High titre preparation of phage

High titre preparation of Streptomyces phage

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

• Streptomyces coelicolor with ΦC31, ΦBT1, ΦHau3, ΦR4
• Streptomyces lividans with ΦC31, ΦBT1, ΦHau3, ΦR4
• Streptomyces venezuelae with ΦSV1

High titre preparation of phage

High titre preparation of phage (phage lysates) can be used for any experiment requiring specific bacteriophages (e.g. for phage typing of strains, generalized transduction or any other experiments).

A single plate (from a Streptomyces Bacteriophage plaque assay) generates several millilitres of a high titre (~10⁸ pfu/mL) phage lysate.

Protocol

Materials needed:

1. Plates containing the bacteriophage grown on a permissive host strain (see Streptomyces bacteriophage plaque assay)
2. Liquid DNB medium
3. 0.45µm syringe filter

Instructions:

1. Take plates from the Streptomyces Bacteriophage plaque assay
2. Take the dilution(s) that show almost confluent lysis (You should still be able to distinguish the shapes of individual plaques, but the plate should display as much lysis as possible - to harvest the maximum number of phage. Often it pays to look at the edges of the plates where it looks like there is no growth of the indicator strain – these often display confluent lysis).
3. Flood the plates exhibiting almost confluent lysis with 5 ml of Difco Nutrient broth (DNB), and leave at room temperature for 2 hrs. Plates can be gently rocked to increase the phage titre.
4. Pour the broth off the plate into a Bijoux bottle (or other suitable container).
5. Filter the DNB through a 0.45 µm syringe filter. The resulting filtrate is your high-titre phage preparation and should be around 10⁸ pfu/mL.
6. This phage preparation (phage lysate) should be stored at 4°C.

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.