Have you tried King B medium?

simply by culturing on boor mineral medium or you can use plate count agar...

Dear Bahare,

I am a little surprised by the difficulty you seem to have with the stability of your Pseudomonas stored at -80°C. It is a standard way to maintain many different species of bacteria, including pseudomonads - and I have stocks in my -80°C freezer that are older than some of my students.

I make -80°C stocks by adding 300 µl 50% (v/v) sterile glycerol to 700 µl of over-night culture. I quickly mix by inverting the cryotube and freeze immediately at -80°C. Most strains are easily recovered by re-streaking onto an agar plate and incubating at the appropriate temperature - 20°C or 28°C for the strains I use.

I prefer incubating strains on King's B medium, but the Proteose Peptone No. 3 in the original formula is very expensive, so we now make 'KB*' using standard Proteose Peptone. This has had no obvious impact on our work (see Kusmierska & Spiers, dx.doi.org/10.1155/2016/4846565), but you need to be careful if you are no seeing the colony phenotype and growth you expected.

I wonder if the real problem you are having is with the growth of your pseudomonads after recovery from the -80°C stock. The expression of siderophore requires low iron concentration and usually happens latter in the growth phase - so if your media has a high level of Fe3+ because you are not using a medium like King's B or Pseudomonas selective media (PAS), or perhaps because your water supply is not so good, you may not see siderophore expression when expected.

For our model pseudomonad, P. fluorescens SBW25, I would not expect to see siderophore expression in an over-night (18 h, 20°C) shaken culture or in colonies incubated at 20° for 2 days. However, if you leave the cultures or colonies to incubate for another day, I'd expect to see the green-yellow fluorescence characteristic of this species.

perhaps you should set up some liquid cultures and plates and incubate them at 20°C or 28°C for up to 5 days. You can leave the cultures statically on the bench with loose lids (lots of oxygen) and check for fluorescence each day. leave them in direct sunlight if you can, as the UV makes the siderophore fluoresce - this is not as obvious under ordinary lights.

Andrew

which media whould you use for preparing stock culture bacteria in -80 degree?

thanks for your complete answer