Dear all, I have been purifying phages for nearly two months and have made no progress, which is frustrating. I obtained some phage mixtures from Klebsiella pneumoniae. I used the phage plaque assay to purify the phages. Every time, I used the 10 uL tip to pick up one PFU and dip it into the SM buffer. I then serially dilute this buffer and add host bacteria to each dilution. After incubation overnight, I chose the PFU that separated the most from different phages and repeated the plaque assay to further purify that phage, but after two months, the plate still contains at least three different phages!! And from what I've heard, it normally only takes a maximum of 1 week to purify the phage. I tried to purify several phages at a time. I'm not sure if this is the problem. Is it because of the cross-contamination?
Can someone please assist me in resolving this issue?
First question is how do you know that you are still getting 3 different phages? Maybe these are just plaque variants of the same phage. If you have done more than 2-3 rounds of plaque isolation (and the plaques are at least 1cm from the neighbors) then you should have a pure stock.
However it could be true that you are introducing contaminants. You may wish to clean your pipets and be sure that you are using barrier tips otherwise you will get phage in the pipet barrel.
Also you may wish to use a freshly autoclaved bottle of SM buffer, sometime you can get contamination spreading from the buffer.
Michael J. Benedik Dear Professor Benedik, Thank you for your insightful response. Regarding your question, I thought the same phage would only cause one type of plaque. And I saw three different plaque sizes on one plate. That is why I thought I was still receiving three different phages? I had no idea that even one phage could produce plaques of varying sizes. I think my next step would be to use TEM and sequencing to characterise them in order to determine whether they are the same phage.
I would suggest something much simpler. Pick one from each of the 3 types of plaques you see. Make serial dilutions and plate them out for single plaques. Then if all 3 produce the same 3 plaque types you will know they were the same. Then you can just pick one and proceed.
I would add that at the same time you can do a negative control experiment to show that it is not some contamination. Pick a plug from a section of the lawn where there are no plaques and treat it identically to the plaques and try to titer that. You should see no phage plaques at any dilution with that. If you do see plaques then you have a contamination issue.
Michael J. Benedik Dear Professor Benedik. This is a really good point, something I never considered. I will try your easier techniques to see if my phages are sufficiently pure.
hello dear. in my opinion, if you are picking on plaque and you are purifying the same plaque many times I think it is purified phage. plaque size depends upon many factors you cannot decide on plaque size. furthermore, if you further want to confirm you can use restriction enzymes, sequencing, or electron microscope examination
Muhammad Shahzad Rafiq Dear Muhammad, many thanks for your insightful suggestions.
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