Development of an improved medium for primary isolation of Flavobacterium psychrophilum, cause of bacterial coldwater disease.



Cliff Starliper

USGS National Fish Health Research Laboratory

11649 Leetown Road, Kearneysville

WV. 25430

Ph: 304-724-4433


Sue Marcquenski

Wisconsin Department of Natural Resources

101 S. Webster St.

Madison, WI. 53707

Ph: 608-266-2871


Andrew Noyes

New York Department of Environmental Conservation

8314 Fish Hatchery Road

Rome, NY. 13440

Ph: 315-337-0910


August 2007




Cold Water Disease (CWD) is a serious disease and cause of mortality to salmonid fish species in the northern United States. The causative bacterium of CWD is Flavobacterium psychrophilum. Primary recovery of the pathogen and diagnosis of CWD is problematic because of the difficulty in isolation of F. psychrophilum using cytophaga medium (AO), a medium currently used by many fish diagnostic laboratories.  On AO, 3-4 days are required to produce F. psychrophilum colonies 2-4 mm in diameter.  This incubation time can allow contaminants to overgrow the primary plates, thus impeding diagnosis of the disease.  Early identification of F. psychrophilum can be greatly enhanced through the use of a bacteriological medium that will improve the bacterium’s growth and retard or inhibit overgrowth by common environmental contaminants.  An improved medium would serve as a useful tool for early treatment of infected fish and reducing overall mortality.  This study evaluated a new developmental medium, #2, in comparison to two other media, AO and EAO+FBS, the latter being the best medium for F. psychrophilum reported in the literature. The ingredients in #2 are 0.5% tryptone, 0.05% yeast extract, 0.05% beef extract, 0.02% sodium acetate, 0.02% calcium chloride, 0.05% magnesium sulfate, and 5% fetal bovine serum. Results from comparative growth studies in broth showed a significant increase (p £ 0.043) in cfu/mL with #2, when compared to AO and EAO+FBS. On agar format, there was no quantitative differences in the cfu/mL yield of the three media; however, colonies on #2 grew more luxuriant, and more importantly, could typically be enumerated one day sooner, compared to AO. The individual medium components of #2 were varied and evaluated to optimize the recipe; significant responses were noted with various serum supplements. Optimum performance was with fetal bovine serum. Isolates of F. psychrophilum and representative contaminants collected from primary isolation plates that were cultured from coldwater diseased fish were screened against antimicrobials to identify potential selective agents to incorporate into #2. Although fetal bovine serum does add cost to the medium, the other ingredients are all very inexpensive, particularly considering the small amounts used of each component. We feel the cost of serum is justified given the excellent colony growth of our large number of F. psychrophilum isolates, and the reliability that the #2 medium demonstrated; we did not have one culture that did not grow.