When determining titer of T7 phage by plaque assay, I get a gradient of plaques where one side of the plate is clearing and the other half of the plate is a lawn of bacteria. Anyone experience this issue?
If there is a reason to expect that cell density will be uneven across the gradient (because, for instance, the plates were not poured on a perfectly leveled surface) then the more populated area of the lawn will reach stationary phase faster. Many phages -lambda, for instance- cannot infect stationary phase cells or do so poorly. If such is the case for T7, that might be one possible explanation.
Hope it helps!
Are you sure that the soft agar overlay was distributed evenly across the plate?
Thanks Alejandro and Kathryn. I only see this problem happen on bigger 15 cm dishes where I only use 10 mL top agar/phage/cell solution. Never see it on the smaller standard sized plates (6 cm i think) which I use 3 mL of the top agar solution. It may be that I need to "swirl" the dish a lot to get complete coverage of the agar on the big plate since so it is inherently uneven. Will test this. Thanks!
Three comments
1. You might try vortexing the top agar bacteria phage tube before pouring. Not too hard, i.e. you don't want bubbles but enough of a spin to get a complete mix. Or you might simply enter the top agar into the tube with a little more force so as to achieve good mixing instead of counting on the swirling on the plate to accomplish everything.
2. Make the top agar as thin as you can get away with and still get decent gelling. It can vary batch to batch with the agar. I generally use 0.5% top agar. You can go lower. Handle the plates gently and incubate them upside up. Pay attention also to the temperature of the top agar. Warmer gives a better spread but not to kill the bugs. I usually use 50 but think you can go up to 55 since it cools quickly once poured.
3. You are right to consider using more top agar. Play around with that. There might be be a sacrifice in terms of how uniform the plaques are. You have to play and see
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