I am not clear what you are trying to learn from this experiment. Remember that phage replicate exponentially depending upon the burst size. But some phage might have a burst size of 100 or more, so it only takes a few phage life cycles to generate enough phage to kill off all susceptible cells. The differences among your triplicate samples may just be statistical anomalies (such as how early a resistant mutant arose in the growth) or some other similar issue.

Hi Dr. Benedik,

Thanks for your response. We are trying to use this technique to quantitatively track activity of phage dilutions, similar to kinetic MIC assays done with antimicrobial peptides or compounds. Our thinking was this protocol would be easier to interpret that spot overlay assays. We realize the using phage is unlike assays with peptides, as it is a dynamic system. Our confusion is due to the lack of consistent activity for a given dilution within a set of replicates, as the replicates are from a single tube of phage and cells.

We are interested in how phage activity changes during storage at ambient temperature; is there a protocol you could recommend to best track this?

Thanks.

If you are trying to enumerate phages then I think the traditional overlay method for titers or or spot titers is going to be much more repeatable and meaningful. If you do a full dilution series by spot titers you can get an actual count. I think monitoring growth of the host will prove to be very problematic.