remnant cell
contaminants and mix gently.
4. Centrifuge at 1700 Â g for 30 min at 4
C to pellet chloroforminsoluble material.
5. Filter the supernatant with a 0.22 μm dead-end filter.
3.2.2 Polyethylene Glycol
Precipitation
1. Filter 200 mL of leptospiral lysate (see Subheading 3.1) with a
0.22 μm dead-end filter.
2. Add 8% polyethylene glycol 8000 and 0.3 M NaCl (final
concentrations).
3. Mix thoroughly to dissolve polyethylene glycol.
4. Incubate overnight at 4
C.
5. Centrifuge at 24,000 Â g for 45 min at 4
C.
6. Solubilize the pellet in a minimal volume of TN buffer for
higher yields, and we recommend 5 mL.
3.2.3 Phage
Amplification
1. Grow Leptospira sp. bacterial host in EMJH flasks at 30
C
(we suggest 400 mL).
2. When the optical density of the culture reaches 0.2, divide the
culture in two identical volumes. Induce one culture with 5 mL
of previously extracted phage (see Subheading 3.2.1 or 3.2.2)
and the other culture with the same volume of TN buffer
(control culture).
3. 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 phage amplification.
4. Repeat the PEG extraction and/or isolate phages with double
agar overlay plaque lysis assay (see Subheading 3.3).
Fig. 1 Tangential flow filtration assembly
70
Olivier Schiettekatte and Pascale Bourhy
contaminants and mix gently.
4. Centrifuge at 1700 Â g for 30 min at 4
C to pellet chloroforminsoluble material.
5. Filter the supernatant with a 0.22 μm dead-end filter.
3.2.2 Polyethylene Glycol
Precipitation
1. Filter 200 mL of leptospiral lysate (see Subheading 3.1) with a
0.22 μm dead-end filter.
2. Add 8% polyethylene glycol 8000 and 0.3 M NaCl (final
concentrations).
3. Mix thoroughly to dissolve polyethylene glycol.
4. Incubate overnight at 4
C.
5. Centrifuge at 24,000 Â g for 45 min at 4
C.
6. Solubilize the pellet in a minimal volume of TN buffer for
higher yields, and we recommend 5 mL.
3.2.3 Phage
Amplification
1. Grow Leptospira sp. bacterial host in EMJH flasks at 30
C
(we suggest 400 mL).
2. When the optical density of the culture reaches 0.2, divide the
culture in two identical volumes. Induce one culture with 5 mL
of previously extracted phage (see Subheading 3.2.1 or 3.2.2)
and the other culture with the same volume of TN buffer
(control culture).
3. 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 phage amplification.
4. Repeat the PEG extraction and/or isolate phages with double
agar overlay plaque lysis assay (see Subheading 3.3).
Fig. 1 Tangential flow filtration assembly
70
Olivier Schiettekatte and Pascale Bourhy