After screening for plaque formation, plaques are formed,then I have touched the plaque with sterile Pasteur end and dispersed in in 200ul of PBS and stored it in refrigerator. After repeating this experiment using stored stock , no plaques were formed ... Could anyone suggest an already tested method to store phage from plate for subsequent testing.
Thanks in advance
Hi Kareem, my experience in storing phage that degrade with freezing is to keep them in an Eppendorf tube (~1ml PBS) in the fridge with 50ul of chloroform.
Don't vortex or stir. The chloroform in the tube will kill anything trying to grow in the tube without denaturing the phage. so don't mix the tube or anything. The chloroform at the bottom will do its job if it is kept in peace.
thanxs alot for your consideration Dr. steingrimur and wan .....
but i want to ask if vortex or stirring could disturb my experiment or harm my phage??
Hello Kareem,
I hope your storage problems are solved. If it isn't, you can also try one or both of these methods taken from Chapter 19: Phage Production and Maintenance of Stocks, Including Expected Stock Lifetimes (Louis-Charles Fortier and Sylvain Moineau in Martha R. J. Clokie, Andrew M. Kropinski (eds.), Bacteriophages: Methods and Protocols, Volume 1: Isolation,
Characterization, and Interactions, vol. 501, C 2009).
Mix 0.5ml of phage lysate with 0.5ml sterile glycerol to obtain a final concentration of 50%. Place the cryogenic vials into an ethanol dry-ice slush to snap freeze the phages (5–10 min). Then store the vials at ≤ −70◦C for long-term storage.
Make a 50% glycerol:phage lysate solution and transfer in 0.5 ml into lysophilisation ampoules. Freeze at
Hi Kareem, I would not recommend vortexing M13 or lambda phage cultures.
Thank you so much for your attention... I also noticed that stirring only is more safe
Hello Sofy,
Bacteriophages stored under different conditions for long-term preservation. Bacteriophages may store for 2 years at 4-degree Celsius as broth lysates. During plaque assay, some evidence indicates that glycerol preservation method causes less damage to phage compare than the freeze-drying preservation method.
Up to 2 years, titers of broth lysates generally remaining higher than those of glycerol or freeze-dried preparations. Satisfactory outcomes of the plaque assay of phage as long time stored at 4-degree Celsius showed better titers than those kept at room temperature. However, all titers declined with time regardless of the conditions of preservation.
I suggest you store in 50% glycerol at -80oC. This is the method i used to store my phages.
depends on the phage. my T4 stocks are quite stable in SM without glycerol at 4C for months on end, whereas my phages from C difficile lose PFU/mL on a weekly basis (phage buffer) and are sensitive to being taken out of the fridge (ie warmed to RT) and then put back. As suggested, 1:1 glycerol:buffer at -80 is something of a gold standard for long term storage (months to years), although you will likely lose orders of magnitude upon freezing, so start with a high titre.
Mix your phages with your host strain, incubate them for 10 min at 37C and then add 15-20% glycerol and store them at -80C. If your phage is tailed then you can preserve them at 4C without any preservative in some buffer or just in LB broth. Also read
Alvi, I. A., Asif, M., Tabassum, R., & Abbas, Z. (2018). Storage of Bacteriophages at 4° C Leads to no Loss in Their Titer after One Year. Pakistan Journal of Zoology, 50(6).
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