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Quantitative detection of viable Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease, was evaluated by colony blotting and immunostaining. Bacterial colonies isolated from chum salmon Oncorhynchus keta ovarian fluids on a modified Cytophaga agar plate were blotted onto a nitrocellulose membrane and immunostained with antiserum against F. psychrophilum. Although the blotted colonies were strongly or weakly stained with the antiserum, blots from colonies of F. psychrophilum were distinguishable from those of other yellowish colonies by digital processing of the colony-blotted membrane photograph with an image-analyzing software. It was also confirmed that 12 strains of F. psychrophilum were all positive by the present method, while the subjected six isolates, which formed yellowish colonies but were not identified as F. psychrophilum by PCR targeting gyrB gene, and other reference six strains, F. branchiophilum, F. limicola, F. granuli, Pseudomonas flavescens, P. fluorescens, Chryseobacterium daecheongense, were all negative. From these results, the present procedure using colony blotting and immunostaining is useful for quantitative detection of viable F. psychrophilum from ovarian fluids and kidneys of chum salmon.