Magnetized Carbonyl Iron and Insoluble Zirconium Hydroxide Mixture Facilitates Bacterial Concentration and Separation from Nonfat Dry Milk

Abstract

A mixture of magnetized carbonyl iron and insoluble zirconium hydroxide was investigated for its ability to concentrate various foodborne pathogens from 25-ml samples of reconstituted nonfat dry milk. Each sample was artificially contaminated with 10(3) to 10(6) CFU/25 ml of representative foodborne pathogens (Salmonella enterica serovar Enteritidis, Listeria monocytogenes, and Bacillus cereus spores) and processed for bacterial concentration with high-speed centrifugation for the primary concentration followed by a secondary concentration step involving the carbonyl iron-zirconium hydroxide mixture. Bacterial recoveries, as evaluated on the basis of loss to discarded supernatants, exceeded 75% for all organisms at all inoculum levels and were usually >90%. Recovery was confirmed by direct plating of the immobilized pellet, for which the valueswere similar albeit more varied. Additional experiments confirmed that the magnetized carbonyl iron-insoluble zirconium hydroxide mixture was relatively nontoxic to both Salmonella Enteritidis and L monocytogenes Overall, the entire concentration scheme resulted in a 25-fold reduction in sample volume with the recovery of viable bacterial cells. This novel compound shows promise for facilitating inexpensive, rapid, and effective bacterial concentration in food systems.

Immobilization and Detection of Listeria monocytogenes

“…Immobilization data from detection times confirmed the data obtained by the plate count method with levels of recovery ranging from 96 to 99%. These recovery values are similar to those reported previously for L. monocytogenes and other food-borne bacteria (2,4,8,12). The automated procedure was simple and less time-consuming and gave results sooner (12 to 24 h versus 24 to 48 h).…”

“…Hydroxides of calcium phosphate (hydroxyapatite [HA]), zirconium, hafnium, and titanium are insoluble in aqueous solutions and can act as both affinity agents and solid supports for immobilization of microorganisms (3,11,12). Previous studies have suggested that immobilized cells remain viable, that multiple cells are immobilized on each MOH crystal, and that cell immobilization is nonspecific (2, 11).In previous studies, percentages of immobilization were determined by comparing the optical densities of immobilized cells to the optical densities of control suspensions without MOH (7), directly plating the immobilized cells with the MOH, and comparing the numbers of cells to the initial numbers (4, 12) or, assuming that the immobilized cells were viable, subtracting the numbers of cells left in the supernatants from the total numbers of cells before immobilization, both of which were determined by plate counting (2,4,8,12). The viability of immobilized cells was established by observing oxygen uptake using an oxygen electrode (11), by determining the increase in red coloration of immobilized Serratia marcescens cells during enrichment (11), by fluorescent viability staining of immobilized cells (2), or by directly spread plating and incubating samples of immobilized fractions at different times and comparing the numbers of cells to the numbers of cells in controls containing nonimmobilized cells (4).…”

“…The viability of immobilized cells was established by observing oxygen uptake using an oxygen electrode (11), by determining the increase in red coloration of immobilized Serratia marcescens cells during enrichment (11), by fluorescent viability staining of immobilized cells (2), or by directly spread plating and incubating samples of immobilized fractions at different times and comparing the numbers of cells to the numbers of cells in controls containing nonimmobilized cells (4). Information on the multiplication patterns of immobilized cells is limited.…”

“…C-5267; SigmaAldrich] and zirconium(IV) hydroxide [Zr(OH) 4 ; catalog no. 464171; Sigma-Aldrich] were each prepared by suspending 5 g in 50 ml buffered saline.…”

Strategy for selection of methods for separation of bioparticles from particle mixtures

“…Nevertheless, if the saturation magnetization is neglected and the maximum magnetic field gradient can be applied, the maximal F M that can be reached in biological systems is in the order of 1.5 Â 10 À4 N for 100 mm particles. Even though this force can possibly exceed the centrifugal force, it has been applied in biotechnology mostly on a small scale for the concentration and separation of cells (Comella et al, 2001;Cullison and Jaykus, 2002;McCloskey et al, 2003;Melville et al, 1975;Owen, 1978;Radbruch et al, 1994;Zborowski et al, 2003) and the purification of molecules by their adsorption onto magnetic adsorbents followed by recovery of the magnetic adsorbents (Heeboll-Nielsen et al, 2003;Hubbuch and Thomas, 2003).…”