We are doing experiments with wild-type bacteriophage lambda. Plaque counting is an essential part of the work. Unfortunately, the plaques are rather small and indistinct, which makes it all but impossible to count them when the plate is viewed from the bottom.
Does anyone know any good tricks to improve plaque visibility?
FYI. Recipient strain: c600; plate medium LB agar; agar overlay 1/2 strength LB agar; growth medium: LB; all media supplemented with 10 mM MgSO4.
Procedure: 0.1 ml (diluted) phage lysate added to 0.5 ml exp. phase (OD 0.5) E. coli.Add 5 ml of top agar and pour over plate. Incubate O/N at 37C.
You have picked the right host strain to use at least however be sure to add maltose to the culture while growing up the cells. Lambda attaches to the maltose receptor (LamB) and you will have much higher receptor concentration with maltose induction. You don't need it in the plates, just in the culture of your recipient cells.
Secondly try and change the medium and use tryptone broth plates (10g tryptone and 5 g NaCl), this is the classic lambda media. The cells grow a bit more slowly and this helps good plaque formation. You might also reduce the agar concentration, historically 1% agar instead of 1.5% was used for the plates. I'm not sure how big a difference that makes but you can try. Lastly, be sure your plates are fairly fresh and wet, very dry plates inhibit nice plaque formation.
Another thing to consider is whether you really need to use wild type lambda or can you use one of the clear plaque forming mutants, those plaques are much easier to see and count. But that depends upon the experiment you are doing.
Andrew Jenkins I'm curious if you were able to get better plaques and what worked for you?
We haven't tried yet. However, one thing that seems to help is to view the plates on a dark background.
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