Abstract:
A process for isolating microorganisms from samples, particularly  Shigella  spp. from food samples, and a system, apparatus and composition therefor are provided. Magnetic particles are used to capture microorganisms and a system having separate magnetically-based apparatuses for collecting, concentrating and retrieving is used to isolate the magnetic particles having bound microorganisms. The apparatus for concentrating magnetic particles utilizes a small magnet assisted by vibration to concentrate collected particles at a localized region on the bottom of a container. The process, system and apparatus of the present invention are simple and inexpensive providing improved magnetic particle recovery adaptable to large scales.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. provisional patent application U.S. Ser. No. 60/924,001 filed Apr. 26, 2007, the entire contents of which is herein incorporated by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to a process for isolating microorganisms from samples, particularly food samples, and a system, apparatus and composition therefor. 
       BACKGROUND OF THE INVENTION 
       [0003]    The use of magnetic particle technology, particularly antibody-coated magnetic beads (immunomagnetic beads), for the selective isolation of microorganisms in microbiology in general and in food and environmental microbiology in particular is becoming more widely used. Different systems and individual pieces of equipment have been developed to assist in the use of magnetic particles. 
         [0004]    Many systems have been developed for collecting magnetic beads from small scale volume samples. Such systems typically handle samples of volumes from 1 ml (Eppendorf tubes, e.g. MagneSphere Technology Magnetic Separation Stand, Promega Cat. # Z5331, Z5332, Z5333 (two-position), Z5341, Z5342 and Z5343 (twelve-position) up to about 50 ml (Falcon tubes, e.g. PolyAtract System 1000 Magnetic Separation Stand, Promega Cat. # Z5410). Magnets are used to concentrate the magnetic beads at the side of the tubes and a pipette is used to either remove supernatant liquid or remove the beads directly. Magnetic pipette&#39;s, for example the PickPen™ product, may be used to remove the magnetic beads directly. Such systems are not well adapted for large sample sizes and large volumes of medium, thereby limiting their usefulness in the isolation of some types of microorganisms, e.g.  Shigella  spp according to Health Canada, Compendium of Analytical Methods for Food and Drug Administration, Bacteriologic Analytical Manual. 
         [0005]    Automated systems, for example the Pathatrix™ system from Matrix MicroSciences, peristaltic pumps, tubes and in-line filters to minimize human handling of samples. Such systems are very expensive, have problems with bead loss on the filters due to the formation of bio-films, and are prone to spillage when transferring the beads from the system. 
         [0006]    Other systems, for example the Kingfisher™ system, are based on the use of electromagnetic pins for capturing magnetic beads from an array of small-sized tubes (&lt;2 ml) and transferring the beads to new tubes for further processing. The electromagnetic pins may be used to hold beads while exchanging tubes, and then to release the beads into the new tubes. Their applications are limited to purification of DNA from PCR products or from gels. 
         [0007]    Various other systems use magnets in various ways to process magnetic beads. For example, United States Patent Publication 2005/0013741 published Jan. 20, 2005 discloses a device for immobilizing and re-suspending magnetic particles during washing and elution steps. The device comprises two permanent magnets which are movable along the side of a tube containing the magnetic particles in a liquid. 
         [0008]      Shigella  spp. presents a greater challenge to its rapid detection as these microorganisms generally contaminate food samples at much lower concentrations than other species of microorganisms and are poor competitors. We believe the detection and/or isolation of  Shigella  spp., especially from food samples, requires processing samples on a larger scale, preferably assisted with specific antibodies coated on magnetic beads. 
         [0009]    There remains a need for simple, inexpensive and flexible processes, systems and apparatuses for selectively isolating microorganisms from samples, especially food samples, particularly rapidly and on a large scale, and particularly for  Shigella  spp. 
       SUMMARY OF THE INVENTION 
       [0010]    There is provided a process for selectively isolating microorganisms from a sample, the process comprising: providing a container containing a medium for selectively enriching the microorganisms; adding the sample containing the microorganisms to the medium; adding to the medium magnetic particles adapted to bind the microorganisms of interest; magnetically collecting the particles with bound microorganisms at a bottom of the container; aided by vibrating the container, magnetically concentrating the particles with bound microorganisms at a localized region on the bottom of the container; and retrieving the particles from the localized region with a magnetically assisted pipette. 
         [0011]    There is further provided a system for recovering magnetic particles from a mixture in a container, the system comprising: a magnetic particle collector having a first magnet for attracting the magnetic particles to a bottom of the container; a magnetic particle concentrator having a second magnet for concentrating the magnetic particles collected on the bottom into a localized region on the bottom, the concentrator having means for vibrating the container to assist in movement of the magnetic particles to the localized region; and, a magnetic particle pipette having a third magnet for retrieving the magnetic particles concentrated in the localized region. 
         [0012]    There is further provided an apparatus for concentrating magnetic particles into a localized region on a bottom of a container, the apparatus comprising: a support structure for supporting the container; a magnet positioned below the container when the container is supported on the support structure, the magnet having a magnetic field localizable at the region on the bottom of the container to concentrate the magnetic particles into the region, the magnetic field removable from the region to permit retrieval of the magnetic particles without interference from the magnet; and, means for vibrating the container to assist in movement of the magnetic particles to the localized region. 
