Rapid and highly sensitive detection of Flavobacterium psychrophilum using high gradient immunomagnetic separation with flow cytometry

Abstract The bacterial cold water disease caused by Flavobacterium psychrophilum is a concern not only for the aquaculture industry, but also the environment. We developed a novel method for detecting F. psychrophilum using high gradient magnetic separation (HGMS) with flow cytometry (FCM). HGMS is a magnetic separation method in which the magnetic force is strengthened by introducing a magnetic gradient between the magnetic filter and nearby column. F. psychrophilum cells that had reacted with immunomagnetic beads were loaded onto the column containing the magnetic filters. When the column was placed inside a neodymium ring magnet, the cells became stuck to the magnetic filters. When magnetic separation of a 500 ml sample was performed, more than 80% of the beads were trapped by the magnetic filter. The collected cells were easily removed from the filter by turning off the magnetic field. The cells were reacted with fluorescein isothiocyanate-labeled antibody, and then fluorescence was measured using FCM. The detection sensitivity was three orders of magnitude higher than that of an FCM assay without HGMS. The cell numbers determined by FCM correlated with those obtained using the colony counting method in the range of approximately 10 1 –10 5 colony forming units per milliliter. One FCM assay could be completed within 60 s and the total assay time, including sample preparation, was less than 2.5 h. The proposed method using HGMS and FCM allows for rapid and highly sensitive detection of F. psychrophilum .