Streptomyces Bacteriophage plaque assay: Difference between revisions
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# Solid [[DNA]] medium | # Solid [[DNA]] medium | ||
# Molten [[SNA]] medium | # Molten [[SNA]] medium | ||
# Liquid [[DNB]] medium | |||
#1M MgSO<sub>4</sub> solution (sterile) | #1M MgSO<sub>4</sub> solution (sterile) | ||
#0.8 M CaCl<sub>2</sub> solution (sterile) | #0.8 M CaCl<sub>2</sub> solution (sterile) | ||
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*Melt Difco soft nutrient agar ([[SNA]]) and store in 50oC water bath. | *Melt Difco soft nutrient agar ([[SNA]]) and store in 50oC water bath. | ||
*Dilute phage suspension (10-fold serial dilution) using Difco nutrient broth (DNB | *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). | ||
*Pipette 0.1 ml of each phage sample | *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 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 | *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 30oC). | *Incubate the plates overnight at the required temperature (usually 30oC). | ||
Revision as of 09:17, 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
- Molten SNA medium
- 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)
- Melt Difco soft nutrient agar (SNA) and store in 50oC water bath.
- 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 30oC).
- 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 plauques) ma be lost to overgrowth. Proceed to the high-titre phage stock preparation or single plaque isolation.
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 C31 parameters are suitable for isolation.
Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.