         [0013]    There is further provided a composition for selectively enriching  Shigella  spp. comprising: water, tryptone, potassium phosphate dibasic, potassium phosphate tribasic, sodium chloride, glucose, polyethyleneglycol sorbitan monooleate, and Crystal Violet. 
         [0014]    The process, system and apparatus of the present invention are particularly useful for determining the presence of and/or assaying the amount of food pathogens in a food sample. Food pathogens include, for example, bacteria, parasites and viruses. Microorganisms are of particular interest, for example, bacteria. Microorganisms include, for example,  Aeromonas  spp.,  E. coli, Shigella  spp.,  Salmonella  spp.,  Listeria  spp.,  Campylobacter  spp.,  Clostridium  spp.,  Vibrio  spp.,  Staphylococcus aureus , and  Yersinia enterocolitica . The process, system, apparatus and composition of the present invention are particularly useful for the isolation, detection, measurement and/or enrichment of  Shigella  spp.  Shigella  spp. includes, for example,  S. boydii, S. dysenteriae, S. flexneri  and  S. sonnei.    
         [0015]    The process, system and apparatus are particularly useful on a relatively large scale. Most prior art processes, systems and apparatuses are adapted for tubes having volumes on the order of a few milliliters, e.g. up to about 50 ml, and to small sample sizes. Small volume and sample size limits the efficiency and effectiveness of microorganism growth, detection and measurement, reduces capturing yield of the microorganism and contributes to the difficulty of handling samples and the possibility of false negative results. In Canada, (Health Canada, Compendium of Analytical Methods) and the USA (Food and Drug Administration, Bacteriologic Analytical Manual), the legal sample size is 25 g and the volume of enrichment broth is 225 ml. Further, tubes typically have rounded bottoms and are therefore more difficult to handle. In contrast, the present invention can be practiced at much larger scales with containers having flat bottoms. Containers having volumes of over 100 ml, or even over 250 ml, for example about 500 ml, can be easily handled. Use of flat-bottomed containers, for example Erlenmeyer flasks, beakers and evaporating dishes are preferred. Samples having masses over 10 grams can be used, for example 25 grams. 
         [0016]    The process of the present invention involves providing a container containing a medium (e.g. an enrichment broth) for selectively enriching microorganism of interest and adding a sample containing the microorganism to the medium. The sample is preferably a food sample and the sample is preferably mixed with the medium initially before incubation. Microorganisms are then allowed to incubate for a time of from about 6 to about 24 hours. The microorganisms of interest will selectively grow on the medium, out-competing other microorganisms which may have been present in the sample. The medium is preferably a liquid (e.g. a nutrient broth). Media for enriching microorganisms are commonly known in the art, for example as disclosed in Health Canada, Compendium of Analytical Methods or Food and Drug Administration, Bacteriologic Analytical Manual. A liquid medium is preferred since a solid medium will need to be mobilized in a liquid prior to adding magnetic particles. 
         [0017]    For enriching  Shigella  spp.,  Shigella  broth and a broth containing the composition of the present invention (known as Shiga broth) are preferred. The composition of the present invention advantageously contains Crystal Violet which suppresses the growth of  Bacillus , common interfering bacteria in  Shigella  assays which are not eliminated on currently available media used to selectively grow  Shigella  spp., e.g.  Shigella  broth. The composition of the present invention may also include other antibiotics, for example novobiocin. 
         [0018]    Before adding the magnetic particles to the medium, it is sometimes desirable to filter the enriched medium. It is particularly desirable when a very evident bio-film has formed. When filtering is desired, the filter preferably does not inhibit the microorganisms of interest. Any suitable filter may be used. In one embodiment, a filter assembly may be used to filter the entire medium, the filter assembly comprising a Millipore Express Plus 0.22 μm filter in which the 0.22 μm filter is removed and replaced with a foam pad from Filtaflex Ltd. that does not inhibit microorganisms. 
         [0019]    The magnetic particles added to the medium are adapted to bind the microorganisms of interest, preferably by means of specific antibodies conjugated to the magnetic particles. Preferably, the magnetic particles are added in an amount of about 50-100 μl per 250 ml of medium, for example about 100 μl per 250 ml. Preferably, the magnetic particles are added to the medium with mixing and then the medium incubated to allow the microorganisms to bind to the magnetic particles. Incubation time is preferably about 15-30 minutes. 
         [0020]    The magnetic particles may be spherical or non-spherical. Spherical particles are preferred as non-spherical particles may kill microorganisms. Some examples of magnetic particles include Cortex Megacell™-Streptavidin magnetic particles, Cortex Megabeads™-Streptavidin CM3454 (8.8 μm particle size and coated with magnetizable polystyrene/iron oxide particles), Cortex Megabeads™-Streptavidin CTM-CM019 (15.6 μm particle size and coated with polystyrene copolymer/iron oxide particles), Dynabeads™ M-280-Streptavidin (3-4 μm particle size), and Genpoint BugTrap™ magnetic beads. 
         [0021]    Cortex Megabeads™-Streptavidin CTM-CM019 (15.6 μm particle size and coated with polystyrene copolymer/iron oxide particles) conjugated to  Shigella  antibodies (monoclonal and/or polyclonal) and Genpoint BugTrap™ magnetic beads which are universal for capturing gram positive and negative bacteria, have diameters in a range of about 15 μm. These are preferred over the non-spherical Cortex Megacell™-Streptavidin magnetic particles. More preferable yet are the BugTrap™ binding beads from Genpoint AS, Oslo, Norway, which have diameters in a range of about 2.5-15 μm. The Genpoint BugTrap™ binding beads can be used even when a bio-film is present in the medium, and these beads are in a ready to use kit and are coated with a ligand for capturing Gram positive as well as Gram negative pathogenic bacteria (Canadian Patent Publication 2,397,067 published Jul. 26, 2001). 
