Isolation of phages from single plaques

Isolation of phages from single plaques

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

• Streptomyces coelicolor with ΦC31
• Streptomyces venezuelae with ΦSV1

Isolation of phages from single plaques

It is often very useful to isolate phage from single plaques (this is the equivalent of working with single colonies of bacteria). At an appropriate multiplicity of infection (MOI), each plaque will represent a single infection event (with all phage progeny being, in theory at least, genetically identical to the "parent" phage.)

Thus, when isolating novel phage from the environment, it is essential to go through at least one round of isolation from a single plaque.

Protocol

Materials needed:

1. Plates containing the bacteriophage grown on a permissive host strain (see either Streptomyces Bacteriophage plaque assay or Isolation of phages from environmental samples)
2. Liquid DNB medium
3. 0.45µm syringe filter
4. Sterile yellow pipette tips, Bijoux bottles, etc.

Instructions:

1. The bacteriophage of choice should be grown on a permissive host strain, following either the enrichment procedure or bacteriophage plaque assay protocol.
2. Identify the plaque(s) you wish to isolate. Ideally, these should be well-separated from any other plaques.
3. Using a ‘yellow pipette tip’ pick the plaque from the SNA overlay and place it into 1 mL of DNB broth in a Bijoux bottle.
4. Shake at room temperature for 2 hrs.
5. Filter sterilise using a 0.45 µm syringe filter, and store at 4°C.
6. This phage lysate can be used to generate high-titre preparations and for host range studies.

Notes

1. The phage lysates remain viable for long periods when stored at 4°C (I have recovered phage from filtrates ~10 years old!).
2. It is not necessary to add chloroform to the phage lysates.

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