Hello
I am doing phage propagation using s. aureus for the first time but the phage titer is very low! then when I did the EOP no plaques were observed, I use 200 μl of both phage lysate and bacterial culture. Any suggestion for increasing the phage titer?
Did you also do serial dilutions in addition to using your phage directly? Sometimes if you have too many phage you don't see any plaques because the entire plate is a lawn of phage or if your MOI is too high you might not get good results.
What is the titer of the starting phage stock you wish to propagate and does it form nice plaques on your host?
Thank you for your reply, i used phage diluted 10^1, 10^2 and 10^3 and all the plates contained too many plaques that i could not count.
That may not be enough of a dilution. A good phage stock might have 10e10 pfu so I would make dilutions at least to 10e6 or 10e7
Do the lawns on these plates all look very smooth and dark or are they very spotted? If the first, then it may be you have no phage at all. If the second then your probably have too many phage. A no phage control plate is usually useful here.
Sultanah Ab
Hi, You may need to enrich your existing phage stock first. For this, you can add 100 microlitres from your purified phage stock to your grown host culture (3 to 5 ml). Incubate overnight. Centrifuge the suspension to pellet the bacteria, collect the supernatant and filter sterilize it. This would usually enrich initial phage stock to 100-1000 times. You can try plating from this at higher dilutions as suggested by Michael J. Benedik .
I have solutions of your problem, follow it then you will get the plaques
1.Do spot assay
2.Next day scrap that clear area and mix with host bacteria and broth
3.Next day centrifuge and filter this preparation
4.You will get plaques without any problem.
5.If still you are not getting it..let me know I have many solutions for this problem.
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