Streptomyces Bacteriophage plaque assay: Difference between revisions
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Materials needed: | Materials needed: | ||
# Solid [[DNA]] medium | # Solid [[DNA]] medium (approx. 8 plates/phage being assayed) | ||
# Molten [[SNA]] medium | # Molten [[SNA]] medium (approx 20-30 mL/phage being assayed) | ||
# Liquid [[DNB]] medium | # Liquid [[DNB]] medium | ||
#1M MgSO<sub>4</sub> solution (sterile) | #1M MgSO<sub>4</sub> solution (sterile) | ||
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*Pour agar plates with Difco Nutrient agar ([[DNA]]) and allow to set and dry. | *Pour agar plates with Difco Nutrient agar ([[DNA]]) and allow to set and dry. | ||
**For ΦC31, the [[DNA]] plates should contain 10 mM MgSO<sub>4</sub> and 8 mM CaCl<sub>2</sub> (to 100 mL of medium, add (1ml of 1M MgSO<sub>4</sub> and 1ml of 0.8M CaCl<sub>2</sub>) | **For ΦC31, the [[DNA]] plates should contain 10 mM MgSO<sub>4</sub> and 8 mM CaCl<sub>2</sub> (to 100 mL of medium, add (1ml of 1M MgSO<sub>4</sub> and 1ml of 0.8M CaCl<sub>2</sub>) - see note. | ||
*Melt Difco soft nutrient agar ([[SNA]]) and store in | *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]]): 10<sup>0</sup>-10<sup>8</sup> should cover most preparations of phage from high-titre lysates through to enriched environmental samples). | *Dilute phage suspension (10-fold serial dilution) using Difco nutrient broth ([[DNB]]): 10<sup>0</sup>-10<sup>8</sup> should cover most preparations of phage from high-titre lysates through to enriched environmental samples). | ||
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*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. | *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 | *Incubate the plates overnight at the required temperature (usually 30°C). | ||
*The following morning inspect the plates for plaques | *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. | |||
====Notes==== | ====Notes==== | ||
#Each phage with have an optimal divalent cation requirement for propagation – this is generally in the range of 0-10 mM | #Each phage with have an optimal divalent cation requirement for propagation – this is generally in the range of 0-10 mM MgSO<sub>4</sub> and 8-25 mM CaCl<sub>2</sub> but will need to be optimised for each phage. For isolation of novel phages, the standard ΦC31 parameters are suitable for isolation. | ||
Revision as of 09:24, 5 May 2020
Streptomyces bacteriophage plaque assay
This protocol has been confirmed to work for the following organisms:
- Streptomyces coelicolor with ΦC31
- Streptomyces venezuelae with ΦSV1
Bacteriophage plaque assay
Protocol
Strains needed:
- Streptomyces strain sensitive to the bacteriophage
Materials needed:
- 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)
- 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.
- 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 – 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.
Notes
- 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.
