I'm performing double layer agar assay for my phages. The protocol I'm following is
1- culture the bacteria in LB broth until the exponential phase is attained
2- Make dilutions of the phage i-e, take 900ul of LB broth in 10 eppendrofs. Pipette out 100ul of your phage lysate and start diluting it. (vortex before next dilution)
3- pipette out 100ul of diluted phage lysate and 100ul of exponential phase bacteria in a falcon tube and incubate them at 37°c for 20 mins
4- After 20 mins, pour 4ml of semisolid agar in falcon tube and pour it on N-agar plates.
5- Incubate them at 37°c overnight
Next day I found these results.
No plaques were observed.
It sounds like you have the right dilutions (10 tubes of 10-fold dilutions). So either you don't have any phage in the lysate or else the bacterial strain is not a suitable host for your phage. It also could be your phage make really tiny plaques that are hard to see.
Michael J. Benedik there is an attached photo of what my plates look like after overnight incubation
The problem is with the bacterial lawn. I can't tell from the pictures whether you did not have enough cells to make a good lawn or something else is the problem. One possibility is that the culture was not dense enough before using it. A second possibility is that the top agar was too hot and so you killed most of the bacteria from the heat (the agar should be about 55-60C). A third possibility is that the lawn is fine but something was heavily contaminated and you have contaminant bacteria growing on top of the plate. If you can see a nice turbid lawn between the colonies, then the latter is the most likely explanation.
Try to do a few controls 1) cells only, 2) phage dilution buffer only, 3) phage stock only and plate each of them with top agar and see what you get. I strongly suspect it is contaminants.
Yeah Michael J. Benedik , you were absolutely right. My bacterial culture was not dense enough to form a lawn.
Now, i'm getting some results
Thankyou so much for your help.
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