Hello,
I got a question concerning the double layer agar method for plaque assay:
1) Do I really have to use two agar layers, wouldn't it be enough to use one pre-inoculated soft-nutrient agar layer? For me a second agar layer despite from the soft agar makes no sense.
2) If I use the while-still-liquid-inoculated soft agar to get a bacteria lawn, do I have to incubate the culture before adding the phage or should I directly add the phage in or on the agar plate?
3) I have read about people adding the phages directly into the medium with the host bacteria and about some just putting a drop of the bacteriophage suspension on top of the agar, which works better for phage assay?
I think a pro for dropping on top is the higher motility of the phages but on the other hand, the bacteria growing insider the agar will remain turbid, so it might be harder to spot the clear plaques
Thank for your answer,
best regards,
André Leonhardt
Hello André!
We have been working with Phage Display for quite some time with pretty cool results so maybe I can be of help.
As far as I know (im attaching NEB's phage display protocol) the composition of the double-agar layers are different. In our case, bottom agar contains IPTG and Xgal (which permits us blue/white screening), while top agar only contains tryptone, yeast and NaCl.
With respect to questions 2 and 3, we usually incubate bacteria until they reach 0.5 OD, separate them in microtubes, add different phage dilutions to each tube (after adding incubate for 5 min at room temp), then transfer this mix to still-liquid soft agar and vortex briefly. Then, we immediately pour this into pre-warmed agar plates and let it spread evenly.
If you have any other questions or if I can share anything else, let me know.
Victor
Precisely. This is done through diffusion between the two layers in a more controlled manner.
1) Do I really have to use two agar layers, wouldn't it be enough to use one pre-inoculated soft-nutrient agar layer? For me a second agar layer despite from the soft agar makes no sense.
Yes, typically your regular solid media has an agar content of ~1.5%. This doesn't provide enough permeability for the phage to move and interact with your bacteria. The bottom layer solid layer is crucial for an even bacteria lawn, which the phage will grow on. The softer, semi-solid media allows for (like Victor mentioned) better diffusion, or an easier medium for the phage to interact with the bacteria. In my experience you can add indicator reagents to either layer.
2) If I use the while-still-liquid-inoculated soft agar to get a bacteria lawn, do I have to incubate the culture before adding the phage or should I directly add the phage in or on the agar plate?
The happier your bacteria are when you're using to inoculate the soft agar, the better your lawn will be. Typically, I grow an overnight culture, dilute it out 100-fold the next morning and grow to A600 of 0.2 to 0.4. Then, I incubate (15 min, room temperature) 100 uL cells with 100 uL phage (at various dilutions depending on what you're doing), and inoculate 3 mL heated soft agar with the entire 200 uL volume of phage-cell mixture, vortex lightly (to avoid bubbles) and pour onto pre-warmed solid agar plates.
There is another method - the spot titer method. Again it depends on what you're trying to do it. If you're screening for something, I suggest the prior method. If you want a rough idea of what your phage titer is, this method is okay. In this method, you'll just directly inoculate the 3 mL soft agar with 100 uL bacteria cells, let it set and then spot 3-5 uL of phage from neat to around 10^-7.
3) I have read about people adding the phages directly into the medium with the host bacteria and about some just putting a drop of the bacteriophage suspension on top of the agar, which works better for phage assay?
It depends on your final goal.
Hope this helps! Please let us know how it goes. :)
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