I have a question, I try to isolate phages from environmental samples, and during this process, I face with a clear glass tubes after overnight incubation (host+phage), but I spot nothing on the agar plate after titration process (bilayer agar method). after several days of amplification, I keep seeing the same results; clear glass tubes, no plaque on the agar. Do you have any idea what is happening?
You don't provide enough information to answer your question, here are some things we would need to know in order to help diagnose.
1) is your environmental sample actually growing? A clear tube could mean nothing grew and not that you saw lysis.
2) what indicator strain (strain used to make a lawn) are you suing for the bilayer agar plate. Remember that phages are mostly very strain specific and will only show plaques on the right strain.
3) Are you doing appropriate dilutions from the overnight? It could also be that you have so many phage you are not diluting enough to see single plaques.
Thank you Michael J. Benedik for your attention. About your first question, I have to say that yes, when I compare my bacteria+(probable) phage tube, with my bacteria control tube, I see the difference. when my bacteria control tube is turbid, my phage+bacteria tube is totally clear, meaning that lysis is actually occurring (I had this phenomenon several times so I am pretty sure that there is something in my tube that lysis the bacteria).
To answer your second question I have to say that I use the same strain as the one I use in liquid media tube. In detail, I incubate bacteria+phage in fresh liquid media (TSB in my case) and incubate them in shaker incubator overnight, then, I centrifuge and filtrate the lysate and use the same bacteria strain for spot test, but I see no plaque even though I have a clear tube after overnight incubation.
And about the dilutions, yes, I make serial dilutions, so I have 100 to 10-5 dilutions on my spot test plate respectively. I also have to mention that I use psychrophilic bacteria strain.
It is a bit worrisome that your phage growth tubes remain totally clear, usually with phage you see lysis debris. It could be you have lots of phage and are killing the cells early, or it could be there are inhibitors in your stock. A few things to try:
1) make dilution of your "probable phage" before adding to the culture tube with cells and see what happens
2) use the "probable phage" stock in spot tests on lawns of your cells. If there are enough phage to lyse the culture tube there should be enough to show plaques or lysis spots. I would also do a few dilutions of this as above.
3) Do your bilayer plates really have very nice smooth lawns so that you can see phage spots (a picture might help). Sometimes having an uneven bilayer masks whether you can see things or not.
Michael J. Benedik Thank you for your response, but I have to say that in fact I have cell debris at the bottom of my clear tube (I see them after centrifugation), meaning that the bacteria cell were dead by the effect of phage lysis. I will test your recommendation (1) to see the results, thank you. The problem is, even though there seems to be enough phage to lyse the bacteria in tube, but they do not show the plaque on agar plate (when they are confronted by the same bacteria strain, with the same growth condition as they were in the liquid tube). Also, I kind of have always good bacteria lawn on the plates, but the plaques in this especial case do not appear.
If you see debris then it does sound like you should have phage.
Could you post a picture of one of the plates, maybe someone will have an idea
Michael J. Benedik unfortunately no picture with me now, but I will post it definitly in my upcoming manipulations. thanks for your recommendation.
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