Streptomyces Bacteriophage plaque assay
Streptomyces bacteriophage plaque assay
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
Bacteriophage plaque assay
The bacteriophage plaque assay can be used to calculate (titer) the number of plaque-forming units (pfu) present in a sample, i.e. a phage lysate or an environmental sample suspected to contain bacteriophage.
It is also useful for the preparation of phage lysates (see the protocol, High titre preparation of phage) which can be used e.g. for generalized transduction or other experiments.
It can also be used to isolate phage from single plaques (the equivalent of working with single colonies of bacteria), as at an appropriate multiplicity of infection (MOI), each plaque will represent a single infection event. (See the protocol, Isolation of phages from single plaques.)
- Streptomyces strain sensitive to the bacteriophage
- suitable phage lysate or sample suspected to contain phage particles
- Solid DNA medium (approx. 8 plates/phage being assayed)
- Molten SNA medium (approx 20-30 mL/phage being assayed)
- Liquid DNB medium
- 1M MgSO4 solution (sterile)
- 0.8 M CaCl2 solution (sterile)
- Sterile Eppendorf tubes, pipet tips, etc.
- Pour agar plates with Difco Nutrient agar (DNA) and allow to set and dry.
- For ΦC31, the DNA plates should contain 10 mM MgSO4 and 8 mM CaCl2 (to 100 mL of medium, add 1ml of 1M MgSO4 and 1ml of 0.8M CaCl2) - see note 1.
- Melt Difco soft nutrient agar (SNA) and store in 50°C water bath. Work carefully with the molten SNA: if it is too hot, it will kill some of the Streptomyces spores; if it is too cold, it will solidify.
- Dilute phage suspension (10-fold serial dilution) using Difco nutrient broth (DNB): 100-108 should cover most preparations of phage from high-titre lysates through to enriched environmental samples).
- Pipette 0.1 ml of each phage sample onto the centre of an agar plate.
- Add spores to the SNA to give a slightly turbid suspension (roughly around 0.1 mL of 1 x 108 spore stock per 10 mL of SNA) – it is often useful to do this in universal bottles as it makes pouring on to plates easier.
- Add 2-3ml of the SNA/spore mixture to the surface of the plate and swirl immediately to cover the whole plate. Leave to set for 10-15 minutes.
- Incubate the plates overnight at the required temperature (usually 30°C).
- The following morning, inspect the plates for plaques. Plaques can be visible within 10-12 hours, be careful not to allow the plates to overgrow as some lytic phage plaques may appear turbid and temperate phage (turbid plaques) ma be lost to overgrowth.
- Proceed to the High titre preparation of phage or Isolation of phages from single plaques protocol.
- Each phage with have an optimal divalent cation requirement for propagation – this is generally in the range of 0-10 mM MgSO4 and 8-25 mM CaCl2 but will need to be optimised for each phage. For isolation of novel phages, the standard ΦC31 parameters are suitable for isolation.
Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.