3.5.2 One-Step Growth
Kinetics
1. Grow Leptospira sp. bacterial host in EMJH flask at 30
C.
2. When the culture reaches exponential phase, standardize dilute
with EMJH to 10
8 bacteria/mL in a final volume of 10 mL.
3. Infect this culture with a final concentration of 10
7 bacteriophages per mL (MOI of 0.1).
4. Incubate at 30
C for 30 min.
5. Centrifuge the mixture at 6000 Â g for 30 min to remove
non-adsorbed phages.
6. Resuspend the supernatant in 100 mL EMJH.
7. Each 30 min, sample 1 mL of each culture or control, and filter
immediately with a 0.22 μm dead-end filter.
8. Count the lytic bacteriophages with the double agar overlay
plaque lysis assays.
9. The number of phages should remain approximately 10
6 /mL
(considering the dilution at step 6) as long as the latency period
lasts. The burst is observed when this number grows, and the
percent increase corresponds to the number of phages produced per infected cell (burst size).
4 Notes
1. To date, leptophages have only been isolated from water
sources contaminated with Leptospira species [1]. But soils
[7] and organs of infected animals should also be considered.
2. For environmental samples, ultrafiltration can handle larger
volumes than PEG purification with higher yields.
3. If no band is found, or if bacteriophages are not recovered from
the bands, a systematic sampling and TEM observation of
different layers of the gradient may be necessary.
4. The double agar overlay plaque lysis assay is required to isolate
phage clones, but samples can be pretested in a fresh liquid
culture as a preliminary assay.
5. Fast dropping is a quick counting or screening method, especially when testing numerous samples, but can only be used to
elucidate the order of magnitude of the virus concentration.
Acknowledgments
This work was part of the Ph.D. thesis of O.S. who received
financial support from “Universite ´ Paris Diderot” and “Sorbonne
Paris Cite ´.” We thank Soizick Lucas-Staat and Nicolas Dufour for
technical assistance during leptophage extractions, Olivier Gorgette
for technical assistance during the TEM observations, and Robert
A. Gaultney for proofreading the manuscript.
74
Olivier Schiettekatte and Pascale Bourhy
Kinetics
1. Grow Leptospira sp. bacterial host in EMJH flask at 30
C.
2. When the culture reaches exponential phase, standardize dilute
with EMJH to 10
8 bacteria/mL in a final volume of 10 mL.
3. Infect this culture with a final concentration of 10
7 bacteriophages per mL (MOI of 0.1).
4. Incubate at 30
C for 30 min.
5. Centrifuge the mixture at 6000 Â g for 30 min to remove
non-adsorbed phages.
6. Resuspend the supernatant in 100 mL EMJH.
7. Each 30 min, sample 1 mL of each culture or control, and filter
immediately with a 0.22 μm dead-end filter.
8. Count the lytic bacteriophages with the double agar overlay
plaque lysis assays.
9. The number of phages should remain approximately 10
6 /mL
(considering the dilution at step 6) as long as the latency period
lasts. The burst is observed when this number grows, and the
percent increase corresponds to the number of phages produced per infected cell (burst size).
4 Notes
1. To date, leptophages have only been isolated from water
sources contaminated with Leptospira species [1]. But soils
[7] and organs of infected animals should also be considered.
2. For environmental samples, ultrafiltration can handle larger
volumes than PEG purification with higher yields.
3. If no band is found, or if bacteriophages are not recovered from
the bands, a systematic sampling and TEM observation of
different layers of the gradient may be necessary.
4. The double agar overlay plaque lysis assay is required to isolate
phage clones, but samples can be pretested in a fresh liquid
culture as a preliminary assay.
5. Fast dropping is a quick counting or screening method, especially when testing numerous samples, but can only be used to
elucidate the order of magnitude of the virus concentration.
Acknowledgments
This work was part of the Ph.D. thesis of O.S. who received
financial support from “Universite ´ Paris Diderot” and “Sorbonne
Paris Cite ´.” We thank Soizick Lucas-Staat and Nicolas Dufour for
technical assistance during leptophage extractions, Olivier Gorgette
for technical assistance during the TEM observations, and Robert
A. Gaultney for proofreading the manuscript.
74
Olivier Schiettekatte and Pascale Bourhy