METHOD FOR THE RAPID SEPARATION AND IDENTIFICATION OF MICROBIAL CONTAMINANTS FROM A COMPLEX MATRIX

Abstract: A method is provided for the separation and identification of a target microorganism from a complex matrix such as food. The method employs an aqueous two-phase polymer separation system to selectively enhance the number of the target microorganisms with respect to the background flora, followed by selective growth and identification of the target.

Description: METHOD FOR THE RAPID SEPARATION AND IDENTIFICATION OF MICROBIAL CONTAMINANTS FROM A COMPLEX MATRIX FIELD OF INVENTION The present invention relates to the field of microbiology and methods for the separation and detection of specific microorganisms from contaminated complex matrix. More specifically the invention relates to the use of an aqueous two phase polymer system to isolate members of a specific target microorganism. BACKGROUND Food borne bacterial contamination is a prominent health hazard in developed as well as developing countries. For example, two to three million cases of Salmonella food poisoning are estimated to occur each year in the United States. Acute symptoms of this disorder include nausea, vomiting, diarrhea, cold chills, fever, and exhaustion. About 2,000-3,000 (0.1%) people die each year in the United States from Salmonella poisoning, these victims usually including infants, the sick, and elderly. Poultry, red meat, seafood, eggs and any foods which contain these products particularly have the potential to carry bacterial pathogens. The challenge to a food microbiologist is to be able to recover small numbers of these select pathogens from food products which often contain large numbers of a variety of other bacteria. Recovery of sublethally injured bacterial pathogens is even more difficult. Heating, freezing, chemicals and other processing steps can sublethally injure or debilitate pathogenic bacteria in foods. It is estimated that over 5 million Salmonella analyses are run annually in the United States. Traditional detection of bacterial pathogens in clinical, food, dairy, environmental, and water samples involves growth of the particular microorganism of interest in at least two types of culture enrichment broths. Initially, the sample is added to one type of broth (pre-enrichment) and then allowed to incubate for a certain period of time at a particular temperature. A volume of this broth will then be added into a second type of (selective) enrichment broth and then incubated for a certain period of time at a particular temperature. The purpose of the enrichment process is to permit the growth of extremely low levels of a pathogen in a sample to sufficient levels for detection. For example, there may be only one viable Salmonella cell in a 25-gram food sample. Thus, there is a need to allow for growth of the Salmonella to high enough concentrations to permit detection in assays, such as pure culture assays, immunoassays, motility immobilization assays and hybridization probe assays. The most sensitive immunoassays and hybridization assays generally require the presence of at least 100,000 cells per ml and preferably 10,000,000 cells per ml in an enrichment culture. Typically, identification of food-borne bacterial pathogens involve 3 basic steps: 1. Non-selective or pre-enrichment growth: Generally a food sample is first homogenized into non-selective, liquid medium (pre-enrichment broth) , using a stomacher or blender. Food samples contain a diverse spectrum of bacteria, not all of which are pathogens. These non-pathogenic flora are referred to as background organisms while the pathogenic species to be detected are known as the target organisms. Often the target organisms contained in the food sample are injured in the food processing environment and require the non-selective broth for resuscitation. Depending on the food, the ratio of target-to-background organisms at the end of pre-enrichment can range from 1:1 to 1:100,000. A typical pre-enrichment medium for Salmonella is lactose broth with slight variations for different foods as described in the Bacteriological Analytical Manual. 6th Edition, Association of Official Analytical Chemists, Arlington, VA (1984) . 2. Selective Enrichment: Next the sample is incubated in a media which promotes selective growth of the target microorganism. This step allows the sample to be further enriched in growth-promoting medium which may additionally contain selectively inhibitory reagents. A continued increase of target organisms is achieved while simultaneously restricting proliferation of background bacteria. 3. Selective plating: This step employs solid selective media that restricts growth of background bacteria and eventually allows visual recognition of pure, discrete colonies suspected of being the pathogenic target. The relative numbers of isolated "suspect positive" colonies depends on the food matrix, and also the selective properties of the liquid and solid media. Hopefully there are adequate numbers of isolated suspected target colonies in the presence of a larger number of background colonies to allow statistical selection of the targets for early biochemical confirmation, i.e., presumptive positive or negative identification. 4. Presumptive iden ification: Suspect positive colonies are further inoculated in appropriate differential agars. If these tests are positive, the colony is deemed presumptively positive, and is then submitted to a battery of biochemical and serological tests for final confirmation and identification. Thus, with the conventional process, a suspect positive call can be made within 3 days, a presumptive positive within 4-5 days, and a confirmation and serological identification with 5-7 days. Because of the time involved for a positive identification there exists a need for a method of positively identifying food-borne bacterial pathogens much more rapidly. It would be desirable, for example, if one of the growth steps currently needed for pathogen determination could be eliminated or shortened allowing for a more rapid identification. Applicant has discovered that aqueous two phase partitioning has the potential for selectively enhancing the target to background ratios of bacterial contaminants from a food matrix, thus, in effect, substituting for step 2 of the prior art method discussed above. The technique of aqueous two-phase partitioning has been long applied to the separation of particles (Bronsted, J. N., Z. Phys . Chem. Abt . A 257, (1931)) . Per-Ake Albertsson was one of the first to realize the potential of the method for the separation of biological entities (Per-Ake Albertsson, "Partition of cell particles and macromolecules", 3rd edition, J. Wiley & Sons, (1986) . Since the work of Albertsson, partitioning in aqueous two-phase systems has been used to elucidate and determine cell surface properties, and separate proteins, enzymes, and nucleic acids, for example. U.S. Patent No. 4,980,065 teaches the use of aqueous two-phase system useful for the separation and purification of biochemicals and " optical isomers. PCT Patent No. WO 90/05768 describes use of polymeric two- phase systems for removing microbial contaminants to purify industrial lubricating agents. Aqueous two-phase partitioning has also been used in a variety of methods for the separation of biological cells. For example Johansson et al, (Anal . Biochem, 155, 215, (1986) ) present a review of the use of aqueous two-phase systems for the partitioning of a variety biological entities such as enzymes, nucleic acids, cell organells, eukaryotic cells, and bacterial cells including strains of E. coli, Salmonella, and Shigella . Smilarly, Tortoli et al., (Eur. J. Clin . Microbiol . Enfect . Dis . 