         [0022]    Preferably, the particles are immunomagnetic particles, more preferably immunomagnetic beads, comprising one or more monoclonal and/or polyclonal antibodies that specifically bind to an antigen on the microorganisms of interest. A mixture of immunomagnetic particles comprising different antibodies specific for different species of the microorganism genus of interest may be used. Some examples of species specific antibodies for  Shigella  spp. are: monoclonal anti- Shigella sonnei ; clone 1028/437 cat. # MAB755 from CHEMICON-Millipore; Polyvalent D from Danka Seiken Co. for  S. sonnei ; anti-Shigella IgG with biotin from Cortex Biochem for  S. boydii, S. flexneri , and  S. dysenteriae  (cat. # CR1243RB); and, polyclonal antibody to  Shigella  spp. with biotin from Acris Antibodies GmbH ( S. boydii  ATCC #8700,  S. flexneri  ATCC #29903 and  S. dysenteriae  ATCC #13313). Antibodies to other specific species may be raised by known methods and incorporated into an immunomagnetic particle. 
         [0023]    Immunomagnetic particles typically comprise a core magnetic particle coated with an avidin (e.g. streptavidin), in turn coated with biotin. The biotin is in turn coated with the antibody or antibodies. Methods for constructing immunomagnetic particles are generally known in the art (e.g. Safarik, I. and Safarikova, M. “Magnetic techniques for the isolation and purification of proteins and peptides.”  BioMagn. Res. Technol.  2 (2004) 7). 
         [0024]    The magnetic particles with bound microorganisms are then recovered by magnetically collecting the particles at a bottom of the container, magnetically concentrating the particles at a localized region on the bottom of the container, and retrieving the particles from the localized region with a magnetically assisted pipette. To accomplish this, the system and apparatus of the present invention are preferably employed. 
         [0025]    The system comprises a magnetic particle collector, a magnetic particle concentrator and a magnetic particle pipette that cooperate to recover the magnetic particles from the medium. 
         [0026]    The magnetic particle collector is used first and comprises a magnet that attracts the magnetic particles to the bottom of the container that contains the medium. The magnet is preferably large, preferably having a surface at least as large as half the area of the bottom of the container. The magnet may be at least as large as the bottom of the container or larger than the bottom of the container. The magnet preferably has a large and extensive magnetic field to attract magnetic particles as far away as the upper surface of the medium in the container. Thus, magnetic particles throughout the entire medium are attracted to the bottom of the container. The magnet may be any suitable shape, for example cylindrical. Thickness of the magnet is preferably at least one-fifth that of its diameter. The magnet may be, for example, a permanent magnet or an electromagnet. A block magnet such as the Huge-Field Magnet from Filtaflex Ltd. is one embodiment of a suitable magnet. The magnet may be made of any suitable material, for example, neodymium-iron-boron alloy or samarium-cobalt alloy. The magnetic particle collector may have a protective cover for storing it when not in use. An enclosure around the magnetic particle collector may be used to protect it from magnetic objects drawn to it from the surroundings. A label warning users about possible injury if magnetic objects are brought too near the magnet may also be affixed to the magnetic particle collector. 
         [0027]    The magnetic particle concentrator is then used to concentrate the magnetic particles into a localized region on the bottom of the container. A preferred embodiment of the concentrator is an apparatus of the present invention. 
         [0028]    To effect movement of the particles into the localized region, the apparatus comprises a magnet that is positioned below the container when the container is in the apparatus. The magnet has a magnetic field localizable at the region on the bottom of the container. The magnet may be a permanent magnet or an electromagnet. The magnet is preferably small having a surface significantly smaller than the bottom of the container, with the size of the magnet determining the size of the localized region. The magnet preferably has a magnetic field large enough to attract particles from the furthest edge of the bottom of the container. The localized region is preferably at the center of the bottom of the container and the magnet is preferably positioned under the center of the bottom of the container. 
         [0029]    The magnetic field generated by the magnet is removable from the region once the magnetic particles have been concentrated there in order to permit retrieval of the magnetic particles without interference from the magnet. The magnet may be physically removed from the region to lessen or eliminate the effect of the magnetic field at the region. The magnet may be moved, for example, by use of a handle conveniently located on the concentrator, for example on a side or front. If the magnet is an electromagnet, the magnetic field may be removed by switching the electromagnet off. 
         [0030]    To further assist movement of the particles to the localized region, the apparatus advantageously further comprises means for vibrating the container. Any suitable means for vibrating the container may be used, for example vibrating arm or arms, vibrating base, sonicator, etc. If a sonicator is used, care should be taken not to kill the microorganism. Vibration raises the particles slightly off the bottom thereby making it easier for the magnet to move the magnetic particles through the medium to the localized region without re-suspending the particles in the medium as a whole. Preferably, lateral vibrations are applied to the container. Vibrations should not be severe enough to shake the container thereby re-suspending the particles throughout the medium as a whole. 
         [0031]    The apparatus further comprises a support structure for supporting the container. The support structure may comprise a base on which the container sits. The support structure may be composed of any suitable material, for example plastic (e.g. polycarbonate), metal (e.g. aluminum) or a combination thereof. To assist in determining whether the magnetic particles have all been concentrated into the localized region, all or part of the support structure, particularly the base, may be transparent. Further, the apparatus may further comprise one or more mirrors to assist in observing the bottom of the container from underneath the container. Furthermore, the apparatus preferably further comprises controls and gauges for controlling and displaying various operational parameters such as vibration time and speed. 
