Feb 08, 2016

Public workspaceObtaining pure cyanophage stocks (plaque purification)

DOI
dx.doi.org/10.17504/protocols.io.dqq5vv
  • Mathias Middelboe1,
  • Amy M. Chan1,
  • and Sif K. Bertelsen1
  • 1Manual of Aquatic Viral Ecology
  • VERVE Net
  • Suttle Laboratory of Marine Molecular Microbiology and Virology
Amy Chan
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.dqq5vv
External link: http://www.aslo.org/books/mave/MAVE_118.pdf
Protocol Citation: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen 2016. Obtaining pure cyanophage stocks (plaque purification). protocols.io https://dx.doi.org/10.17504/protocols.io.dqq5vv
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 02, 2015
Last Modified: November 09, 2017
Protocol Integer ID: 1520
Make a dilution series of the lysate (assume 104 to 105 PFU per mL in the plaque lysate)
Note
Use this to perform a second round of plaque assays to purify the phage.
Repeat the plaque purification procedure 2 more times to ensure that the cyanophage isolated is clonal.
Repeat the plaque purification procedure again.
Finally, prepare a primary cyanophage stock using lysate from the final purification via method A "Liquid Amplification" OR method B "Plate Amplification"
Method A: liquid amplification
Protocol
Liquid Amplification
NAME
Liquid Amplification
CREATED BY
Amy Chan
Add some of the lysate to target host in liquid culture.
After the culture has lysed, remove cell debris via centrifugation.
Filter sterilize the stock.
Store at 4°C until further analysis.
Method B: plate amplification
Protocol
Plate Amplification
NAME
Plate Amplification
CREATED BY
Amy Chan
Prepare plaque assays with a dilution series of lysate from the final purification.
Note
Plates with confluent lysis of the host lawn (typically ca. 104 PFUs) can then be used to obtain cyanophage stocks by elution of phages from the plates.
Add 5 mL sterile seawater to the plate.
Scrape off the top agar layer into the seawater.
Leave at 4°C overnight.
Duration18:00:00
Remove agar and cell debris by centrifugation.
Filter sterilize the stock.
Store at 4°C until further analysis.
Titer the final stock via plaque assay.
Cyanophage stocks stored at 4°C in the dark are stable for at least a year.