Isolation of phages from environmental samples

Isolation of phages from environmental samples

This protocol has been confirmed to work for the following organisms:

• Streptomyces lividans strain TK24
• Streptomyces coelicolor strain J1929 (pgl^- strain)

Isolation of phages from environmental samples

It is often useful to isolate bacteriophage from environmental samples, to study bacteriophage diversity or to isolate new phages that can be used as genetic tools (i.e. for generalized transduction with a specific Streptomyces host strain or for isolating new phage recombinases to develop useful plasmids.)

This protocol allows for the enrichment of bacteriophage that are able to infect a specific Streptomyces indicator strain (see note 1). Note that any Streptomyces phage unable to infect the indicator strain, will not be enriched, so this protocol will not offer a comprehensive survey of all Streptomyces phage present in the environmental sample.

Protocol

Strains needed:

1. suitable Streptomyces indicator strain(s)

Materials needed:

1. Soil or environmental sample from which you wish to isolate bacteriophage
2. Liquid DNB medium
3. 0.45µm syringe filter

Instructions: Enrichment -

1. Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
2. Add 500 µl of a dense spore preparation (~1 x 10⁹) from a Streptomyces indicator strain of your choice - see note 1.
3. Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your Streptomyces indicator strain.
4. After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.
5. Decant the enriched DNB and filter through a 0.45 µm syringe filter.
6. The resulting filtrate can be used in the standard Streptomyces Bacteriophage plaque assay, using the same Streptomyces indicator strain, to isolate new phage.

Notes

1. Often host range is a key parameter in phage isolation, so it is worth looking at a number of strains. Streptomyces lividans TK24 has been used extensively in the past.
2. New phage should be purified using the Isolation of phages from single plaques protocol.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.