Hi all!
My thesis groupmates and I are working on purifying an E. coli bacteriophage. The phage was handed down to us by the previous thesis group, but they only managed to purify it to about 60%, so we are aiming to increase its purity. However, we have recently encountered an issue where we are getting a bacterial lawn with no plaques present or sometimes faint plaques, and we are really lost right now.
In the second week after receiving the phage (which the previous group had stored in the refrigerator for almost 3 months), we began purifying it. Despite our unfamiliarity with the DLA technique, we initially got good results. Here’s what we did to achieve those results:
- Used an underlay (with a composition of 20 g/L broth and 15 g/L agar) and an overlay (with a composition of 20g/L broth and 8 g/L agar).
- Added 5% glycerol and MgCl2 to get visible and better plaque size.
- Made serial dilutions from 10^0 to 10^-9 (900 μL LB broth: 100 μL bacteriophage).
- From our serial dilution, we took 400 μL and transferred it to a microcentrifuge, then added 400 μL of a 24-hour cultured OP50 bacteria.
- Immediately added this mixture to a tube of 4.5 mL hot still-molten overlay, then poured it onto the bottom agar. However, we didn't mix the overlay well.
- It took us several hours to dry the overlay, and some areas were still wet, but we got good results (see top picture below).
After this slightly successful DLA, we attempted the DLA again to improve some aspects of our process. Note that we used the same samples. Here’s what we changed:
- Made serial dilutions from 10^0 to 10^-9 (500 μL LB broth: 100 μL bacteriophage).
- Took 200 μL instead from our serial dilution and transferred it to a microcentrifuge, then added 200 μL of a 24-hour cultured OP50 E. coli bacteria.
- Before pouring it to the overlay mixture, we incubate the phage + bacteria to 37 degC incubator for 50 minutes
- Also, note that the 24-hour cultured bacteria were grown in 9 mL LB broth + 1 mL bacterial culture, and this time we added ampicillin to ensure only the OP50 (ampicillin-resistant strain) was growing.
- We did not immediately add the phage + bacteria to a tube of 3 mL overlay. Instead, we let the overlay cool down to a point where it was still molten but cool enough before adding the phage + bacteria mixture.
- Poured the overlay onto the bottom agar, mixing the overlay well on the top of the underlay.
- It took only a few hours to dry. Unfortunately, the results were not good (see bottom picture below).
Please give us some advice or techniques. Where might we be going wrong?
Maybe this can help:
Article Simple Method for Plating Escherichia coli Bacteriophages Fo...
The plates not drying is probably due to the 5% content of glycerol. Try lowering it to 0.5%.
Good luck!
A few things. First be sure your top agar (overlay agar) is at the right temperature. It should be around 48oC (keep it in a water bath). If too hot you will roast your plating cells. Secondly, make sure your plates are warm so that you have a minute to spread the top agar. I like to warm them at 37C for an hour before using them.
Third, your plates look like they have contamination. This might come from the top agar if used too often or stored too long, or from your pipets (if you are using pipettors and non-sterile tips. From the picture you have lots of white colonies growing on top, that is what I think are contaminants but I only have the one picture to work from.
Lastly, you are keeping your phage/cell adsorption mixture too long at 37 before plating. Depending upon the phage you around the time for one complete burst cycle. Usually 10-20 minutes is plenty of time for absorption before plating. Although 37 is optimal, I have never seen much difference if doing 20 minutes at Room Temp.
Michael J. Benedik
We maintain our top agar in a water bath at approximately 52°C, following a protocol from a journal we read. And yes, we suspect that frequent use of the same top agar may be contributing to contamination issues, so we plan to start preparing fresh batches regularly. Additionally, we are re-evaluating the composition of both the top (1.5 lb agar) and bottom agar (0.7 lb agar). It's possible that the agar-to-broth ratio is impacting the diffusion of the bacteriophage. Would it be more effective to use bacteriological agar instead of LB agar? Given the faint plaques we’re observing, we supplemented the underlay with 5% glycerol, CaCl2, and ampicillin, and the overlay with 5% glycerol, CaCl2, and MgSO4. Despite these adjustments, we continue to see faint or even no plaques. Are there other supplements or methods we could try? Lastly, we are concerned that our phage concentration might be low. In this case, is it still necessary to perform serial dilutions, or could we directly mix 100 μL of undiluted phage with the bacteria?
52oC should be fine for the top agar. If it is at all cloudy then discard. I usually would melt and use for my experiment, then remove from the water bath and the next day melt again, that way the high temperature remelting would kill off any few bugs that might have gotten in.
Your agar concentrations are ok, but some folks think that 1.0 or 1.2% agar for the plates is better. I'm not sure I've seen much difference. Likewise I doubt the agar type matters, but the media might. With many coliphages we often used tryptone plates instead of LB, being less rich the cells grew more slowly and allowed larger plaques. But I think the most important is to be sure your plates are not too dry. So don't use month old plates.
I doubt glycerol will have much effect, adding a small amount of MgSO4 and CaCl2 is fine and pretty typical. You don't need to add to both, if you put in the plates then you don't need in the top agar (or vice versa). There is plenty of diffusion.
You might also try different temperatures, you might get better plaques at somewhat lower temp like 30 or 32. Again most of the time it shouldn't matter but you can try.
The problem with not doing dilutions is that if you have too many phages you also fail to see plaques. I would usually do a few 100-fold dilutions, so undiluted, 10-2, 10-4 and 10-6 should be plenty. And hopefully you see something on one of them.
Have you tried a control phage and do you get nice plaques with that?
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