New culture medium enhances the isolation and culture of Flavobacterium psychrophilum, 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.