New culture medium enhances the isolation and culture of <i>Flavobacterium psychrophilum</i> , causative agent of bacterial coldwater disease and Rainbow Trout fry syndrome

OBJECTIVE: Flavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and Rainbow Trout fry syndrome (RTFS) in salmonids, resulting in significant economic losses worldwide. Bacterial culture remains a widely used method for F. psychrophilum diagnostics and is core to BCWD and RTFS research; however, some isolates grow poorly on existing media. The primary goal of this study was to develop a solid culture medium that improves the in vitro recovery of F. psychrophilum across a diverse set of multilocus sequence typing variants. METHODS: Initially, CFU yields of 165 diverse F. psychrophilum isolates were compared across three established media, and the best-performing medium was selected for optimization. A Plackett-Burman screening design was used to identify influential medium components, after which two new media (F. psychrophilum medium-A [FPM-A] and F. psychrophilum medium-B [FPM-B]) were formulated and compared with tryptone yeast extract salts medium (TYES) under laboratory and field conditions. RESULTS: Five medium components (e.g., casamino acids, CaCl2, MgSO4, tryptose, and fetal bovine serum) significantly influenced CFU yield, guiding the formulation of FPM-A and FPM-B, which increased CFU yield by over 2.4-fold relative to TYES. During surveillance of 240 spawning-age salmonids (four species, four sites), FPM-A and FPM-B significantly improved F. psychrophilum recovery in comparison with TYES. CONCLUSIONS: The new media improved the recovery of diverse F. psychrophilum variants and improved culture-based detection from naturally infected fish. Both FPM-A and FPM-B performed similarly; however, FPM-A has fewer components, is less expensive to produce, and thus is recommended to support future BCWD/RTFS diagnostic and research efforts.