I conducted a plaque assay using the double agar overlay method for 46 hours, but wasn't sure if this was long enough for the phages to maximize their plaque frequency and diameter. The strain of phage used was T4r and the type of bacteria was E. Coli strain B. Is it possible that the number/diameter of plaques observed would have increased if they were incubated for a longer period of time? Some literature I have read has recommended a five day incubation period.
Hey Ritvik Viniak,
Plague size depends on the phage more than the incubation period. Usually, you can achieve the best plague size in 24-48 hours of incubation until your phage has a long latent period. From my experience, a Longer period of incubation will diminish your plague. I would recommend using soft agar with (.4-.6% ) and a bit higher salt concentration to ease the infection.
Cheers!
I would say the for E. coli B and T4 phage you should see the plaques at their maximum just overnight, 8-12 hours, assuming you are putting the plates at 37C. Slightly longer if incubating at lower temperatures but not more than 24 hrs. Once the lawn is fully formed then the cells are no longer growing and you won't get new plaques with most phages. Longer incubation might be needed for some slow growing bacteria but not E. coli.
Hi all,
Yes, indeed, overnight incubation is sufficient for most phage/bacteria couples that currently seem to be relevant for humans ;)
Very important is to use a good protocol. It will reveal more than the number of active phages.
Article Enumeration of Bacteriophages by Double Agar Overlay Plaque Assay
Best wishes,
Jean-Paul
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