I was doing an overlay for bacteriophages and bacteria, the OD values for the bacteria are more than 0.6 that indicates the log phase, however after I mixed bacteria and bacteriophage together, then incubate for 24hrs, I got isolated bacteria colonies for most of the plates.
I mixed the bacteria and phages together in the top agar by vortexing.
What would be reason for this result?
Hi Liu
Increase the conc of the bacteria.
Usually, we use (three) serially diluted bacterial culture.
After melting the agar, put the agar on pre-set (40-45C) water bath.
Hope these will help you getting good result.
Mohammad
Shahnaij Mohammad Hi Mohammad,
Thanks for your help. The thing is every time I got the same optical density for the host so I do not think that is going to be the problem of concentration of the host. However, I will try to increase the volume I added into the top agar next time and see how it would change the result.
Thanks
Siman
Deart it seems that after mixing bacteria and phage you incubate for more time and your phage lysate has high titer, most of the bacterial cells got lysed before spreading, you only get the colonies of the cells which were non infected. i will suggest after mixing bacteria and lysate incubate for just 5-7 mins, and dilute your phage lysate. hopefully this will work
Siman Liu Yes. mix bacteria and the phage in some tube lets say eppendorf tube, place this mixture for 5-7 minutes before pouring so that the phage got attached to the bacterial receptors (if any). sometime due to the high concentration of phages in the prep it seems that some (very less) colonies have grown and no lysis or plaque is seen to solve this problem you should also go for the dilutions of your phage prep. i will advise you to use 5-12th serial dilutions for plaque visibility. Best of luck. do share the pictures of your plates, it will be more helpful to sort out a solution after visualizing plates.
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