It looks to me that you have phage all over the lawn (all the little plaques everywhere) suggesting that your host strain or some thing you used to make the top agar layer had phage contamination. Then the large spots also look like they could be phage as well, although it could also be anything that is inhibitory to bacterial growth.

Hello Marutpong Panya ,

From the picture that you have uploaded one thing is for sure that these inhibition zones are a result of bacteriophages. But it is puzzling to see these plaques at locations other than where you had spotted them. I agree with Michael J. Benedik, seems like your bacterial culture itself got contaminated with the phage sample. Based on your information, I would suggest that you undertake a double-layer agar assay (agar overlay). Prepare soft agar tubes with 0.75% agar (~10ml per tube if working with 90mm plates). Grow a fresh culture of your bacteria and add 150ul of this culture (OD600 ~ 0.15-0.2) and 150ul of your test sample (phage specimen) to molten sterile soft agar (warmed to 50-55 degrees), mix gently my swirling/shaking and pour/cast it over a nutrient agar plate.

Further, if you want to test the host range of your phage against various isolates of C. jejuni/coli, you should undertake a spot assay (the one you performed).

I would like to thank Ali Mahmoudpour Michael J. Benedik Jatin Chadha for your kind suggestion. I will perform the experiment as your suggestion, and I will inform all of you later.

Regards.

These all are phage clear zones,just now pick out these clear zones and mix with host and propagate ,then perform plaque assy.u will get the plaques