Hi Karrar, a bit more information would be helpful to troubleshoot your question. We don't know the cells or the phage you are using. Plaques form from phages lysing the cells, which means that they will form less clearly if the bacterial growth is high. It is good that you stored the samples and phages at a lower temperature, but you may need to increase the temperature and wait long enough for the plaques to form when looking for titer - some species form plaques exclusively at higher temperatures, others at lower temperatures. It is also important that the bacteria are in the right growth phase for the phage to infect. Some phages like bacteria in the logarithmic growth phase, others when the culture is already in stationary phase. You don't mention if you have seen plaque formation with the bacterial host you are using - are you sure that the bacteria are susceptible to the phage? Do you have a positive control? If they are resistant, you may need to wait much longer for plaque to form, if they do at all. Finally, have you tried increasing the amount of input into your culture? You may not have enough phage particles on your plate to visualize plaques consistently.

These are some points that I would consider thinking about off the top of my head, but this article (http://www.dairyscience.info/index.php/enumeration-of-lactococcal-bacteriophages/factors-affecting-plaque-formation.html?showall=1&limitstart=) goes into more detail about plaque formation, at least in regards to phage screening in the dairy industry. It may be a good place to look at for preliminary information, but you will also need to find an optimized protocol for your experiment's strains.

 Hi Dr Johan Jaenisch  thank you very much for your answer,it was useful  to me .I mention I  have seen plaque formation with the bacterial host iam using because i took the  the electron microscopic image for phages that isolated from plaque  ..

 my problem when  i incubated my phage and their host in the 30 c and i took plaque when i isolated the plaque and wanted to take  special Pfu in same conditions that i took primary plaque from, but  i did not  see any plaque  in my plates ...what do you think is the problem? 

Ah, sorry - I didn't see that this was after the primary isolation. Indeed, then it is surprising that they didn't form again. You mentioned that you kept your isolated plaques at 4°C after the first round - did you keep them in a phage buffer during storage? I remember that I once forgot to add divalent salt to my buffer after preparation and none of the phages grew. If they were stored properly, then the only thing I can imagine is that your phage count may be lower than you think from a single phage. It has been a while since I have worked with phages so I can't remember if I used more input from the buffer that I stored my isolated plaque in... Intuitively I would guess you need more of the buffer that you put your agar plug in so that you have a higher concentration of phage on the plate. Have you tried that?

Thank you very much Dr.Johan Jaenisch for the recommendation.  i kept my isolated plaque at 4°C after  the first round but  did not keep them in phage buffer i keep them in a Liquid  media that i use it for isolate phage and their host .

I have got same problem any one can help me

I got plaque but when i tried to propagate the phage with bacteria and do double agar layer there is no growth even after make serial dilution and repeat the procured triplicate