Immunomagnetic separation using carbonyl iron powder and flow cytometry for rapid detection of Flavobacterium...

Bacterial cold water disease, caused by Flavobacterium psychrophilum, is a serious problem in the aquaculture industry worldwide. Several methods to prevent and treat cold water disease have been studied. Although detection at the early stage of F. psychrophilum infection is very important for the prevention and treatment of cold water disease, an effective detection method has not yet been developed. The use of flow cytometry (FCM) for the rapid determination of bacterial cell numbers with high sensitivity is beginning to attract attention. Immunomagnetic separation (IMS) has also been used to detect F. psychrophilum. The purpose of the present study was to develop a method to quickly determine the number of bacterial cells by combining the FCM and IMS methods. Because samples can be more effectively concentrated using smaller magnetic beads and stronger magnetism, we used carbonyl iron powder as the magnetic beads for the IMS. The detection level of F. psychrophilum using FCM combined with IMS was 5 orders lower than that using FCM without IMS. The values determined using FCM combined with IMS strongly correlated with those obtained using the colony-counting method, in the range of approximately 10-10(8) 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 h. The combined method of FCM with IMS developed in this study can be used reliably for the rapid detection of F. psychrophilum.