Dear Victor,

I've never encountered such a problem before and have not done much phage work in my time. However, if you are convinced that the problem is really the result of significant contamination with endolysin in your phage preparation (rather than someone contaminating their pipetter or a similar silly mistake), you could start by trying to semi-purify the phage particles using PEG precipitation, or possibly by concentrating the suspension using a high-molecular weight cut-off filter (e.g., nominal MWCO of 100 kDa would retain the phage but lose the endolysin in the filtrate). There are some other old-fashion techniques which would be rather interesting to do – run the phage preparation out in a non-denaturing agarose gel and then over-lay this with your host bacteria (you would expect that a zone of lysis to occur at a lower apparent MW than for a phage particle). Similarity you could use a HMCO dialysis membrane to separate the phage suspension into LMW autolysis and HMW phage, and to test the two solutions on a bacterial lawn.

Regards, Andrew

Dear Andrew,

Thanks a lot for your suggestions! I will certainly try some of them, if not all.

The funny (?) part is that we only encounter this problem with two specific siphoviruses infecting Klebsiella pneumoniae.........

I actually doubt that any endolysin would be sufficiently concentrated to give you lysis at those dilutions. My guess is that you have phage contamination somewhere, either in the indictor strain or the dilution buffers or your pipets. You might only be seeing on certain hosts because those are the hosts for that particular phage.

Distinguishing between plaques of lysis caused by bacteriophages (phages) and those caused by endolysins can be challenging but can often be accomplished through careful experimental design and analysis. Here are some strategies to differentiate between the two: