I have isolated bacteriophages from river water and it is showing complete lysis when it is spotted on the lawn of bacterial host isolated from the same water sample. But, when plaque assay is performed for the same phage and host, it is not showing any plaque.
The same problem is coming when I inoculate overnight grown pure culture in culture broth containing phages, no phage population was recovered.
Suggest the solution or any change in protocol or any special treatment.
There are lots of possible reasons so it is hard to advise without further information. A few possibilities might be 1) the plaques are so tiny that you just don't see them (but they are there). This can often be changed by changing the growth conditions (richer or less rich media, changing the tempurature, etc. 2) You are not using the right dilution of the phage stock to see single plaques, if you have too many phages or too few you won't see any plaques.
I presume you are spotting a few microliters of your sample on a bacteria lawn to see the spot. You could try creating a series of 10-fold dilutions of the stock and spotting those to see if you can see individual plaques on one of the dilutions.
Keep in mind also the temperature of the soft-agar before adding it to the bacteria-phage mixture. Sometimes, if it is to high, it can demage the phages. It has been shown also that the percentage of agar in the soft-agar-media can also influence the size of the phage plaques.
The most probable reason for that is lysis from without. In other words, if you used very high phage titer they attack bacterial cells from outside and cause lysis so you observe lysis upon spotting even the used bacteria is not a host.In contrary, in plaque assay u are using diluted phage so if the bacteria is not possible host the phage couldn't adsorb and cause subsequent infection. My advice to u is to use spot assay of different dilutions of phage stock.
I have solution for such cases just follow my two following methods
1.Do ten fold dilutions till 10 raise to power -10 i.e in 10 tubes
2.Do your spot assay, next day scrap that clear zone and put into 1 ml SM buffer and vortex then centrifuge at 10000 RPM and use your supernatant as phage preparation
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