I don't know about endotoxin and metabolite free. I have used PEG precipitation to purify phage. Basically grow your phage, spin down, and filter the supernatant (0.45 um). Add 20% PEG 2.5 M NaCl solution 1:4 to supernatant and incubate on ice. Spin at 3200xg for 30 min, discard sup, and resuspend pellet in 5ml PBS. Add 1ml of same PEG solution from above and incubate on ice. Spin again same as above, discard sup, and resuspend pellet in PBS. Check at this point for endotoxin and any other key contaminate. You may be able to continue washing with PBS and precipitating with PEG. Good luck

Thanks, Todd :) And how should I remove residues of PEG from PBS?

Concentrate your phage by PEG precipitation, then use cesium chloride density gradient ultracentrifugation.

After desity gradient ultracentrifugation you can dilute out the gradient and ultracentrifuge again. All the phage should be in the pellet and then you can resuspend in any buffer. That could work. I would still carefully check the final product before putting it into animals.

If your standard requirements are very high you can use monolith chromatography. It performs very well and It is easily scalable. The purity of the final product is excellent.

My first question is have you isolated the phage in chloroform (standard procedure)? If yes, then the next point is to enumerate the phage on a suitable agar medium seede with K. pneumoniae. Count the particles as plaque forming units. concentrate the chloroform solution till you have the desired number. Mind you, this is not so easy if you are trying your hands on any lysogenic phage. Your question is not very clear in this regard..

Dear Jai Ghosh, well I know how to grow and check the titre of phages. This is quite easy routine method. I was asking how to concentrate phages from large volumes of broth cultures and purified only viral particles in high purity.

Thanks to all of you for such useful tips. I hope they will work.

Dear Povilas

If you see my first reply, you will see that I told you to concentrate the chloroform suspension till you get the desired concentration. Now why chloroform? that is because when you will grow the phage in presence of the host, all nutrients of the host will be water soluble, and to get rid of most of these and certain metabolites of the host, you must separate it in chloroform. Now you can easily concentrate the phage in chloroform by simple evaporation preferably in vacuum. Its all that simple.

Oh see now. Thank you :))

After purification of concentrated bacteriophage as stated above , you can test for endotoxins contamination by chromogenic LAL .

According to my experience, after desity gradient ultracentrifugation you can dilute out the gradient and ultracentrifuge again. All the phage should be in the pellet and then you can resuspend in any buffer. That could work for sure. 

agree with Pierre Béguin's advice · first concentrate the phage by PEG precipitation, then run CsCl2 equilibrium gradient ultracentrifugation. Phage will be visibly banded, then easily recovered using syringe with fine grade needle to puncture the centrifuge tube and withdraw the banded phage particles.

See:

Shore et al Proc.Nati.Acad.Sci.USA Vol.75,No.1,pp.400-404,January1978

Diana et al J Mol Biol. 1978 Dec 15;126(3):433-45.

40.20 · 337.99 · Institut Pasteur

Concentrate your phage by PEG precipitation, then use cesium chloride density gradient ultracentrifugation.