If you see individual colonies after incubation of the soft agar method, I think you need to increase bacteria concentration. 10^7 cfu/ml is ok. You can add 3 or 4 ml melted top agar+100ul bacteria stock (10^7 cfu/ml ) + bacteriophage ( it depends on concentratiion).

-You can check top agar plate have the right antibiotics. do a control test if everything ok, then check your page stock,

Note: Always prepare fresh E.coli culture for amplification.

Thank you so much everybody for your answer. The problem is that when I infect phage with E.coli on day 1, the plaques form pretty well on top agar. But after amplification, it does not. So the cause does not lie in E.coli concentration or top agar. It works fine at day 1. I even put more E.coli (which is fresh) in day 2 and 3 than day 1. That is why I am so frustrated.

The resulting 'new' phages have an altered pIII tail. I would suspect this altered tail interferes with the infectivity of the phage.

It is often seen with fusion proteins of pIII.

Dieter Vandenheuvel Thank you so much! Then is it impossible to amplify this kind of helper phage? Or is it just this stock's problem?

Also I have tried this several times, so I don't think it makes sense in that if its a mutation problem, the results won't come out the same every time I try. Every time the first day plaque forms well, but not the last day (end of amplification)

Well, according to the regular protocol - and how I'm understanding what you are doing -, you use the native VCSM13 phage to infect E.coli TG1 which contains a plasmid with an altered gene for the pIII-fusion protein. This fusion protein could interfere with the infectivity of the resulting VCSM13 phage with the pIII-fusion protein.

To complicate matters... You also wouldn't expect plaques to show up. If your altered phage isn't infective, it shouldn't be able to make plaques. But, I'm just trying to figure out what your problem might be.

Hi Hye nice question.