12, 425, (1993) disclose an aqueous biphasic system for the isolation of mycobacteria from clinical specimens. The mechanism responsible for the ability of two- phase systems to separate different biological cells relies on the variation found in the biochemical composition of the cell walls or cell membranes exposed to the liquid systems. Biological cells and particularly bacterial cells have a wide variety of structures and biochemical macromolecules, e.g., fimbriae, flagella, proteins, acidic carbohydrates and lipopolysaccharides which are expressed on the cells surface. The type of structures and biochemical compounds and their degree of expression and concentration on the cell's surface define the physicochemical surface characteristics. It is well established that cells belonging to different genera, species or strains have distinct physicochemical surface characteristics which distinguish and differentiate them from other bacterial groups. A general set of surface properties for example, may be common to all bacteria, but the degree that these properties vary differentiates the cell populations from one another. These properties include surface charge, hydrophobicity/hydrophilicity, differences in cell density, size, and shape as well as "surface energies", which define surface forces coming into play when cells contact other cells and/or other types of surfaces/interfaces (solid, liquid or air) . Biphasic systems are uniquely qualified to capitalize on the difference between surface properties of bacterial cells to effect separation of even closely related strains. For example Levive et al., (J " . Liq. Chromatogr. , 7, 403, (1984)) teach the use of countercurrent partitioning chromatography to separate cells of Salmonella typhimurium whose surface properties were identical except for the proportion of lipopoly- saccharide molecules with long vs..short chain poly- saccharide chains. One of the most popular aqueous two-phase polymer systems comprises Polyethylene glycol (PEG) and dextran. PEG-Dextran systems have particular utility in the separation of bacterial cells. For example WO 9002948 demonstrates that polymeric two phase systems are useful for the quantitative or semi-quantitative determination of fungal spores or bacterial cells in grain products. Further, Xiu et al., (J. Gen . Microbiol . , 129, 3075, (1983) disclose the use of aqueous biphasic partitioning in dextran-polyethyleneglycol to separate Salmonella belonging to different serogroups, and Stendahl et al., (Acta Pathol . Microbiol . Scand. Sect . B Microbiol . , 85, 334, (1977)) make use of the difference in surface charge characteristics of smooth and rough Salmonella typhimurium to separate that bacteria using a biphasic system containing 6.2% (wt/wt) dextran 500 and 4.4% (wt/wt) poly(ethyleneglycol) 6000 followed by free zone electrophoresis. Although the above methods are useful for the separation of closely related bacteria there has been no effort to apply aqueous two phase systems to the problem of the isolation and detection of pathogenic bacteria from food or other complex matrices. Also, to date, aqueous two-phase systems have been used to separate related bacterial strains from each other, rather than for the separation of all members of a bacterial genus from a varied background bacterial population. Applicant has discovered that aqueous two-phase polymer systems may be adapted to separate and concentrate all members of a particular target bacterial genus from background microorganisms. The ability of the instant method to be both inclusive with respect to the target pathogen and exclusive with respect to background organisms and other elements of the complex matrix is unique in the art. Additionally, since biphasic separations require only minutes to perform, this method results in a considerable time savings over traditional methods which require selective growth procedures needing significant incubation time. Further, Applicant has discovered that use of the two-phase separation system allows for the detection of certain strains of pathogens not detected by conventional methods. Hence, the use of an aqueous two-phase polymer separation technique for the separation and identification of pathogens from a contaminated matrix represents a significant advance within the art of the microbial detection. SUMMARY OF THE INVENTION The present invention provides a method for the separation of a target microorganism contained within a complex matrix, comprising the steps of: i) homogenizing said complex matrix and incubating said complex matrix in the presence of a pre- enrichment media to resuscitate and grow said target microorganism whereby the number of said target microorganisms in said complex matrix is enhanced; ii) emulsifying a sample of said enhanced complex matrix of step i) in at least one aqueous polymeric two-phase separation system; and iii) allowing said emulsified system to separate into a top phase and a bottom phase, whereby a preponderance of said target microorganisms from the sample is found in either said top phase or said bottom phase. The invention also provides a method for the separation and detection of a target microorganism contained within a complex matrix, comprising the steps of: a) homogenizing said complex matrix and incubating said complex matrix in the presence of a pre- enrichment broth to resuscitate and grow said target microorganism whereby the number of said target microorganisms in said complex matrix is enhanced; b) emulsifying a sample of said enhanced complex matrix of step a) in at least one aqueous polymeric two-phase separation system; c) allowing said emulsified system to separate into a top phase and a bottom phase, whereby a preponderance of said target microorganism from the sample is found in either said top phase or said bottom phase; and d) removing an aliquot from that phase of step c) containing a preponderance of said target microorganism and detecting the presence of said target microorganism within the aliquot. In its preferred embodiments, target microorganisms are bacteria, the complex matrix is food, and the aqueous two-phase system is PEG/Dextran. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a typical phase diagram for any two- phase liquid polymer system. Figure 2 is a typical phase diagram for any two- phase liquid polymer system demonstrating methods of determining the polymer compositions of the top and bottom phases. Figure 3 is a phase diagram for PEG(6000) and Dextran(500) system. Figure 4A is a plot of a two-phase separation of an E. coli strain in PEG and Dextran in containing varying amounts of NaCl. Figure 4B is a plot of a two-phase separation of a Klebsiella oxytoca strain in PEG and Dextran in containing varying amounts of NaCl. Figure 5 illustrates the results of two-phase partitioning of E. coli and Klebsiella oxytoca with varying amounts of NaCl in the feedpoint composition. Figure 6 illustrates the single two-phase separation method for the isolation of target pathogenic microorganisms from contaminated complex matrices. Figure 7 illustrates the bifurcated two-phase separation method for the isolation of target pathogenic microorganisms from contaminated complex matrices. Figure 8 is data showing the reduction in target bacteria to background bacteria ratios of S. typhimurium in the presence of a mixed population of £". coli, E. cloacae and C. freundii from the time of growth of the mixture in pre-enrichment media to the end of the two-phase separation scheme. Figure 9 is data showing the reduction in target bacteria to background bacteria ratios of S. typhimurium inoculated into ground beef from the time of growth of the mixture in pre-enrichment media to the end of the two-phase separation scheme. Figure 10 is data showing the reduction in target bacteria to background bacteria ratios of S. typhimurium inoculated into dried milk from the time of growth of the mixture in pre-enrichment media to the end of the two-phase separation scheme. Figure 11 is data showing the reduction in target bacteria to background bacteria ratios of a