Some general comments, first of all lambda titers are usually in the 10e10 or 10e11 range (or less if something went wrong) but never 10e19. If you are getting numbers like that then you have a phage contamination issue.

Media and host organism have a big impact on plaque size and ability to see. Traditionally most lambda labs used tryptone broth plates (TB) which is much less rich than LB or other media and the slightly slower growth of the cells gives you nicer plaques. Also use fairly fresh plates that are a bit wet and not dried out.

I think either of your host strains is ok but be aware that ER2738 is E. coli K12 and BL21 is E. coli B, so they are quite different. Since both are deleted for their restriction system that should not be a problem but if you move to a different host be aware of possible restriction issues. Are you working with wild type lambda or some special mutants which might have certain host requirements?

Your procedure seems fine, just be sure that the top agar is 55-60 C and not much hotter than that otherwise you risk killing your plating cells.

You might not be seeing plaques because your lysate really doesn't have phage, or you have the dilutions wrong. You can do a quit spot test to rapidly check lysates, add 100ul cells to top agar and pour onto your plate. let dry a few minutes. Then spot 5ul spots of different 10-fold phage dilutions. This lets you do an entire range of dilutions and even test a few different lysates rapidly on one plate.

You asked about adding maltose, it can be helpful to have maltose in the growth medium when you are growing your plating or host cells so they have maximal expression of the receptor protein LamB. But there is no need to have maltose on the plates afterwards.

When you prepare your phage lysates, are you seeing good lysis? I suspect the issue you have may be you just are not getting good lysates to begin with. I usually added 10e5-10e6 pfu to 100ul cells, let adsorb for 10 min, then add 5-10ml TB or LB and grow shaking vigously at 37 for a few hours until you see growth followed by lysis debris. It should be very visible.

I agree with Michael Benedik: a titer of 10e19 must be due to contamination of the dilution buffer or you used the same pipette tip for sequential dilutions, with carry-over as a consequence. A titer of 10e19/ml would amount to 1 kg of phage/ml !

Pierre Béguin I never thought about the mass, but yes that would around 100uM phage stock!!!

Binyamin Kerman are you perhaps using pipettors and tips, and if so you MUST use barrier tips when working with phages otherwise you will almost certainly get cross contamination.

Thank you @Michael J. Benedik and @Pierre Béguin. In response to some of your suggestions I think you are right that we do not always use barrier tips to do the dilutions, that would explain the impossibly high results.

The phage we use are standard lambda but some of the cells are engineered for protein expression in which we also lowered the growth and lysis temperature to 25c instead of the optimal 37c because we are trying to balance protein solubility and phage activity.

I think that the lysis is probably a source of the current issue, we don't always see strong drop in OD sometimes more of a platue but we go ahead with phage recovery anyways because we don't want to continue the cells' protein expression longer to avoid inclusion bodies.

There are definitely parts of our protocols that can be improved to get around some of these issues. I'll try your ideas.

Thanks again

Binyamin Kerman if you would like to better explain what you are trying to do that might make it easier to offer some suggestions or advice. There are numerous lambda mutants out there, some of which might work better for your experiments (unless you have already optimized for that).

Binyamin Kerman It have been performed plaque assay 1000 times , but could not get this much high titer ,probably there are some mathematical errors.