         [0032]    The magnetic particle pipette is then used to retrieve the magnetic particles from the localized region. In one embodiment, the magnetic particle pipette is similar to commonly available micropipettes (e.g. Eppendorf, Gilson, PickPen™) with some differences. The magnetic particle pipette useful for the present invention is larger than either the Eppendorf, Gilson or PickPen™. Also, unlike the Eppendorf, the magnetic particle pipette is also equipped with a magnet to retain magnetic particles. Unlike the PickPen™, the magnetic particle pipette has a central plunger for particle retrieval and a side lever for tip removal, which reduces accidental loss of particles due to inadvertent activation of the side lever. The magnet may be a permanent magnet or an electromagnet. 
         [0033]    After recovering the magnetic particles with bound microorganisms, the magnetic particles may be washed. Washing is preferably accomplished with TALON™ binding and washing buffer in a small volume container (e.g. an Eppendorf tube). Preferably, a buffer having a pH in a range of from about 7.5 to about 8.0 is used to wash the particles. The wash solution may be removed, for example with a pipette, after collecting the particles using the magnetic particle collector of the present invention or any other magnetic particle separation technology (e.g. MagneSphere Technology Magnetic Separation Stand, Promega Cat. # Z5331, Z5332, Z5333 (two-position). The magnetic particles with bound microorganisms may be assayed directly or frozen for storage at −80° C. until later analysis for downstream needs, e.g. isolation, serology, ELISA, DNA extraction, PCR, hybridization, etc. Freezing may be accomplished using, for example, a CryoStor™ (Innovatek Medical Inc., Vancouver, British Columbia, Canada). 
         [0034]    Any suitable analytical technique may be used to detect and/or measure the microorganisms of interest that have been bound to the particles. For example: the particles may be plated on a medium (e.g. agar) and the microorganisms cultured; DNA may be extracted from the microorganisms on the particles and amplified with PCR; serology may be performed directly from the beads (e.g. add  Shigella  specific antibody and observe clumping); or an assay (e.g. ELISA) may be performed directly from the beads. Such techniques are well known in the art. 
         [0035]    The collection, concentration and retrieval of magnetic particles in the process of the present invention have been divided into separate steps using separate apparatuses in a system. As a result, the present invention has a number of advantages. For example, in comparison to prior art, microorganisms may be isolated on a much larger scale, there are fewer problems with contamination, microorganism capture is more efficient and most or all of the magnetic particles may be retrieved, leading to more consistent and reproducible results. Since, the apparatuses used in the present invention are much less complicated, lower in cost and more amenable to scale-up than equipment required in many prior art processes, the entire process of the present invention is lower in cost than prior art processes. Further, the invention is particularly adaptable for effectively isolating  Shigella  spp., which are usually present in samples only at very low levels and heretofore have been difficult to isolate. 
         [0036]    Further features of the invention will be described or will become apparent in the course of the following detailed description. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0037]    In order that the invention may be more clearly understood, embodiments thereof will now be described in detail by way of example, with reference to the accompanying drawings, in which: 
           [0038]      FIG. 1  is a side schematic view of a magnetic particle collector for collecting immunomagnetic beads on a bottom of an Erlenmeyer flask resting on the collector; 
           [0039]      FIG. 2A  is a schematic front perspective view of a magnetic particle concentrator for concentrating immunomagnetic particles into a localized region on a bottom of an Erlenmeyer flask resting in the concentrator; 
           [0040]      FIG. 2B  is a schematic rear perspective view of the magnetic particle concentrator depicted in  FIG. 2A ; 
           [0041]      FIG. 2C  is a schematic top perspective view of the magnetic particle concentrator depicted in  FIG. 2A ; 
           [0042]      FIG. 2D  is a schematic bottom perspective view of the magnetic particle concentrator depicted in  FIG. 2A ; 
           [0043]      FIG. 3  is a front schematic view of a magnetic particle pipette for retrieving immunomagnetic beads from a localized region at a bottom of an Erlenmeyer flask; 
           [0044]      FIG. 4A  is a schematic front perspective view of a second embodiment of a magnetic particle concentrator of the present invention; 
           [0045]      FIG. 4B  is a schematic rear perspective view of the magnetic particle concentrator depicted in  FIG. 4A ; 
           [0046]      FIG. 4C  is a schematic top perspective view of the magnetic particle concentrator depicted in  FIG. 4A ; 
           [0047]      FIG. 4D  is a schematic bottom perspective view of the magnetic particle concentrator depicted in  FIG. 4A ; 
           [0048]      FIG. 4E  is a schematic side perspective view of the magnetic particle concentrator depicted in  FIG. 4A ; 
           [0049]      FIG. 5A  is a side schematic view of a second embodiment of a magnetic particle collector; 
           [0050]      FIG. 5B  is a side schematic view of a third embodiment of a magnetic particle collector; 
           [0051]      FIG. 6A  is a schematic plan view of a tilting frame of a third embodiment of a magnetic particle concentrator of the present invention; 
           [0052]      FIG. 6B  is a schematic view of a section along A-A of the tilting frame of  FIG. 6A ; 
           [0053]      FIG. 6C  is a schematic plan view of a chassis of the third embodiment of the magnetic particle concentrator; 
           [0054]      FIG. 6D  is a section view along C-C of the chassis of  FIG. 6C  together with an electric motor; 
           [0055]      FIG. 6E  is schematic side view of a vibrating assembly of the third embodiment of the magnetic particle concentrator; 
           [0056]      FIG. 6F  is a schematic plan view of the chassis together with the vibrating assembly of the third embodiment of the magnetic particle concentrator; and, 
           [0057]      FIGS. 7A-7C  depict schematic cross-section views of a second embodiment of a magnetic particle pipette. 
       