multiplicity of Salmonella species inoculated into ground beef, chicken, ground turkey, and pork sausage from the time of growth of the mixture in pre-enrichment media to the end of the two-phase separation scheme. DETAILED DESCRIPTION OF THE INVENTION As used herein the following terms may be used for interpretation of the claims and specification. The term "matrix" or "complex matrix" will refer to any organic or inorganic material that will support the growth of a variety of microorganisms. The matrices of the present invention will be complex in nature and will comprise of a variety of different organic growth- supporting substances. Typical matrices include components of food matter, biological tissues, organic waste products, fecal material, blood, soil, ground water, sewage and the like. As used herein the term "stomacher" or "homogenizer" or "blender" will be used interchangably and will refer to an instrument capable of thoroughly blending and mixing microorganisms and food matrices. To "homogenize" means to physically blend or mix, such that the complex matrix is broken into small fairly uniform pieces of matter. In cases where the matrix is already finely-particulated, the "homogenizing" step is accomplished by simply stirring or mixing of the matrix to achieve uniform distribution of microorganisms. The term "aqueous two-phase system" or "aqueous polymer system" or "biphasic system" will refer to the combination of any two water soluble polymers which upon vigorous mixing will separate into two phases on the basis of differences between the molecular structure, ionic and electrostatic properties of the two polymers. A common two-phase aqueous polymer system is comprised of polyethylene glycol and Dextran although other polymers are possible and often used. Other typical biphasic systems may include but are not limited to: hydroxypropyl starch - PEG/H 2 0; ficoll - PEG/H 2 0; ficoll- Dextran/H 2 0; polyvinyl alcohol - Dextran/H 2 0; sodium carboxymethyl dextran - PEG - NaCl/H 2 0; PEG - potassium phosphate/H 0; and polyampholytes acrylic co- polymers . Additionally other combinations are possible, where either one or both polymers are either nonionic or polyelectrolytes, or where one of the polymers can be substituted with a low molecular weight component. Multi-phase, preferably two-phase aqueous systems may be used either singularly or as part of a sequential "countercurrent" system. As used herein the term "countercurrent separation" will refer to two-phase aqueous partitioning where at least two stages of an aqueous two-phase system are used and where the relative wt % composition of the polymers in each stage is the same after emulsification has been carried out and equilibrium has been attained. In this fashion, a higher selectivity for the desired target organism is obtained. The term "target microorganism" or "target organism" or "target bacteria" will refer to a micro- organism to be isolated and identified by the present method. Target microorganisms may be either prokaryotic, or eukaryotic and may be members of defined mixed cultures, or exist as contaminants in complex matrices. Target microorganisms of particular interest are food-borne bacteria. The term "background microorganism" or "background bacteria" or "background organism" will refer to any organism that is found in the presence of the target organism but is not the target organism. Like target organisms, background organisms may be either prokaryotic or eukaryotic, and may or may not be related genetically or biochemically to the target organism. Those background organisms of most interest in the contest of the present application are food-borne bacteria. The term "preponderance of target microorganisms" of a sample means the majority of the target micro¬ organisms from that sample. The term "selective growth media" will refer to either a solid or liquid growth media specifically formulated to enhance the growth of the target organism and discourage the growth of background organism. Additionally, selective growth media may contain components designed to facilitate the detection of target organisms, generally by visual inspection. The term "pre-enrichment media" will refer to either a liquid or solid media designed to encourage the growth of both target and background microorganisms. Microorganisms are inoculated and incubated in pre- enrichment media to enhance the numbers of all micro¬ organisms present and to resuscitate any target organism that may have been damaged in initial sample processing. The pre-enrichment media of the present invention is buffered to allow for the variations in pH of a variety of different food matrices. The term "enriched" or "enhanced" culture will refer to the culture obtained after incubation in pre- enrichment media, wherein the number of target and non- target microorganisms has grown. The term "feedpoint composition" will refer to a starting composition of at least two liquid phase polymers, prior to mixing and emulsion and calculated on the basis of total wt % of each polymeric component present and on the basis of a phase diagram. The present invention provides a method for the rapid isolation and identification of target micro¬ organisms from a complex matrix. A sample of the matrix, suspected of containing the target microorganism is first homogenized and then inoculated into a pre- enrichment medium. Incubation in the pre-enrichment medium serves to enhance the numbers of both background microorganisms and the target microorganism to be detected. After a sufficient incubation time a sample of the enriched culture is subjected to separation via a biphasic countercurrent two-phase aqueous polymer system, typically comprising PEG and dextran. Separations preformed in the two-phase system serve to isolate (i.e., preferentially select) the target organism over the background and facilitate the identification procedure. The identification process can be accomplished, as in the instant preferred embodiment, using selective plating and visual identification. Alternative methods of identification, however, could include, for example, genetic amplification techniques such as the polymerase chain reaction to yield sufficient target DNA for homogeneous or hybridization assays; or antibody detection methods using Elisa-type assays, for example. Target enrichment; The minimum industry standard for the detection of food-borne bacterial pathogens is a method that will reliably detect the presence of one pathogen cell in 25 g of food matrix. In order to meet this stringent test enrichment methods and media have been developed to enhance the growth of the target pathogen cell in order to facilitate it's detection by biochemical, immunological or nucleic acid hybridization means. Typical enrichment procedures employ media that will not only enhance the growth and health of the target organism but will also promote the growth of any background or non-target microorganisms present . For example the U.S. Food and Drug Administration endorses a Salmonella assay procedure described in Andrews et al. , "Isolation and Identification of Salmonella Species," Chapter 7 in Bacteriological Analytical Manual. 