Hi.. i have to perform " lysis profile of host bacteria after phage infection". In a paper, they have taken 0.1 MOI in this assay. I want to ask on what basis they have selected only one MOI because generally we perform the "lysis profile of host bacteria after phage infection" at different MOIs. I'm a bit confused . Should i also go for one MOI or take different MOIs? and if i have to select one MOI, which MOI should i pursue with?
please guide.. it will be a huge help
Dear Saleha,
The basis for using a multiplicity-of-infection (MOI) of 0.1 is that enough bacterial cells will become infected in your assay to be easily detectable when you plate them out, but the likelihood of a single cell being infected by more than one phage at the same time is low.
The likelihood of a single cell being infected by more than one have at the same time is probably easy to calculate, and will depend on the concentration and total numbers of cells and phage, and I suspect that some has done this sort of thing in the past and concluded that a MOI of above 0.1 was too risky.
Understanding why the MOI is not a lot lower probably is based around the fact that the fewer phage added the fewer cells will be infected, and the fewer plaques you will see when you plate things out. Effectively, the higher the MOI, the more progeny phage will be produced, so you will need to make more dilutions and plate out these - or, the other way around, the lower the MOI and the more concentrated the samples will be that you need to plate out in order to see any plaques being formed. Obviously, there is a limit to how much o the sample you can put into your soft-agar overlay before it no longer sets.
You've also touched on another interesting question - why should you do what one group of people do (MOI of 0.1) but not what others do (a range of MOIs). The answer to this is a little bit complex. If determining the phage burst size is only part of your work and it is not something of great interest, then clearly you want to do the least amount of work yet still have the results recognised by your research community. If this is the case, then find an important review or research paper in your field, and do what they did!
If, on the other hand, you have more time, because this is the first time you have done this, or because you are really interested in the replication of your phage, testing a range of MOIs (e.g. 1, 0.1, 0.01) might give you a better data set to see small differences in burst size that may reflect differences in the host or phage progeny.
I hope this helps. My main point (which I have been thinking about recently for other reasons) is that good research should not simply be about applying the protocols others have used: it should be about thinking about what is the best protocol you should use in the context of your own project. There are many variations-on-a-theme for protocols, and unless you are prepared to test each protocol side-by-side, you will not learn which is the best for you; your strains etc. may be slightly different from other's and perform differently, and the aim of your work may be different too, which impacts on what protocols, controls and reference strains you choose.
Regards, Andrew.
Thankyou so much Andrew J Spiers for such a detailed answer.
That's so kind of you.
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