I am propagating bacteriophage phi x 174, with its host bacteria E.coli C strain. I prepared E.coli C strain broth culture of OD600 0.4. Then I took 100 micro litres of E.coli and added to soft agar along with 100 micro litres of phage lysate. Mixed well using a pipette and overlaid on agar and incubated for 18 hrs. But after incubation separated bacterial colonies obtained without any plaques. I tried with serially diluted phage also. Again the result was same. How can I overcome this?
As you are getting separated bacterial colonies there are a few explanations that are most likely:
- There is something wrong with the overlay itself? Do you run the overlay without phage as a negative control; if not try that. You might not be adding enough bacteria, or you might be killing the E. coli (if the overlay was too hot for example).
- Are you adding too many phage to your overlay? You do not mention the dilution series you did for the phage - what was your highest dilution?
Other things to consider. (a) Do you add a cation to help phage absorption? Can't remember the requirements for phiX174, but I think it might be Ca2+ ions (though that would prevent phage reproduction). Try spotting the phage dilutions (5 - 10 µl spots) on the agar overlay rather than adding to the cells in the overlay.
Vincent Mulholland 's answer provides very good insight, and his suggestions are absolutely worth investigating. I will add my suggestions here to supplement Vincent's.
OD600 is not always a reliable measurement, and by that I mean it doesn't necessary correlate with the number of viable bacteria. It does seem like your colonies are growing, but there may be too many or too few viable bacteria at the same OD. Also, I recommend plating the bacteria on some hard agar (no soft agar) to make sure it grows and that it produces homogeneous colonies.
In addition to Vincent's idea of doing phage dilutions, if no plaques form, keep incubating over another day or even two. If the phage are there, you should eventually see plaques, which would mean your phage concentration is lower than you think.
There is also a question of phage viability. Did you purchase the phage or did you isolate and concentrate them yourself? How certain are you of your starting concentration? Were they purified by ultrcentrifugation/dialysis?
It may be worth running an SDS-PAGE experiment to see if the MW of the phage proteins roughly matches the standard.
The last thing I'll add is that both my major suggestions have to do with the concentration of the phage and the host. The efficacy of propagation is related to the multiplicity of infection (MOI), which is the ratio of virions:bacterial cells. Every phage/host pair has a unique, optimal MOI which produces the best chance for propagation. I recommend looking up the literature value for your pair and then trying to match that MOI for best change of propagation.
Good luck!
The answer that Vincent Mulholland and Kirk R Jensen provide are both good. A few minor other points.
Using 100ul of an E. coli culture at that density should give you a very nice lawn. Did you do a control plate without any added phage to see what that looks like? If you don't have a very nice lawn then you have a procedural problem such as the top agar was too hot and killed the cells.
If you are getting a nice lawn, then try your serial dilution series again but be sure to use the entire range. If you add too many phage then most of the cells making up the lawn will be killed and you will have some discreet survivors that form colonies which are most likely mutants resistant to the phage.
Michael J. Benedik Kirk R Jensen Vincent Mulholland
Thank u so much for your valuable suggestions. I furthermore done dilutions of phage lysate and now am able to get plaques. As mentioned by Vincent Mulholland Michael J. Benedik earlier I was plating too much phage lysate. Now from 10^6 dilutions onwards plaques are forming.
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