6th Edition, Association of Official Analytical Chemists, Arlington, VA (1984) . In this procedure, at least two enrichment steps are described. The first step is a "pre-enrichment step" wherein the food sample is enriched in a nutritious, nonselective broth medium to restore injured Salmonella cells to a stable condition and to promote growth. Next, a sample of the pre- enrichment broth (i.e., first enrichment broth) is added to two selective enrichment broths (i.e., second enrichment broth) , wherein the sample is further enriched in a growth-promoting medium containing selective reagents. Each selective enrichment broth allows a continued increase of Salmonella organisms while simultaneously restricting proliferation of most other bacteria. The present invention has improved the above target enrichment methods by using specially formulated pre- enrichment media particularly adapted for the detection of bacterial pathogens from food matrices. Two different media have been developed for use in preferred embodiments of the present invention, one specifically for Salmonella detection and the other for Listeria . These media are highly buffered to allow for the variations of pH of a variety of food matrices. The Salmonella medium consists of lactose broth, potassium phosphate, mannitol, magnesium chloride and peptone. The Listeria medium consists of brainheart infustion broth, glucose, lithium chloride, ferric citrate, polymeric acid and potassium phosphate. The selective enrichment step of the bacterial target, typical in standard methods is eliminated and replaced in the present invention with the two-phase aqueous separation system described below. An indication of the effectiveness of the defined pre-enrichment media of the present invention is seen when growth of Salmonella and Listeria in the instant media are compared against the media suggested by the FDA and described in the Bacteriological Analytical Manual. 6th Edition supra . Comparison of the Salmonella pre-enrichment broth (BBSM) and FDA's BAM lactose broth can be made by comparing the specific growth rates of pure Salmonella strains in both media. Given the S-shaped growth curve that organisms follow when growing in liquid media, the exponentially growing part of the curve can be described by the following equation: where N = cell concentration at any time [=] cells/ml. No = initial cell concentration at time 0 it = specific growth rate (of the organism) [=] 1/hr. t = growth time [=] hr. The specific growth rate (it) is also related to the doubling time (tv) or mean generation time, if one sets N=2No, i.e., one doubling: t D = (1/Ψ) ln2 = 0.69 (1/ιt) The specific growth rates of Salmonella strains growing in BBSM medium compare favorably with specific growth rates in FDA's BAM lactose broth. The specific growth rates are compared below: It is evident that BBSM is at least comparable to lactose broth. Aσueous Two-phase Polvmer Separation Systems: Liquid polymer phase separation is particularly well suited for the separation of biological entities as it is a very gentle method of fractionation, yet is able to take advantage of the vast array of surface and physico-chemical properties that differentiate biological materials . The simplest systems involve a mixture of two immiscible aqueous based polymers in a specified ratio. Vigorous mixing of the polymer systems will result in first a turbid emulsion and finally the separation of the polymers into two distinct phases. Selection of the appropriate two-phase system for the problem at hand is imperative. For the separation of biological entities, (and particularly of biological cells where it is important to maintain cell viability throughout the separation process) the phases should be mild and consideration must be given to water content, ionic compositions, osmotic pressure and denaturing effects. Most of these considerations eliminate phases containing organic solvents and leave primarily aqueous based polymers. The basis for separation by two-phase systems is the selective distribution of substances between the phases . For soluble substances distribution takes place mainly between the two bulk phases, and the partition is characterized by the partition coefficient K, C-t C b where Ct and C b are the concentration of the partitioned substance in moles per liter of top and bottom phase respectively. The partition coefficient is not dependant on the volume of the phases but is affected by properties of the two phases, properties of the partitioned substance and by temperature. The mechanism governing partition is not fully understood, however it appears to be the composite result of interactions of hydrogen, ionic and hydrophobic bonds of the particle and the two polymer phases. in general, properties having the greatest effect on partition are surface charge of the particle, size of the particle and particle hydrophobicity. For a complete review of the theory governing the distribution of particles in a two- phase system see Per-Ake Albertsson, supra or U.S. 5,110,733 herein incorporated by reference. Single stage separations are often sufficient for the separation of particles with uniform partitions coefficients such as DNA of bacterial spores. Other materials with varying partition coefficients will require multistage procedures such as countercurrent distribution. Countercurrent two-phase systems employ at least two stages of an aqueous two-phase system where the relative wt % composition of the polymers in each stage is the same after emulsification has been carried ' out and equilibrium has been attained. In counter- current techniques a sample is first subjected to separation in a first stage. After the two phases come to equilibrium, a fixed amount of the target-rich phase is transferred into the second stage. The second stage contains the specific balance of polymers that, once mixed with the transferred target-rich phase, result in relative wt. % composition of the polymers which is equal to that of the first stage. As the number of stages increases to the order of tenths or hundreds, the "countercurrent" system begins to behave like a chromatography column in separating mixtures via variable residence time. Repeated dividing and remixing of the phase gives further and further purification. Typical countercurrent methods often require 50 to 60 separate extractions to obtain acceptable purity of a particular substance. In theory, any number of stages can be used in the countercurrent embodiment, however for the purpose of the present invention a three stage method is preferred, and where necessary, for Salmonella isolation, for example, the three stage method can be bifurcated. A bifurcated two-phase separation method refers to a method incorporating two distinct and different sets of aqueous two-phase systems, each having three stages and being carried out in a countercurrent fashion. In the bifurcated approach a first portion of the sample is subjected to separation in the first two-phase system, while a second portion of the sample is subjected to separation in the second two-phase system. Upon completion of the third stage of both systems, samples for the target rich phase of each two-phase system is plated onto separate selective growth media. The target rich phases contain an enriched amount of the target microorganism or reduced amounts of the background microorganisms or a combination of both. In this fashion, a higher selectivity of the desired target organism is obtained. Phases that will become target rich are pre-determined based on simple-stage partitioning experiments of pure cultures conducted using the appropriate aqueous two-phase systems. Suitable Polymers: A variety of polymer systems consisting of at least two aqueous polymers are suitable for the present invention and may include but are not limited to: polyethylene glycol/dextran; polypropylene glycol/dextran; polyethylene glycol/polyvinyl alcohol; polyethylene glycol/Ficoll (r) (copolymer of sucrose and epichlorohydrin, from Pharmacia Fine Chemicals, Uppsala, Sweden) ; polyvinyl alcohol/dextran; methyl cellulose/dextran; polypropylene glycol/polyvinyl pyrrolidone; charged polyethylene glycol/dextran; polypropylene glycol/methoxypolyethylene glycol; polypropylene glycol/polyvinyl alcohol; polypropylene glycol/hydroxypropyl dextran; polyethylene glycol/polyvinyl pyrrolidone; polyethylene glycol/starch; and polyvinyl alcohol/methyl cellulose polyampholytes-polyvinyl alcohol. Additional combinations of these and other polymers are also suitable and well known from the literature (Per-Ake Albertsson, supra) . The polymer system preferred