I am working on isolation of phages from sea water. I have isolated phages and kept in oxalate-EDTA buffer. Since, suspension has unknown phages, I am infecting this suspension with bacterial strains isolated from sea water. After infection, lysate supernantent is precipitated with 8% PEG or 12% PEG + 2.5M NaCl. But none of the solution is able to precipitate out phages.
What to do ?
Dear Atif,
There could be several reasons, However can you tell us if you are using plate propagation or liquid propagation method?
Problem 1: it could be that your phages are not alive or phage titer is too low...!
I assume you are infecting the known bacterial isolates with unknown suspension of isolated phages, which are stored in Oxalate-EDTA buffer (right), Have you tried using alternate buffers like store your isolated phages in an oxalate buffer with subsequent transfer to modified SM (MSM) phage storage buffer (0.4 M NaCl, 0.02 M MgSO4, 0.05 M Tris, pH 7.5). Long ago I was working with phages and we were always storing our phages in 1x SM buffer with no or negligible drop in titter.
Problem 2: in case you are not using the plate propagation method, where you can see zones of lysis (ideally a web pattern), you wont be able to say that your phages are specific to any of the bacterial isolate and that they infected or even lysed the bacterial cells.
In case your phages did infect the bacterial cells, how can you be sure that they lysed the cells? In this case I would recommend to add 1% chloroform at the end of your experiment and incubate for another 5-10min while shaking to lyse any non-lysed bacterial cells inorder to remove trapped phages. and then proceed with the PEG precipitation. We usually used 10% w/v PEG6000 and 1M NaCL.
I hope it might help you!
BR, A
- Badges
- Science method