Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2002
Publication Date: 12/27/2002
Citation: IRWIN, P.L., FORTIS, L.L., CHAU, L.I., GEHRING, A.G., TU, S. EVIDENCE FOR BUOYANCY-DEPENDENT IMB-SALMONELLA COMPLEX LOSSES DURING MAGNETIC PHASE SEPARATION. JOURNAL OF RAPID METHODS AND AUTOMATION IN MICROBIOLOGY. 2002. Interpretive Summary: The contamination of foods with pathogenic bacteria may lead to substantial food poisoning epidemics. To minimize outbreaks of food poisoning, effective isolation and detection procedures should be developed. Since the level of bacterial contamination in foods is usually very low (e.g., only a few cells in a large volume) one must first capture and concentrate them prior to detection. The most rapid process for achieving this goal involves capturing the pathogen onto small magnetic beads which have proteins on their surface which specifically bind to the pathogen. Once captured, the bead-bound pathogen can be concentrated by a permanent magnet. However, one problem associated with the magnetic bead protocol for pathogen isolation/concentration is that a significant number of captured cells are lost during each separation step. In this report we show that most of these losses are related to the increased bouyancy of the ebead-pathogen complex (e.g., the cells are less dense than the beads and thereby change the bouyancy). These magnetic separatory losses can be greatly lessened by the use of either a greater magnetic field or a longer isolation time. This information is useful for microbiologists in the detection of pathogenic bacteria in foods using virtually any detection method.
Technical Abstract: One question associated with the immunomagnetic bead (IMB) protocol for pathogen isolation/concentration is why a significant number of captured cells are lost during each magnetic separation step (MS). For instance, we observe an average IMB-Salmonella concentration decrease of 5 % per MS even at extremely low Salmonella Enteritidis cell densities. An [IMB-S.E.] change of -5 % per MS could be quantitatively significant since, in practice, more separation steps would be necessary when isolating organisms from environmental samples (e.g., more washing steps involved). These apparent losses in pathogen activity are not due to the injury/decease of IMB-bound bacteria inasmuch as numerous MS steps, accomplished without changing the supernatant, result in no significant diminution in bound Salmonellae. In this manuscript we show that these losses increase, up to about 15 % per MS, with [S.E.]:[IMB]. This latter observation argues that MS-dependent pathogen losses are related to changes in the bouyancy of the IMB S.E. complex since, at elevated pathogen levels, we show that as many as 4-6 Salmonella cells bind per IMB and decreases the IMB S.E. complex's density by about 8 % or 0.1 g per square cm.