for the present isolation comprises the polymers of polyethylene glycol (PEG) and dextran (Dx) . PEG is a linear synthetic polymer available in a variety of molecular weights . PEG is quite stable in solution, in the dry powdered or flake form in which it is sold. Common fractions of PEG supplied by Union Carbide Corporation used in the present phase partitioning process include PEGs having a molecular weight in the range of about 300-50,000 preferably in the range 300-30,000 such as PEG 300, PEG 400, PEG 3,400, PEG 8,000 and PEG 20,000. Other commercial names for PEGs include polyglycol E (Dow Chemical) , carbowax (Union Carbide) , and pouracol E (BASF) (Wyandotte Corp) . PEG stock solutions of 30-40% by weight are made up by accurate weighing of the powder and dissolution in water at room temperature. PEG dissolves readily on stirring for a couple of hours and fresh, properly stored PEG powder usually contains less than 0.5% water. The stock concentration can be measured by refractive index. Dextran, predominantly poly( alpha-1,6 glucose) is commercially available in a range of molecular weight fractions from approximately 2,000 to 2 million. Similar molecular weights for derivatives are also useful. Stock solutions of dextran of 20-30% by weight are made by mixing the powder into a paste with an equal weight of water, then adding enough water by weight to yield an apparent concentration approximately 10% higher than desired in order to allow for water in the dextran. Dextran stock solutions are stirred 2-3 hours at room temperature, but the time may be shortened with heating. Concentrations of dextran stock solution can be determined by polarimetry. Adding salts, such as potassium phosphate, sodium chloride, ammonium sulfate, magnesium sulfate, copper sulfate, sodium sulfate and lithium sulfate, mono- saccharides and disaccharides to the water soluble polymer to enhance the solubility, phase separation or chirality are also considered to be within the scope of the present invention. The aqueous two-phase system may be prepared from the stock solutions of polymer. The stock solutions may contain known buffers to control the pH and salts to increase the tonicity of the solutions. Preferably, potassium phosphate buffer is used to adjust the pH. These buffers and salts are selected according to the nature of the material to be separated and can be readily determined by one skilled in the art using the criteria set forth above. For example, since the water soluble polymers are generally ion-free and have large molecular weights, their contribution, at high concentrations, to the tonicity of a solution is small. If a mixture to be separated comprises a bacterial cell which would be destroyed by low ionic strength solutions, the addition of salts would be indicated. The tonicity of the phases can be measured by using a vapor pressure osmometer. Additives such as salts, buffers, sugars and polymers may be added to enhance phase separation and separation factor. Suitable additives include potassium phosphate, glucose, glycerol, butylcellusolve, propyl- alcohol and sodium chloride. Additives which can increase the difference in density or hydrophobicity between the two phases, reduce the viscosities of either phase, or increase the interfacial tension between the two phases, will enhance phase separation and separation factor. The temperature and pressure can also be manipulated to enhance phase separation and partition coefficient. Suitable temperatures span the range from where the aqueous two-phase system begins to freeze up to the boiling point of the water, in general from about -10°C to 100°C. Because viable target microorganisms are desired, however, the preferred temperature of the invention is from 0°C to between 37°C and 43°C. The range of pressure includes all pressures which result in a two-phase system, in general from 0.01 to 1,000 atmospheres. The preferred pressure is ambient pressure. Although a variety of PEG and Dextran of varying molecular weights are available, preferred PEG is PEG(8000) or PEG (3400) and preferred dextran is DxT500. Selection of Two-Phase System: Selection of the appropriate polymers for a specific separation is facilitated by use of the phase diagram. In a mixture of two polymers and water a two- phase system will arise when the constituents are present in a certain range of proportions. The constituent composition at which phase separation occurs may be represented in phase diagram such as shown in Figure 1. In Figure 1 the concentration of the two polymers is plotted as wt % of the polymer in water. The curved line separating the two areas is called the binodial locus of points. All mixtures which have compositions represented by points above the line give rise to two-phase separation whereas mixtures represented by points below the line give a homogeneous solutions. Two-phase systems that are in equilibrium are illustrated by the phase diagram of Figure 2. If point A on line C-B in Figure 2 represents the composition of the total system then the composition of the bottom and top phases will be represented by points B and C respectively. Point A represents the feed-point composition which is the composition of the components of the polymer systems added to the system before emulsion. After emulsion and settling the relative compositions of the top and bottom phases changes, and it no longer reflects the feed-point parameters. Thus, phase diagrams may be used to determine the compositions of both phases if the composition of the feed-point is known. For a complete discussion of the theory governing phase diagrams and their use in determined the composition of two-phase system see Per-Ake Albertsson, supra or U.S. 5,110,733 herein incorporated by reference. The present invention provides an improvement over standard techniques used for two-phase separation of biological entities to effect the isolation of a specific population of microorganisms from a complex matrix contaminated with background microorganisms . Applicant's method takes advantage of the parameters of microbial cell surfaces, and two-phase system properties, and has successfully modeled the dynamics of cell/two-phase systems to develop effective specific two-phase protocols for particular target micro¬ organisms. A suitable stepwise procedure for determining the appropriate feed-point compositions for the isolation of the desired target organisms is as follows: a) select the type of polymers, molecular weight, poly er-ligands, electrolytes, buffer capacity, settling time and temperature to be used on the basis of the cells or biological entities to be separated; b) construct the two phase diagram of the above system, either by using Gel Permeation chromatography or turbidity as the transition indicator between one phase and two phases and vice versa as described by Albertsson supra; c) select a feed-point well within the binodial curve and select several target and background microorganisms that are likely to be encountered in the samples; d) grow pure cultures of these microorganisms in a pre-enrichment medium to be used as part of the microorganism isolation process so as to achieve a balance of target and background organisms typically presented to the isolation procedure; e) conduct single-stage partitioning experiments with each of the above pure cultures by determining cell numbers of target and background cells in the culture using techniques and methods well known in the art (i.e.. Coulter Counter device, optical density, plating on non-selective agar etc.); f) sample top, bottom and also the emulsified (mixed) phase for target and background cells; g) determine the cell fraction in top and bottom phases and interface by writing appropriate mass-balance equations; h) compare the partitioning results of all target and background organisms where maximum separation of targets from background in either the top