I have conducted plaque assay to know the concetration of MS2 (a type of phage).
The host was E. coli (strain K12), and the sample was PBS spiked with MS2.
After dilution of the sample, I incubated at 37 C over night.
Then, I found a lot of white colonies instead of plaques (picture).
Since the agar was almost transparent, I thought the concentraion of the phage was too high and most E. coli were eaten. Some survivors started to form the colonies.
Is my assumption correct?
There are so many possibilities that it is difficult to form an opinion without further information.
It is possible that you've added too many phage, but there are other reasons which could account for your results. What did the lawn of bacteria look like in the negative control (i.e. where no phage were added)? What strain of E. coli K12 did you use - did it contain the F plasmid? Did you do a dilution series of the virus? Did you use a soft agar overlay?
I agree with Vincent Mulholland , there are many possible reasons. You do need to do a dilution series if you really want to count plaques and get an accurate CFU/ml. But from the picture it looks like most the cells were killed, probably too many phage or too hot top agar. The small colonies are probably either resistant cells or contaminants (which by definition are also resistant cells, just not E. coli).
You need to make dilution.. it looks like an over dose of phages was put in the soft agar..
the white colonies perhaps resistant.
Dear all experts,
I appologize for my late reply.
Based on all of the kind advice, I found the problem happend on the host cells.
I cultured them too long; they passed the exponential growth phase and lost F pili.
After I controlled the enrichment time, the experiment went well.
With your comments, I was able to deepened my understanding about the plaque assay.
I was impressed for all of the answers.
Thank you very much!
Glad that you solved the problem. However strains do not lose the F plasmid very easily, in fact it is really hard to cure an E. coli strain of F. So even if the culture had grown a bit too long it would not have lost F. I suspect the problem was something else entirely, but the good news is that if you solved it then it doesn't matter so much what it was.
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