Francisella noatunensis subsp. Orientalis (FNO) is a facultative intracellular bacterium and the causative agent of franciselliosis, a disease that affects tilapia production in cold water temperatures. Due to the intracellular nature of franciselliosis, it is difficult to diagnose and treat in production operations. Thus, necropsies are a valuable tool for diagnosis. To isolate the bacteria, different methods, media and supplements are utilized. This current trial aimed to optimize the conditions (assay I) and preservation of FNO (assay II) and verify the effects of the inactivated bacteria on the proliferation of leukocytes in vitro (assay III). In assay I, treatments groups were two nutritional supplements, Nile tilapia blood serum (TBS) and fetal bovine serum (FBS); two concentrations of these supplements (3 and 10%) and bacteria cultured at four different times (12, 24, 48 and 72h). In assay II, the treatments were stored at varying temperatures (-20°C, -80°C, and -196°C) to examine viability. Müller Hinton culture medium was supplemented with 1% glucose and 0.1% L-cysteine hydrochloride monohydrate, along with 3% TBS or FBS. This media was used as a standard, and experimental treatments consisted of different glycerol concentrations in the culture medium (either 20, 30, or 50%). In assay III, FNO was inactivated with formaldehyde, and the impact of this inactivation was evaluated with leukocytes from different organs. No FNO growth performance differences were discerned compared to 72 hours of cultivation. However, hemocytometer counts showed that after 12h of cultivation, the treatment with FBS at a concentration of 10% showed a higher amount of cells/µl than the other treatments (P=0.002). In 24 hours of culture, only the treatment with FBS of 10% showed a significant difference from the treatment with TBS of 3% (p= 0.0196). After 48 h of culture, FBS at a concentration of 3% showed a significant difference with TBS at 3% with the treatment of TBS at 10% (P= 0.622). For cryopreservation, a better performance was found using 20% glycerol in FBS (P = 0.297). However, after 7 d, there was a statistically significant difference in glycerol concentrations. When analyzing the temperature, the TBS with 30% glycerol at -80°C showed a significant difference between the FBS with 50% glycerol at -196°C (P=0.010). Concerning in vitro leukocyte responses at different bacterial concentrations, cell proliferation was examined following exposure to inactivated FNO. The leukocyte proliferation values of groups provided Concavalin A and 104 cells/μL were different (P= 0.014) compared to the 106 cells/μL group. In the spleen cell culture, there was a significant difference between the concentration of bacteria in the 105 cells/μL dilution in relation to the unstimulated control group (P=0.046). However, a significant difference was found between multiple comparations in liver cell culture. These investigations have provided additional insight into FNO storage, handling, and viability methodologies for fish health researchers and diagnosticians.