or bottom phase is optimal; i) if separations are inadequate, select a feed point closer to the binodial curve and repeat steps d) through h) ; j) if the operating conditions render the two- phase unattractive, go to step a) . Two-phase polymer system for the separation of Salmonella and Listeria : The present two-phase systems useful for the separation of Salmonella and Listeria are comprised of PEG and Dextran which were chosen on the basis of their prior use for cell separation (P. A.Albertson supra) . A starting feed point composition of PEG and dextran was determined on the basis of the phase diagram shown in Figure 3. Combinations of between 5 and 6.5% Dextran in water and between 3.5 and 6.5% PEG in water were used based on partitioning data of pure cultures of target and background microorganisms using the stepwise feed- point selection method described above. After the polymer system was selected the concentration of electrolytes and buffers was determined using pure cultures of six bacterial strains. The concentrations of electrolytes was determined on the basis of the partitioning behavior of pure target and background microorganisms in response to varying electrolyte concentrations, and also with the knowledge that NaCl phosphate buffer is carried into the two-phase system when fixed volume inoculum from the pre-enrichment broth is added into it. Additionally PEG-bis (amine) (a positively charged PEG molecule) is added to the two- phase system to take advantage of the cell surface charge characteristics and enhance the partitioning of the target and background microorganisms. Bacteria were subjected to two-phase partitioning in 4% (w/w) PEG (MW 8000) in 0.012 potassium phosphate buffer and 5% (w/w) Dx (MW 500,000) in 0.012 potassium phosphate buffer containing varying amounts of NaCl. Figure 4A illustrates the results for one such partitioning with E. coli . As can be seen in Figure 4A increasing concentration of NaCl in the feed point composition of the two-phase system resulted in a shift of E. coli found in the top phase first to a majority of the bacteria found in the bottom phase and finally, at high salt concentrations to a predominance of the cells at the interface of the two phases. This general pattern held true for Klebsiella oxytoca (Figure 4B) and all bacterial strains tested, however, the relative concentrations of cells in each phase varied at different rates. For example at 0.04M NaCl and cell concentrations of 5.8X109 and 1.2 xl08 for E. coli and K. oxytoca respectively indicated that 88% of the E. coli cells and 96% of the K. oxytoca cells were concentrated at the top and bottom phases respectively. Next, tests were run on two-phase partitioning of mixed binary cultures with varying amounts of NaCl. Figure 5 illustrates the results of two-phase partitioning of E. coli and Klebsiella oxytoca with varying amounts of NaCl in the feed-point composition. Mixed culture data varied from the pure culture in that only 46% of the E. coli cells moved to the top phase at 0.04M NaCl while 95% of the K. oxytoca cells stayed at the bottom phases, and 52% of the E. coli cells migrated to the interface. Based on these and similar results from other mixed cultures it was decided that countercurrent separation was a preferred method for the separation of bacterial cells. Therefore, on the basis of empirical tests with potential target bacterial pathogens and using knowledge of cell surface properties Applicant has developed a method to tailor a countercurrent two-phase separation system for each target microorganism. Figure 7 outlines the bifurcated three-stage countercurrent two-phase separation system for the Salmonella genus. One ml samples of pre-enrichment cultures were added to two different two-phase separation systems, A and B. System A is designed to partition the target bacteria into the top phase while system B is designed to partition the target bacteria into the bottom phase. The counter¬ current procedure is conducted as described above for only three stages at which point samples of the target containing phase are plated on selective differential media and incubated for detection. It should be noted that in experiments which included food matrices the food matrix generally and unexpectedly partitioned into the interface. Thus by the end of the third-stage, little or no contaminating food matrix remains in the system. Development of the bifurcated process was necessary to allow for the isolation of all possible strains and species of the Salmonella target bacteria. Empirical data suggested that there was sufficient difference in the partition coefficients of Salmonella strains that not all strains could be reliably partitioned into the same phase without a very high number of partitions and without loosing genus inclusivity. For example, the Salmonella genus is very diverse, consisting of about 2,000 strains. The present method had to be inclusive for all Salmonella strains and enable one to isolate all Salmonella from any food matrix. Thus to reduce the number of partitions needed and to allow for total inclusivity of all target bacterial strains the bifurcated process was devised. Because there is less variation of cell surface properties among Listeria species and genera than with Salmonella, and species strain variations within the genus of Listeria is less than Salmonella, only a single, three-stage aqueous two-phase system was needed to partition Listeria . The Listeria targeting aqueous two-phase system was based on partitioning experiments of pure Listeria and other competitive microorganisms and is also tailored to the Listeria pre-enrichment medium. Food matrices: In order to test the present method for application to field situations, a broad variety of raw and processed foods were mixed with various strains of Salmonella and Listeria and the mixtures were subjected to two-phase separation systems. Food matrices were divided into two categories, raw and processed. Raw food included ground beef, ground turkey, chicken, chicken backs, ground chicken, pork sausage and eggs. Processed foods included dry milk, whole milk, cocoa. salad dressing, cottage cheese, coarse pepper, and ice cream. Each food type presented different parameters that could potentially affect the separation process. For example many raw foods have high concentrations of non-pathogenic microbiological flora, which can apply competitive pressure to the growth kinetics of the seeded (or naturally-occurring) Salmonella or Listeria target strains. Consequently, it was seen that many target strains grew at concentration levels which were three to four orders of magnitude less than the background concentration due to the competitive pressure of the background bacteria. Additionally, processed foods often contain injured background, non-pathogenic bacteria which may not specifically challenge the growth of seeded target strains but may contain growth inhibitors that will interfere with target cell growth. Fermented products, often found in processed foods, have starter, native bacteria valuable for the fermentation of the product but inhibiting to the inoculated or naturally occurring target strains. Finally, the very presence of the food matrix itself and the numerous solute compounds emanating from it presents a problem to be overcome, since food debris interferes with the plating and selective growth of bacterial cultures . Unexpectedly the preferred three stage counter-current two-phase partitioning system eliminated virtually all of the food debris from the system. In the present aqueous two-phase systems, food debris quickly migrated to the interface thus reducing the amount that was being transferred into the second stage. When the same process was repeated again, virtually all food left in the targeted phases was removed and was prevented from interfering with transfers and/or with cell plating. Selective growth: The selective differential agars used in the present invention for identification are standard in the field of microbiology and routinely used with many reference methods. For the purpose of the present invention Hecktron Enteric (HE) and Xylose Lysme Desoxycholate (XLD) agars were used. Both agars are based on the bacterial utilization of lactose and/or sucrose. The identification of Salmonella on these agars is facilitated by the development of black colonies resulting from the capture of metabolic H 2 S gas as the insoluble iron-sulfur salt (Bacteriological Analytical Manual. 