I have stocked my laboratory strain of Francisella (F. tularensis sub sp. holarctica LVS) in 100 % glycerol (800 microliter bacterial culture + 200 microliter 100% glycerol ) at -80C refrigerator. Now about 3 years later, I want to do experiment from my stocked bacteria. I am using BHIc culture media with supplementation and CDM media. But I could not reach mid log phage as before (Lower bacterial number than before). I am expecting a suggestion from expert's corner for reviving of Francisella LVS strain ?

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