I have phage aggregation on the surface of certain plates when performing plaque assays.
Dear Narges,
Although it is always rather difficult to uncover problems and provide solutions just by looking at images, I would suggest that perhaps variation within your phage samples is resulting in varied plaque sizes and morphologies.
Although not preparing your dilutions and soft-agar samples prior to pouring-out might lead to some 'aggregation' of plagues (i.e., more plaques appearing in some parts of the plate compared to other areas), it is more likely that you are collecting mutant phage when you prepare your stock solution.
Extra-large plaques may represent fast growing phage, or phage with higher levels of initial attachment, or higher burst sizes.
Transparent plaques, or plages with a grainy appearance, might represent phage with poor infectivity which results in few bacterial cells dying. Depending on whether you have a lysogenic phage or not, this might also be explained by a more virulent phage mutation.
In order to find out what is going on, use a toothpick to remove the software around one of the small plaques, and from around a larger plate (about the same sample size). Vortex this in liquid media, and then add to your host cells and soft agar and pour-out. I would expect that the small plaque will produce small plaques (though perhaps with some large ones), and the large plaque larger plaques (though again with some variation).
Variation in plaque size and morphology could also be caused by variation in the host bacterial cells, so make sure that you are always starting cultures off from good-looking single colonies.
Regards, Andrew.
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