6th Edition, supra) . Partitioning of Yeast and Alσae: Using the teachings of the invention, mixtures of other microorganisms, including various yeasts such as Saccharomyces cerevisiae and Candida tropicalis could be separated in an aqueous PEG/Dextran system (see Examples) . To demonstrate the flexibility of the present invention, the partitioning characteristics of several pure cultures of yeast and algae species were examined using the aqueous two-phase systems developed for Salmonella and Listeria . Separation of mixtures of yeast and algae could be adequately achieved for several species even though the aqueous two-phase systems were unoptomized for these cells. The following examples are meant to illustrate key embodiments of the invention but should not be construed to be limiting in any way. EXAMPLES MATERIALS AND METHODS Polyethylene glycol (PEG) (Sigma Co.) a linear synthetic polymer of various molecular weights and PEG-bis (amine) , (Sigma Co.) a positively charged derivative of PEG, are prepared in 20% w/w and 10% w/w batches respectively, stored at 4°C and used within 3-4 weeks. Likewise, different wt. of dextran (Pharmacia) are prepared in 20% w/w batches, stored at 4°C and used within 3-4 weeks . Hydroxypropyl starch (Reppe AB) also prepared similarly. Microbiological media (broths, selective and differential agars) are prepared in the lab; following established recipes and sterile conditions and are purchased from suppliers like Difco Co. or Becton- Dickson and Co. Bacteria strains are part of internal DuPont collection. Yeast strains are from the American Tissue Culture Collection (ATCC) and the algae strains from Ward's Natural Science Establishment, Live Materials Department. The posititioning behavior of pure cultures in aqueous two-phase systems is determined with the help of Coulter Counter Instrument (Coulter Corp.) and/or direct plating, following decimal dilutions, onto non- selective, nutrient agar. Microorganisms: Strains of microorganisms tested in the following examples consisted of various species of bacteria, yeasts and algae and are listed below: T.iπt.p.ria genus: (internal DuPont database) L . monocytogenes L . welshimeri L . ivanovii L . murrayi L . innocua L . grayi Fungi: Saccharomyces cerevisiae ATCC 2341 Candida tropicalis ATCC 750 Algae: (Ward's Natural Science Establishment) (Rochester, New York) Naviculla pelliculosa (brown diatorus) Chorella pyrenoidosa (green algae) Peridimium sp . (dinoflapellaters; gold color) Anacystis nidulans (cyanobacteria) Calithammion sp. (red algae) Growth conditions: All Salmonella were cultivated in the pre- enrichment media, BBSM, at 37°C, shaking. Listeria cultures were cultivated in the pre- enrichment LBHI broth, at 30°C in a shaker/incubator vigorously agitating at high speeds (300 rpm) . Yeast cultrues were grown in standard Sabaraud dextrose broth, (Difco) at 27°C. The various algae were grown in their respective, specialized media provided by the supplier as follows: Navicula pelliculosa in Bristol's solution (Ward's supra) Chorella pyrenoidosa into Bristol's solution. Peridimum sp . into soil media culture (Ward's supra) Anacystis nidulans in Bristol's solution and/or Erdschreiber's solution (Ward's supra) Calithammion sp . in Erdschreiber' s solution. Algae are cultivated under fluorescent light at 18°C-24°C. Pre-enrichment broth: The compositions BBSM and LBHI are given below: BBSM ( Salmonella) Lactose broth 13 g/1 KH2P04 34 g/1 Mannitol 7.5 g/1 MgC12.6H20 5.0 g/1 Water 1.0 1* *Adjust to pH 7.0 with NaOH LBHI (Listeria) Brain Heart infusion 37.0 g/1 KH2P04 6.8 g/1 Glucose 5.0 LiCl 10.0 g.l Ferric Citrate 20.0 mg/1 Sodium Pyruvate 0.2 g/1 Acriflavin 7.5 mg/1 Cycloheximide 25 mg/1 Nalidixic acid 20.0 mg/1 Charcoal 1.0 g/1 Water 1.0 1* *Adjust to pH 7.0 with NaOH Selective growth: Selective media for the identification of Salmonella is Hecktron Enteric (HE) or Xylose Lysme Desoxycholate (XLD) agar (Difco) . Selective media for the identification of Listeria is a modified oxford agar (MOXA) with and increased LiCl concentration. The composition of MOXA is given below: _!____ Columbia blood agar base 39 g/1 Aesculin 10 g/1 Ferric ammonium citrate 5 g/1 LiCl 15 g/1 Water 1 1* ♦ Adjust to pH 7.0 with NaOH Yeast are selectively grown on BHI agar plates (Difco) . Algae are not plated but are identified on the basis of color when grown in liquid broth. Two-Phase aqueous polymer systems: Feedpoint two-phase aqueous polymer system used in the present invention are listed below in Tables 1-2 Systems were developed for the isolation of Salmonella and Listeria . These compositions were also determined to be useful for the separation of mixed cultures of yeasts and algae. KH2P04 /K2HPQ4 (ecruiTnolar 0 . 012M 0 . 012M 0 . 012M 0 . 012M prg-enrifihrrianr: inoculum: 1 . 0 ml 1 . 0 ml 1 . 0 ml 1 . 0 ml TABLE 2 SEPARATION OF TARGET BACTERIA FROM MIXED POPULATIONS OF PURE CUT.TTTRF.S Example 1 demonstrates the methods used to determine the effect compositions for two-phase aqueous polymer systems for the separation of bacteria. Single- stage aqueous two-phase partitioning experiments were conducted using pure cultures of Klebsiella oxytoca, E. coli (656), Staphylococcus epidermitis and Lactobacillus carnis . Cells were grown overnight in Penassay broth and were harvested the next day by low speed centrifugation and washed twice in 20 mM potassium phosphate buffer. The cells were finally resuspended in 10 mM potassium phosphate buffer. The total volume was 10 ml. The feed point composition of the aqueous two-phase system was 4% w/w PEG (8000), 5% w/w Dextran (T500) in 100 mM potassium with an without NaCl. Mass balance was done by enumerating the cells in the 1 ml inoculum volume, top and bottom phases. Total volume was 10 ml. Partitioning was carried out at room temperature allowing for between 30 and 50 min. settling time. Partitioning behavior of pure cultures was analyzed first and the results are shown in Table 3, below: The data in Table 3 are normalized and represent percentage of cells in each fraction. Based on the pure culture partitioning results, binary mixtures such as E. coli (656) and Staphylococcus epidermitis or E. coli (656) and Klebsiella oxytoca were added into a single-stage aqueous two-phase system of the same composition, and the partitioning behavior of each organism was measured by conducting a mass balance check and the selective differential agars for targeting each organism as describeed above. The single-stage partitioning results of binary bacteria mixtures are tabulated in Table 4 below: The data in Table 4 are normalized and represent percentage of cells in each fraction. As is evident from the data in Table 4 it was possible to adequately separate the binary mixture of E. coli (656) /K. oxytoca in one stage of aqueous two- phase partitioning if one knew, a priori, to select the top phase for E. coli (656) and bottom as for K. oxytoca . Separation of the binary mixture E. coli (656) /S. epidermitis was less ideal since a large portion of both organisms went to interface. However, the remaining portion partitioned to opposite phases, top for E. coli (656), and bottom for S. epidermitis . Given that cells adsorb reversibly at the interface, it was determined that a counter-current multi-stage operation, might effectively separate E. coli (656) and