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     Process, System, Apparatus and Composition 
       [0058]    Referring to  FIGS. 1-3 , a first embodiment of a process of the present invention utilizing a system, apparatus and composition of the present invention is now described. 
         [0059]    A nutrient broth of the present invention specific to  Shigella  spp. is prepared from a basal medium and an antibiotic supplement. The ingredients and their amounts are shown in Table 1. 
         [0000]    
       
         
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
             
             
               
                 Basal Medium (pH = 7.0 ± 0.2) 
               
             
          
           
               
                 Tryptone 
                 20.0 
                 g 
               
               
                 Potassium phosphate dibasic (K 2 HPO 4 ) 
                 2.0 
                 g 
               
               
                 Potassium phosphate monobasic (KH 2 PO 4 ) 
                 2.0 
                 g 
               
               
                 Sodium chloride (NaCl) 
                 5.0 
                 g 
               
               
                 Glucose 
                 1.0 
                 g 
               
               
                 Tween ™ 80 (polyethyleneglycol sorbitan monooleate) 
                 1.5 
                 ml 
               
               
                 Distilled water 
                 1.0 
                 L 
               
             
          
           
               
                 Supplement 1 
               
             
          
           
               
                 Novobiocin 
                 50 
                 mg 
               
               
                 Distilled water 
                 1.0 
                 L 
               
             
          
           
               
                 Supplement 2 
               
             
          
           
               
                 Crystal Violet 
                 1 
                 g 
               
               
                 Distilled water 
                 1.0 
                 L 
               
               
                   
               
             
          
         
       
     
