Detection of immunomagnetically captured, 4 ',6-diamidino-2-phenylindole (DAPI)-labeled E-coli O157 : H7 by fluorescent microscope imaging

Live cells of E. coli O157:H7 were treated with 4',6-diamidino-2-phenylindo le (DAPI), a nucleic acid stain, for observation by epifluorescent microsco py. The treated bacteria. which exhibited minimal growth activity and parti ally retained respiratory function, were captured by goat anti-E. coli O157 serum coated on the surface of polystyrene based immunomagnetic beads (IMB ). The beads with captured bacteria were then concentrated by magnetic sepa rators. The efficiency of this magnetic concentration step was less than th at of using high speed centrifugation. The antibody-captured and IMB-immobi lized bacteria were then applied on HF-treated, bovine serum albumin (BSA)- coated microscope slides mounted on an automated stage, and magnetically al igned before fluorescence distribution was measured by a cooled CCD attache d to an inverted microscope. Since the beads were concentrated and linearly aligned along the edge of the magnetic field, image capture along the edge for a few field widths was sufficient to account for most of captured bact eria. This approach could reduce the enumeration time for and increase the efficiency, of bacteria counting by manual efforts The developed procedure of capturing, concentrating and enumerating E. coli O157:H7 exhibited a sim ilar applicability in buffer, pork carcass wash solution and apple juice as described in this study. The presence of about 100 cells/mL of the bacteri a could be detected in 30 min with developed procedure.