Phage Isolation and Purification Protocol
Version not yet assigned, updated 2026-09-30. https://dustinedwards.info/research/protocols/phage-isolation
- Updated
- 2026-09-30
- Host strain
- Mycobacterium smegmatis mc²155 (ATCC 700084); Microbacterium foliorum NRRL B-24224
Reagents
| Reagent | Amount | Notes |
|---|---|---|
| host culture | 250 µl per plate | |
| filtrate (Direct isolation) | 500 µl | Delivered as 5 x 100 µl with a p200, or with a 5 ml pipette. |
| soil (Enriched isolation) | 10 ml | |
| PYCa (Enriched isolation) | 25 ml | 25 ml PYCa to the 35 ml mark, vortex 1 minute. |
| host added to the filtrate (Enriched isolation) | 250 µl | |
| phage buffer (Purification) | 90 µl or 100 µl | |
| phage buffer (Flooding) | 8 ml per plate |
Equipment
55 °C water bath for molten top agar; plate incubator at 29 °C for M. foliorum; shaking incubator at 220 to 250 rpm; 0.22 µm tube-top vacuum filter units; 1 to 10 µl pipettor for spot titers; light box for counting plaques
Webbed plates and the high titer lysate
Flood the webbed plate from the last purification round to make a low-volume lysate (about 5 ml).
Run a dilution series from the LVL and plate it to find the dilution that webs.
Troubleshooting: Web copies failed.
Pour 5 to 10 replicate plates at that dilution and volume.
Flood them, pool them through one 0.22 µm filter into a 50 ml conical, and titer the pool. That is the HVL.
Plate a 10-fold series of the lysate across whole decades (for example 10^-1 to 10^-4, or 10^-3 to 10^-6), or bracket 10-fold either side of the calculated volume.
See which dilution webbed.
Plate 6 to 8 replicates of exactly that dilution and volume.
Troubleshooting
| Step | Problem | Possible reason | Solution |
|---|---|---|---|
| 2 to 3 | Web copies failed. | They were made from the wrong starting sample instead of the LVL. | Make the web copies from the LVL, as in steps 2 and 3. |