         [0060]    The basal medium (i.e.  Shigella  Broth by Difco™) is prepared by mixing the ingredient and heating the mixture to completely dissolve the various ingredients in the distilled water. The basal medium mixture is autoclaved at 121° C. for 15 minutes, and allowed to cool to 50° C. Supplement 1 is a 0.005% novobiocin solution sterilized by filtration through a 0.45 μm membrane filter. Supplement 2 is a 0.1% Crystal Violet solution prepared by suspending Crystal Violet in sterile distilled water. The two supplements are prepared separately. To prepare the final broth, (“Shiga” broth), 2.5 ml of supplement 1 and 1 ml of supplement 2 are aseptically added to 225 ml of the basal medium in a 500 ml Erlenmeyer flask and mixed thoroughly. 
         [0061]    Immunomagnetic beads are prepared based on magnetic beads coated with streptavidin (e.g. Cortex Megabeads™-Streptavidin CTM-CM019, Dynabeads™ M-280-Streptavidin) or other ligands (e.g. Genpoint BugTrap™ magnetic beads). The coated beads are then conjugated with biotin coated anti-Shigella IgG (e.g. Polyvalent D, a  S. sonnei  specific antibody from Danka Seiken Co., Japan; and/or, anti-Shigella-Biotin IgG from Cortex Biochem, USA, which are specific to  S. boydii, S. flexneri  and  S. dysenteriae ; and/or Acris Antibodies GmbH ( S. boydii  ATC #8700,  S. flexneri  ATC #29903, and  S. dysenteriae  ATC #13313). A mixture of the various types of antibody-conjugated beads may be used in the process. 
         [0062]    A food sample (25 g) suspected of being contaminated with  Shigella  spp. is added to 225 ml of the broth in a Stomacher bag and incubated for 10 min with gentle massage. The homogenate is then transferred into a 500 ml Erlenmeyer flask and incubated at 42° C. for 6 to 24 hours. After incubation, 50 to 100 μl of the mixture of immunomagnetic beads is added and mixed into the broth and the broth and beads mixture is incubated for 30 minutes to allow the  Shigella  bacteria to bind to the immunomagnetic beads. 
         [0063]    Referring specifically to  FIG. 1 , immunomagnetic beads to which  Shigella  bacteria are bound are then collected on the bottom of the Erlenmeyer flask. Erlenmeyer flask  102  rests on magnetic particle collector  100 , which is a large block magnet having upper surface  101  that is larger in surface area than the surface area of bottom  103  of the Erlenmeyer flask. The magnet is a Huge-Field Magnet from FILTAFLEX Ltd. Immunomagnetic beads  105  (only one indicated in the Figure) in broth  104  are drawn down to the bottom of the flask under the influence of the magnetic field of the magnet. 
         [0064]    Referring specifically to  FIGS. 2A-2D , after all of the immunomagnetic beads have collected on the bottom of the Erlenmeyer flask, Erlenmeyer flask  102  is placed in magnetic particle concentrator  200 . Magnetic particle concentrator  200  comprises a support structure for supporting the flask, a magnet positioned below the flask for concentrating the immunomagnetic beads into the center of the bottom of the flask, and means for vibrating the flask. 
         [0065]    The support structure comprises base  202  upon which the flask rests, housing  201  for housing components of the concentrator and a pair of side walls  203  for supporting top strut  204 . Top strut  204  has aperture  215  through which a pipette may be inserted as will be described later. The support structure also comprises arm support  205  for supporting arms  206  which are part of the means for vibrating the flask. 
         [0066]    Movable magnet  207  is mounted on magnet armature  209  and is movable up and down by actuation of handle  208  which moves lever  217  connected to the armature. The handle is connected to the lever. Magnet  207  is positioned just below the center of the bottom of flask  102 . Depressing the handle moves the magnet down away from the flask. Raising the handle returns the magnet to its position just below the flask. 
         [0067]    The means for vibrating the flask comprises a pair of arms  206  between which the flask rests. The arms are curved to accommodate the contours of the flask. The arms are connected to spindles  211  and the spindles connected to rocker bar  214 . The rocker bar is mounted on first pulley  212  and the first pulley is mounted on a drive shaft of electric motor  210 . Second pulley  213  is mounted on the housing and connected to the first pulley by belt  216 . Counterweight  218  is connected to the second pulley by a shaft. Electricity is supplied to the motor from power cord  219  and the motor is switched on and off by actuation of switch  220 . 
         [0068]    To concentrate the immunomagnetic beads at the center of the bottom of flask  102  (shown in phantom in  FIG. 2A ), the flask is placed in the concentrator between arms  206  such that the arms are in contact with the flask, and magnet  207  is raised to its position just below the flask. Motor  210  is switched on and the motor rotates the drive shaft which in turn rotates first pulley  212 . Rotation of the first pulley in turn rotates second pulley  213  by virtue of belt  216 . Rotation of the second pulley is eccentric due to counterweight  218 . The eccentricity of rotation of the second pulley creates a vibration in the first pulley which is transmitted through rocker bar  214  and spindles  211  to arms  206 . Vibration of arms  206  is transmitted to flask  102  and thence to the flask&#39;s contents. Magnet  207  attracts the beads toward the center of the bottom of the flask, and vibration of the flask causes the beads to be slightly raised off the bottom of the flask thereby assisting in movement of the beads by eliminating friction between the beads and the bottom of the flask. 
         [0069]    Referring specifically to  FIG. 3  with further reference to  FIGS. 2A-D , immunomagnetic beads  105  (only one indicated in the Figure) concentrated into center  106  of the bottom of Erlenmeyer flask  102  are then retrieved with magnetic particle pipette  300 , without removing the flask from the concentrator. For clarity, the concentrator is not depicted in  FIG. 3  and reference to the concentrator is in connection with  FIGS. 2A-2D . Movable magnet  207  of concentrator  200  is lowered away from the bottom of the flask by actuation of handle  208  to reduce interference with the retrieval process. The pipette comprises extendable magnetic tip  301  which can be extended and retracted by action of spring-loaded plunger  303 . The pipette also comprises removable rubber tip  302  which may be removed by action of spring-loaded slide  304 . 
         [0070]    The pipette operates as follows. A sterile rubber tip is first fitted over the magnetic tip by inserting the magnetic tip into the rubber tip up to stop  306 . The rubber tip is held in place by friction. Magnetic tip  301  is extended by depressing plunger  303 ; and the pipette is inserted through aperture  215  in top strut  204  of the concentrator and thence into the flask so that the magnetic tip is in the concentrated beads. Immunomagnetic beads  105  collect on the magnetic tip. Keeping spring-loaded plunger  303  depressed, the pipette is withdrawn from the flask with the beads remaining attached to the magnetic tip. The magnetic tip is inserted into a wash solution and the beads transferred to the wash solution by withdrawing the magnetic tip allowing the beads to be pushed off the magnetic tip by the rubber tip. Withdrawing the magnetic tip is accomplished by releasing spring-loaded plunger  303 . Rubber tip  302  is disposed of by depressing spring-loaded slide  304  which pushes the rubber tip off the end of the pipette. 
