7. Observe the grid with a transmission electron microscope at a
magnification of 23,000 for screening and 49,000 for characterization (see Fig. 3b).
3.5 Leptophage
Kinetics
3.5.1 Phage Adsorption
Assays
1. Grow Leptospira sp. bacterial host in EMJH flask at 30
C.
2. When the culture reaches exponential phase, dilute the bacteria
to 10
8 /mL in EMJH.
3. Infect this culture with a final concentration of 10
4 bacteriophages per mL (MOI of 0.0001) or with the same volume of
buffer (control culture).
4. Prepare a control dilution of the bacteriophage at 10
4 bacteriophages per mL (without bacteria).
5. Sample 1 mL of the co-culture every 30 min for up to 6 h, and
filter immediately with a 0.22 μm dead-end filter. Sample 1 mL
of control culture and control dilution at times 0 and 6 h, and
filter with a 0.22 μm dead-end filter.
6. Count the lytic bacteriophages from this sample with the double agar overlay plaque lysis assay (see Subheading 3.3).
7. The number of phages found in the non-infected control culture should stay at 0, and the concentration of phages in the
control dilution should remain close to 10
4 /mL and corresponds to the percent lost in the filter.
8. In the experimental co-culture, the number of recovered
phages should decrease until the first burst. This decrease
corresponds to the proportion of phages adsorbed on host
cells (and retained in the 0.22 μm filter).
Fig. 3 LE4 bacteriophages (a) in a lysate and (b) with the bacterial host
Leptophage Isolation and Characterization
73
magnification of 23,000 for screening and 49,000 for characterization (see Fig. 3b).
3.5 Leptophage
Kinetics
3.5.1 Phage Adsorption
Assays
1. Grow Leptospira sp. bacterial host in EMJH flask at 30
C.
2. When the culture reaches exponential phase, dilute the bacteria
to 10
8 /mL in EMJH.
3. Infect this culture with a final concentration of 10
4 bacteriophages per mL (MOI of 0.0001) or with the same volume of
buffer (control culture).
4. Prepare a control dilution of the bacteriophage at 10
4 bacteriophages per mL (without bacteria).
5. Sample 1 mL of the co-culture every 30 min for up to 6 h, and
filter immediately with a 0.22 μm dead-end filter. Sample 1 mL
of control culture and control dilution at times 0 and 6 h, and
filter with a 0.22 μm dead-end filter.
6. Count the lytic bacteriophages from this sample with the double agar overlay plaque lysis assay (see Subheading 3.3).
7. The number of phages found in the non-infected control culture should stay at 0, and the concentration of phages in the
control dilution should remain close to 10
4 /mL and corresponds to the percent lost in the filter.
8. In the experimental co-culture, the number of recovered
phages should decrease until the first burst. This decrease
corresponds to the proportion of phages adsorbed on host
cells (and retained in the 0.22 μm filter).
Fig. 3 LE4 bacteriophages (a) in a lysate and (b) with the bacterial host
Leptophage Isolation and Characterization
73