Hi we performed a dilution series 1:10 of different amplifications of the same phage library in Xl-blue bacteria, and we see a strange pattern. In the first dilutions of the reamplified library there should be a matt of bacteria but we se few single colonies and then the spot gets progressivly thicker to then beahave like a normal titration. i include 2 pictures one if a library that behaves normally i.e a solid spot of bacteria that graduually gets to songle colonies the furtger down the dilution series and then one that starts off single colonies and increases to a solid spot and then go down.
Has anyone seen this pattern before? has any one any idea of what this can be caused of?
lytic phage infection should not look like this. that would produce lytic placs and just not very few colonies.
does it have anything to do with the helper phage? should very high concentration of phage/helperphage or something else inhibit bacterial growth?
First of all I find this experimental protocol very confusing, I am not sure what you hope to learn from it. But the short answer is that if you have more phage than cells, you will have very reduced numbers (or none) colonies growing in the spot. But once the cells start to outnumber the phage, then you will get spots that look reasonably identical. You are not likely to ever see plaques doing it this way.