         [0071]    Washing the recovered beads is accomplished with a washing and binding buffer solution (e.g. TALON™ binding and washing buffer from Dynal Biotech; B&amp;W Buffer from the BugTrap Bacteria Isolation Kit from Genpoint AS, Oslo) having a pH of 7.5 to 8. The wash solution is removed, for example with a pipette, after collecting the beads using the magnetic particle collector of the present invention. The magnetic particles with bound microorganisms may be assayed directly or frozen for later analysis. 
       Second Embodiment of Magnetic Particle Concentrator 
       [0072]    Referring to  FIGS. 4A-4E , a second embodiment of the magnetic particle concentrator is now described. In this embodiment, magnetic particle concentrator  500  comprises a support structure for supporting the flask, a magnet positioned below the flask for concentrating the immunomagnetic beads into the center of the bottom of the flask, and means for vibrating the flask. 
         [0073]    The support structure comprises base  502  upon which the flask rests, housing  501  for housing components of the concentrator and arm support  505  for supporting arms  506  which help hold the flask in place. The arms are unitized in a single elongated generally U-shaped element with curved ends to accommodate the contours of the flask. Arm support  505  comprises lower block  505   a  and upper block  505   b . Supporting arms  506  are clamped in place between the lower block and upper block with the upper block bolted to the lower block by bolts  505   c.    
         [0074]    Magnet  507  and mirror  517  are mounted on opposite faces of support cylinder  504  which is mounted on magnet support shaft  509 . The magnet support shaft is rotatable by actuation (e.g. rotation) of handle  508  connected to the shaft. Magnet  507  is initially positioned just below the center of the bottom of the flask. Once the magnetic particles have been concentrated, rotating the handle through 180-degrees moves the magnet down and away from the flask and raises the mirror to the position previously occupied by the magnet. The mirror assists in retrieving the magnetic particles by aiding visualization of the concentrated particles from below the flask. Rotating the handle through another 180-degrees returns the magnet to its position just below the flask. Rotation can be clockwise or counterclockwise. 
         [0075]    The means for vibrating the flask comprises base  502  on which the flask rests. The base is a unitized plate having a generally annular portion on which the flask rests and generally rectangular portion  502   a  which extends from the generally annular portion under arm support  505  toward the rear of the apparatus. Proximal the end of generally rectangular portion  502   a , a drive shaft of electric motor  510  (7.4 V, 0.6 A) is engaged with vibration block  503  which sits tightly within an aperture in generally rectangular portion  502   a  of the base. When the motor is switched on, the motor causes the vibration block to vibrate and vibrations from the vibration block are transmitted through the generally rectangular portion of the base which is in contact with the vibration block. Vibrations from the generally rectangular portion are transmitted through the base to the generally annular portion which in turns vibrates the flask resting on the generally annular portion. Vibration isolator  511  reduces transmission of vibrations to housing  501  when the motor is on. Vibrations reaching the generally annular portion of base  502  from generally rectangular portion  502   a  can be controlled by vibration controller  512 . The vibration controller comprises a frustoconical element connected to the housing through the base by bolt  513 . Tightening bolt  513  engages the frustoconical element more tightly against the base thereby restricting motion of base  502  thereby reducing transmission of vibrations from the generally rectangular portion to the generally annular portion. Loosening bolt  513  permits greater freedom of motion for base  502  thereby increasing transmission of vibrations from the generally rectangular portion to the generally annular portion resulting in more vigorous vibration of the flask. The motor is switched on and off by actuation of switch  520 , and electricity is supplied to the motor from power cord  519 , which may be connected to a step-down transformer. 
         [0076]    To concentrate immunomagnetic beads at the center of the bottom of a flask resting on the generally annular portion of base  502 , the flask is placed in the concentrator between arms  506  such that the arms are in contact with the flask, and magnet  507  is raised to its position just below the flask. Motor  510  is switched on and the motor rotates the drive shaft which vibrates vibration block  503  which in turn vibrates generally rectangular portion  502   a  of base  502 . Vibration of base  502  is transmitted to the flask and thence to the flask&#39;s contents. Magnet  507  attracts the beads toward the center of the bottom of the flask, and vibration of the flask causes the beads to be slightly raised off the bottom of the flask thereby assisting in movement of the beads by eliminating friction between the beads and the bottom of the flask. Once the beads have been concentrated in the center of the bottom of the flask, magnet  507  may be rotated away from the bottom of the flask to be replaced by mirror  517 , which assists in visualizing the location of the beads for retrieval. 
       Second and Third Embodiments of Magnetic Particle Collector 
       [0077]    Referring to  FIGS. 5A and 5B , second and third embodiments of magnetic particle collectors are now described. 
         [0078]    A second embodiment of a magnetic particle collector as depicted in  FIG. 5A  comprises a powerful neodymium-iron-boron magnet  405  of a size covering at least half of the surface area of bottom  403  of Erlenmeyer flask  402 . The magnet is attached to and sits on the surface of backplate  406 . 
         [0079]    A third embodiment of a magnetic particle collector as depicted in  FIG. 5B  comprises a powerful samarium-cobalt magnet  415  of a size covering at least half of the surface area of bottom  413  of Erlenmeyer flask  412 . The magnet is recessed in the surface of backplate  416 . 
         [0080]    In both the second and third embodiments of the magnetic particle collector, the backplate has the following features. It comprises a material of high magnetic permeability and susceptibility, for example mild steel or transformer iron. It is larger than the magnet and is approximately the same diameter as the flask. It has a thickness of at least one-fifth of it diameter. The backplate is in good contact with the magnet and is more or less symmetrically disposed around the magnet. The backplate modifies the magnetic field around the magnet to increase the magnet&#39;s strength in an upwards direction toward the flask. The backplate increases by about 3-fold the magnetic field strength experience by contents of the flask. 
         [0081]    Any enclosures and/or protective covers may protect both the magnet and the backplate. 
       Third Embodiment of Magnetic Particle Concentrator 
       [0082]    Referring to  FIGS. 6A-6F , a third embodiment of the magnetic particle concentrator is now described. 
         [0083]    Referring specifically to  FIGS. 6A and 6B , a tilting frame  814 , illustrated in plan in  FIG. 6A  and in section in  FIG. 6B  along line A-A in  FIG. 6A , carries magnet assembly generally denoted at  802 . Tilting frame pivots on pivot  815  allowing magnet assembly  802  to be raised to a raised position (solid lines in  FIG. 6B ) and lowered to a lowered position (dotted lines in  FIG. 6B ) as required so that it is either in contact with the centre of the bottom of flask  801  or far enough away that the magnetic field has a negligible effect on magnetic particles in the flask. Actuation of handle  817  raises and lowers tilting frame  814 . A suitable detent means is be used to keep the tilting frame and therefore the magnet assembly in either the raised or lowered positions. 
