M13 Plaque Assay Problem - (Mar/28/2007 )

Hi All!

I'm routinely doing plaque assays for titering m13 phages (Ph.D.-12 Phage Display Kit from NEB with E. coli ER2738). But since two weeks, the plaques won't grow anymore! All I can see is a dense bacterial lawn, but no plaques... even with the same samples and the same batches of plates (LB IPTG/X-Gal), agar top (LB) and growth medium (LB + Tetracycline) that worked all right three weeks ago! It's like the bacteria suddenly became "resistant" to phage infection! Can anybody help, please?

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hi,
i searched the web and found a similar question, and this was the answer:
Q3: No plaques are visible when titering using the Ph.D.™ Phage Display kit.

A3: Unlike lambda, M13 is a non-lytic phage and does not produce clear plaques. M13 plaques are areas of diminished cell growth, not lysis, and consequently can be difficult to see. Try holding the plate up to a light. Also, since the vector used to prepare the library carries the lacZα gene, plaques will be blue, and easier to see, when using an alphacomplementing strain such as the supplied strain ER2738 and plating on Xgal/IPTG plates.
Also, be sure the dilution range is appropriate for the phage you are titering. For amplified phage, plate 10 µL of 1:109 - 1:1011 dilutions; for unamplified panning eluates, try 1:10 - 1:104 dilutions for early rounds, 1:104-1:107 for later rounds. If the phage is not sufficiently dilute, the plaques will be confluent on the plate and it will look like there are no plaques at all (or a bluish tinge when using Xgal plates). Occasionally after PEG precipitation, the phage will clump and not dilute properly. As a result, you might have a plate containing too many plaques merged together. Make sure to give the phage ample time to resuspend after precipitation (> 1 hour) and vortex each dilution tube very well (~10 seconds).

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Thanks strawberry, but I'm very sure I used the proper phage dilutions etc., because everything worked well for months, until a few weeks ago. I didn't change anything, though. I thought perhaps the agar plates or the media were spoiled, but I prepared new ones and still I can't see anything. I once had a similar problem and could trace it back to tryptone that went bad (intense smell of ammonia). I used new tryptone and voilá - I could see plaques again. But not so this time... I used new chemicals and prepared everything freshly... LB agar, LB top agar, LB medium, streaked e. coli freshly... nothing. The bacteria are growing (and they look and smell like e. coli), they just don't get infected anymore. By whatever phage i throw at them. I'm really desperate...

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well, may be the virus enters a lysogenic phase, or as you said, cells became resistant
i think it's better to start with fresh culture once again..

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If M13 ever went lysogenic, there would be some big questions being asked, believe you me! Same thing if E. coli ever became resistant to a phage like M13. But you could always try the expt with someone else's bacteria.

Dumb question 1: Are you certain about the quality of your M13 stocks? Perhaps a serial dilution of them to see what's happening?
Can you go back to your last plates that worked and purify the RF form of the phage, then test it like any other plasmid? You'd probably have to grow them in a liquid medium in order to have enough to purify and test.

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What swanny said... M13 is non-lytic, you see plaques only because of slower growth of infected bacteria. Regarding the "resistance", there is some recent evidence for this, but I doubt that is the reason for my problem...

In the meantime, I tried again and got it to work once with new bacteria, but failed to reproduce it with the same plates, the same bacteria and the same medium. It's jinxed!



I'm quite certain, yes... I tried a gazillion different M13 clones and stocks in the meantime, even M13 wildtype, with all possible titers ranging from 10e13 to 10e3 pfu/ml... nothing...



I do have some phage DNA, but it's single-stranded. I could add some polymerase and so generate the RF form in vitro perhaps... I'll go and try this... But my gut feeling is that the phage stocks are OK and there is something wrong with the bacteria or the media, but I can't lay my hands on it...

Thanks so far for your thoughts, guys... Of course, more suggestions are always welcome...

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OK, I finally got it!
The bacteria were to blame. In our (non-microbiology-) lab, we unfortunately don't have freezers that get colder than -20 °C, so I store my glycerol stocks there. Apparently, everything went well for ca. 4 months. After that, the E.coli (ER2738 from NEB) still grow, you can plate them and everything. But they don't get infected by M13 anymore! Strange. Well, I ordered new bugs from NEB and voilá - I finally got my plaques! Now the work can go on - at least for another couple of months

Thanks for your help, swanny and strawberry!

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