I tried to revive 3years old Pseudomonas fluorescens from glycerol stocks which were at -20O C , these culture failed to grow on Kings B broth and even common media (Nutrient / LB).
Any better way to preserve them for long storage ?
If you have -80'C freezer in your lab, mix 0.5ml of your overnight culture with 0.5ml of 100% glycerol (final glycerol concentration of 50%) and store the prepared stock immediately into the -80'C freezer. Your glycerol stock will be soft at -80'C (like ice cream). If you don't let it thaw every time you use it, your stock should last at least for 10 years without any problem.
If you do not have deep freezer in your lab, you can store Pseudomonas strains in sterile tap water (about 10E4-10E6 CFU/ml) at room temperature for many years.
You can use Ringer solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2) instead of tap water. But, be sure that strains are not contaminated by other bacteria or bacteriophages.