         [0084]    Magnet assembly  802  produces a strong magnetic field in a direction more or less parallel with the bottom of flask  801  extending to the perimeter of the bottom of the flask. The magnetic field is stronger at its centre so that all magnetic particles in the flask, including those close to or touching the bottom of the flask, experience an attraction toward the centre of the bottom of the flask, while those close to the centre experience an accentuated attraction toward the centre. As illustrated in blow-out B in  FIG. 6B , the magnet assembly comprises disk or cylindrical magnet  818  carrying at its centre hemispherical magnet  819  with backplate  820  of high permeability and susceptibility to increase magnetic strength in an upward direction in a similar manner to that of the backplate illustrated in  FIG. 5 . Both magnets are of either neodymium-iron-boron or samarium-cobalt alloys. Other conformations could be used, for example, a single conical magnet in place of magnets  818  and  819 . 
         [0085]    A step in the process of the present invention is to draw magnetic particles in flask  801  towards the centre of the flask and to concentrate them into an easily removable pellet. Concentration is assisted by vibrating the flask about its vertical axis and above and close to the magnets. 
         [0086]    Referring specifically to  FIGS. 6C and 6D , chassis  808  illustrated in plan and section along line C-C, carries motor  811  of suitable speed. On the shaft of motor  811  and projecting through chassis  808  is an eccentric  812  of suitable eccentricity. Chassis  808  also carries flanged circular bushing  809  and low-friction surface  810  such that vibrating plate  804  can be vibrated about a vertical axis centered on bushing  809 . Means, such as an upper low-friction surface (not shown) that presses down on plate  804  may be used to hold vibrating plate  804  securely close to chassis  808  while it is vibrating. 
         [0087]    Referring specifically to  FIGS. 6C to 6F , vibrating plate  804 , illustrated in plan view in  FIG. 6F  showing planes along D-D and E-E from  FIG. 6E , has circular cutout  805  such that vibrating plate  804  can rotate smoothly on bushing  809 . Slot  813  slidingly accepts eccentric  812  such that the rotating eccentric  812  smoothly transmits an oscillatory motion to vibrating plate  804  in plane D-D. Stanchion  807  on vibrating plate  804  carries springy clamp arms  806  at a suitable height and of a shape to grip the periphery of flask  801  to hold the flask firmly with its axis centered above the centre of bushing  809 . Clamp arms  806  may be covered in resilient high-friction material such as a rubber where they contact the flask so that the oscillation of vibrating plate  804  is transmitted efficiently to the flask causing it to oscillate about its axis. 
         [0088]    To use the apparatus, flask  801  is inserted into clamp arms  806 . Magnet assembly  802  is brought up into contact with the bottom of the flask and motor  811  is switched on. Rapid oscillation of the flask about its axis overcomes forces that cause the magnetic particles to stick to the bottom of the flask thereby allowing the magnetic particles to migrate under the influence of the magnetic field towards the centre of the flask where the magnetic field is most intense. When the particles come close to the centre of the flask the very localized intense central magnetic field from magnet assembly  802  causes the particles to coalesce into a small pellet or button. At this point, motor  811  is stopped, and when the flask has come to rest the button of magnetic particles can be removed. 
       Second Embodiment of Magnetic Particle Pipette 
       [0089]      FIGS. 7A-7C  illustrate a second embodiment of a magnetic particle pipette in which  FIG. 7A  depicts various components of the pipette disassembled into three parts for clarity,  FIG. 7B  depicts an assembled pipette in a rubber tip-attaching configuration and  FIG. 7C  depicts the assembled pipette in a rubber-tip detaching configuration. 
         [0090]    Tubular pipette body  921 , conveniently of circular cross-section and of any suitable material (e.g. metal or rigid plastic), has slot  922  in a higher region of the tubular pipette body such that first knob  926  attached to slider  925  can move freely up and down. A smaller diameter section  935  in a lower region of the tubular pipette body has a diameter such that it can removably grip rubber tip  923 . Rubber tip  923 , fabricated of inert elastic material, is tapered and is closed at its narrow end. The rubber tip is conveniently a commercially available product sold under the trade name PickPen™ Tip by BIOCONTROL System, Inc. WA, USA. Friction plug  924  placed conveniently at the top of tubular pipette body  921  is useful to slidably hold magnet shaft  930  at whatever position a user desires. Slider  925  carrying first knob  926  and push-off wire  927  fits slidably inside tubular pipette body  921  and is conveniently maintained in a raised position by spring  929 , unless the user actuates it by pressing downwards on first knob  926 . Push-off wire  927  is looped (see blow-out F) at its bottom end  928  to loosely encircle the narrowed end of tubular pipette body  921  just above the top of rubber tip  923  when slider  925  is in its raised position. 
         [0091]    Magnet shaft  930  is of a suitable diameter and stiffness to slide up and down in tubular pipette body  921 . At its lower tip, magnet shaft  930  carries a small magnet  932 , preferably of neodymium-iron-boron or samarium-cobalt alloy, seated within a surrounding seat  931  of high magnetic permeability and susceptibility material to increase magnetic field strength in the downward direction (see blow-out G). Magnet shaft  930  also has second knob  933  by which the user can raise and lower magnetic shaft  930 , and stop  934  to prevent the user from raising the magnetic shaft too far. Magnetic shaft  930  is prevented from falling from the raised position by friction plug  924  as needed. 
         [0092]    To use the magnetic particle pipette, the user presses tubular pipette body  921  into rubber tip  923  so that rubber tip  923  remains attached to the body by friction, and then presses down second knob  933  to push magnet  932  into the tip of rubber tip  923  as illustrated  FIG. 7B . When the user then dips the pipette into a button of magnetic particles in the centre of the bottom of a flask, the magnetic particles are strongly attracted to magnet  932  and adhere to the outside of the tip of rubber tip  923  allowing them to be pulled out of the flask and introduced into a receiving vessel for succeeding stages of purification. The user then raises second knob  933  by thumb or finger to draw magnet  932  upwards thereby reducing the magnetic field holding the button of magnetic particles against rubber tip  923 . This allows the magnetic particles to fall off into the receiving vessel. Finally, as illustrated in  FIG. 7C , the user can move first knob  926  downwards causing loop  928  of push-off wire  927  to push the now contaminated rubber tip  923  from the pipette. 
         [0093]    Other advantages that are inherent to the structure are obvious to one skilled in the art. The embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims.