How to measure the density of bacteriophages from a soil suspension like we measure the density of bacterial cells? I would like to know the phage density in the soil suspension to decide the inoculate density. Thanks in advance.
The problem with trying to determine phage numbers in a sample is that it will be entirely dependent on the host you use. So you can enumerate E. coli phages on E. coli, or Pseudomonas phages on Pseudomonas etc. But really the only thing that is going to work is to titer by doing serial dilutions and plaque counts on the host that you are interested in using.
I agree with Michael. Phages are highly host-specific so the standard plaque assay only works for the phages of a given bacterium. Even then, the presence of restriction-modification systems can distort your results, so it would be wise to use a restriction-deficient strain, such as E. coli DH5-alpha.
The only alternative would be to perform electron microscopy and count phage particles. I've never done that kind of work, so I'm not sure how practicable it would be.
The problem with using electron microscopy (which can certainly be done) is that you can not distinguish what the virus/phage host is. A given virus that you see could be a human virus, a butterfly virus, a tree virus or a bacteriophage and they could all look the same. Also you can't tell if they are alive or dead.
Thanks for the answers.
My intention is not to focus on only one bacteriophage. So I plan to use the Epifluorescence microscopy to check the abundance of bacteriophage in soil suspension. Hereby I attached the paper I am going to reference.
Cheers
Interesting. We've used the epifluorescence method to count bacteria in water, but I never imagined it could be sensitive enough to count viruses. I can think of several applications. One concern is that the method may not distinguish between viruses and free DNA molecules. I would expect a DNA virus with a 40 Kb genome and a 40 Kb DNA molecule to look the same - i.e. a point of light on the filter.
Hi, Andrew, thanks for your insights. When I extract phage DNA from soil, I use DNase to remove the effects of DNA molecules. But for phage density, I am worried the temperature that inactivate the DNase reaction (65 °C) will affect the results.
I agree with you; heating to 65 °C could easily disrupt some phage particles.
It's possible that the inactivation step is not needed if the phage remain intact throughout the procedure.
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