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

Authors: KNUPP, CHRISTOPHER - Michigan State University; LENNOX, SEAN - Michigan State University; SOTO, ESTEBAN - University Of California, Davis; Wiens, Gregory; LOCH, THOMAS - Michigan State University

Submitted to: Journal of Aquatic Animal Health
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/4/2026
Publication Date: 7/24/2026
Citation: Knupp, C., Lennox, S.M., Soto, E., Wiens, G.D., Loch, T.P. 2026. New culture medium enhances the isolation and culture of Flavobacterium psychrophilum, causative agent of bacterial coldwater disease and Rainbow Trout fry syndrome. Journal of Aquatic Animal Health. 38:197-213. https://doi.org/10.1093/jahafs/vsag011.
DOI: https://doi.org/10.1093/jahafs/vsag011

Interpretive Summary: Flavobacterium psychrophilum is a bacterial pathogen causing disease in salmon and trout that is expensive for fish farmers to prevent or treat. Observations suggest some variants of this bacteria grow poorly on current media formulations, thus impacting our ability to detect this bacteria. Scientists tested different nutrient mixtures that help bacteria grow and discovered that adding specific ingredients like Casamino acids and certain salts made the bacteria grow much better. Two new media (special nutrient mixtures) called FPM-A and FPM-B were developed that helped the bacteria grow over 141% more than standard media. When tested using samples from 300 salmonid fish, the new media allowed detection in 7.9-8.7% more fish than standard media. The new media provides a more effective tool for fish researchers to identify and study this harmful bacteria, which could help prevent disease and protect fish populations.

Technical Abstract: Flavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS) in salmon and trout, resulting in significant economic losses worldwide. Bacterial culture remains a gold-standard method for diagnosing F. psychrophilum infections and is a core component of many BCWD and RTFS research studies; however, observations suggest some variants grow poorly on current media formulations. Therefore, this study sought to develop a plate culture medium that enhanced the in vitro culture of a wide diversity of F. psychrophilum multilocus sequence typing (MLST) variants. Initially, the CFU yield of 165 geographically, temporally, and genetically diverse F. psychrophilum isolates was compared on three published F. psychrophilum culture media (i.e., tryptone yeast extract salts agar; TYES, Oplinger and Wagner medium, and enriched Anacker and Ordal medium supplemented with charcoal and aromatic compounds). The medium yielding the most CFU for the greatest number of isolates (i.e., TYES) was then modified following a Plackett-Burman experimental design, in which eleven nitrogen and two salt sources were assayed for yield effects. Casamino acids CaCl2, MgSO4, tryptose, and fetal bovine serum significantly influenced F. psychrophilum CFU yield. Guided by these findings, two new culture media [F. psychrophilum medium (FPM)-A and -FPM-B] were formulated, both of which significantly increased CFU yield (>141%) compared to TYES. Next, the F. psychrophilum detection capabilities of the new media were compared to TYES during surveillance at four Michigan salmonid spawning-sites. In total, 300 spawning-age salmonids belonging to four different species (i.e., Atlantic salmon, Salmo salar; Chinook salmon, Oncorhynchus tshawytscha; coho salmon, O. kisutch; and rainbow trout, O. mykiss) were analyzed, revealing that the new media recovered F. psychrophilum from 7.9 - 8.7% more fish than TYES. Collectively, results demonstrate that FPM-A and FPM-B support the in vitro culture of many F. psychrophilum genetic variants, improve F. psychrophilum detection from naturally infected fish, and will therefore serve as a resource for enhancing ongoing and future BCWD and RTFS research and diagnostic efforts.