Phage Isolation and Purification Protocol

Version not yet assigned, updated 2026-09-30. https://dustinedwards.info/research/protocols/phage-isolation

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Updated: 2026-09-30
Host strain: Mycobacterium smegmatis mc²155; Microbacterium foliorum NRRL B-24224

Reagents

Reagent	Amount	Source	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	Prepared in the lab	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	Prepared in the lab
phage buffer (Flooding)	8 ml per plate	Prepared in the lab

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 do not web.
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 do not web.	They were made from a sample other than the LVL.	Make the web copies from the LVL, as in steps 2 and 3.