S. epidermitis. EXAMPLE 2 ISOLATION OF SALMONELLA FROM MIXED POPULATIONS OF PURE CULTURES The objective of Example 2 is to demonstrate the ability to separate Salmonella from a mixed population of background bacteria after 20 hrs. of growth in a pre- enrichment broth. The following bacterial strains were grown together to make the mixture to be separated: S. typhrimurium (LT2) target E. coli (925) background bacteria Ent . cloacae (1074) background bacteria C. freundii (894) background bacteria The separation was carried out using the counter¬ current scheme in three stages (Figure 6) . The starting feed-point composition used was as follows: Stage #1: 5% w/w dextran (T500) 4% w/w PEG(8000) 0.025 M MgCl 2 -6H 0 1 ml Salmonella cells The total volume of the first stage was approximately 9.5 ml corresponding to a total mass of 10 gr of two-phase components. Partitioning of stages 1-3 were carried out according to the following protocol: (a) A "master" two-phase system having the same composition as stage #1 minus cells was prepared, emulsified and allowed to settle for about 1 hr. (b) Fixed amounts of the bottom-phase of the stage 1 were pipetted into empty tubes which became stages #2 and #3 of the counter-current scheme. The bottom-phase amounts were approximately 5.3 for stage #2 and 4.0 ml for stage #3. (c) 1 ml of pre-enrichment broth, containing the mixed culture, was added into stage #1 tube. After mixing and settling approximately 5.5 ml of top-phase of stage #1 were transferred into stage #2. The contents of stage #2 were mixed and settled as specified, and then approximately 4.1 ml of top-phase contents were transferred into the tube containing stage #3 components . The mixing and settling was carried out once more. (d) Stage #3 was allowed to settle for 30 min. and the contents of the top-phase from were then serially diluted and plated onto BHI and XLD agars for enumeration. As described above, the BHI agar was used to determine the total bacterial concentration while the XLD agar was used to determine the Salmonella concentration. This partitioning protocol ensured that the feed point compositions of first, second and third stages were close to each other in the phase diagram (Figure 3) and fell well within the two-phase envelope. Any volume combinations are possible, as long as the two-phase systems remained well within the two-phase envelope. The target and background bacteria concentrations were measured via dilution plating on BHI and XLD agar at time 0 hrs, 20 hrs and after the end of the three- stage counter-current partitioning scheme. Given the enumeration, the target-to-background ratio was calculated at each process time and the data is presented in Figure 8. This Example demonstrates that aqueous two-phase partitioning, carried out in a counter-current, three- stage scheme, may be used for the effective separation of the target Salmonella from a mixed population of background organisms. EXAMPLE 3 ISOLATION OF SALMONELLA FROM GROUND BEEF AND DRY MILK FOOD MATRICES Example 3 demonstrates that a three-stage counter¬ current aqueous two-phase system may be used to separate and isolate a single Salmonella species from a food matrix. S. typhrimurium ATCC (1084) cells were inoculated into the Salmonella pre-enrichment broth (BBSM) (225 ml) containing 25 g of the food matrix. The food matrix consisted of either ground beef or dried milk. The native microbiological flora from each food matrix provided the background microorganisms and were not specifically characterized. The Salmonella inoculated food matrix was grown as described above and subjected to the same partitioning protocol as used in Example 2. Samples from stage #3 of the countercurrent system were differentially plated as described in Example 2 and the partitioning results are presented in Figures 9 and 10. Figure 9 gives partitioning data for Salmonella innoculated into ground beef and Figure 10 gives similar data for Salmonella inoculated into dried milk. As can be seen by the data in Figures 9 and 10 the present two-phase system is effective in the isolation of a Salmonella species from a complex food matrix contaminated with a variety of background microorganisms. EXAMPLE 4 ISOLATION OF A BROAD RANGE OF SALMONELLA SP. FROM A DIVERSE SET OF FQQD MATRICES Example 4 demonstrates the ability of the present method to isolate a broad range of Salmonella sp . from a complex food matrix contaminated with a diverse array of background bacteria. 112 strains of Salmonella were tested corresponding to the species listed above in the MATERIALS AND METHODS. Food matrices were divided into two categories, raw and processed. Raw food included ground beef, ground turkey, chicken, chicken backs, ground chicken, pork sausage and eggs. Processed foods included dry milk, whole milk, cocoa, salad dressing, cottage cheese, coarse pepper, and ice cream. All food was obtained commercially from local vendors. As in Example 2, the method of isolation involved a counter-current system, however, in order to allow for greater Salmonella inclusivity the method was modified to a bifurcated, three stage counter-current system as illustrated in Figure 7. The feed point compositions of the two-phase systems employed are referred to as A, A*; B, B* and are described in Table 1 of the MATERIAL AND METHODS section. Cultures of various Salmonella strains were grown overnight in nutrient broth at 37°C in 125 ml flasks in a shaker/incubator shaking at 120 rpm. For each food matrix tested, multiple samples of 25 g were mixed together in a stomacher with 225 ml of Salmonella pre- enrichment broth (BBSM), as per FDA's BAM specification (Bacteriological Analytical Manual. 6th Edition, supra) . Into each of these mixtures a different Salmonella strain was innoculated at a cell concentration of about 0.01 cells/ml. This cell concentration corresponded to the minimum detection level of one Salmonella cell in 25 g of food matrix as required by FDA's specifications (Bacteriological Analytical Manual, supra) . The inoculated broth was then transferred into a sterile 500 ml Erlenmeyer flask and was incubated at 37°C under static conditions, for 20 hrs. A negative control flask containing uninoculated food matrix into 225 ml BBSM broth, was always a part of each set being tested. After pre-enrichment, two 1 ml portions of the pre- enrichment broth were added to aqueous two-phase systems A and B respectively as shown in Figure 7. The two aqueous two-phase systems A or A* and B or B* (Table 1) were determined based on broad screening experiments as described in Example 1 of the single-stage partitioning behavior of 77 pure cultures of Salmonella strains. The protocol used to carry out the three-stage counter-current separation was somewhat different from the one described in Examples 2 and 3. Depending on the total volume of the aqueous two-phase systems (either 10 or 5 ml, of any other value) , a fixed volume of the top- phase for system A (or A*) (4 or 2 ml) or the bottom- phase for system B (or B*) (3.0 or 2 ml) from stages #1 or #2 was transferred into stages #2 or #3 respectively. Stages #2 and #3 contained the appropriate balance of aqueous two-phase components to ensure that upon transferring the appropriate amount and phase, the overall feed point composition was the same as stage #1. Thus if the feed point composition of stage #1 (system A/A* or B/B*) were point A' (Figure 2), upon conducting the transfer of stages #2 and #3 were also the point A' or at minimum a point very close to it. Following two-phase separation 10 μi of the top- phase (third stage, system A/A*) or bottom-phase (third stage, system B/B*) was streaked on either XLD or HE selective different agar as described above. Each plate was incubated at 37°C for 24 hrs. Identification of the target Salmonella colonies was based on the black color formation due to precipitation of H 2 S gas as insoluble FeS salt. Data was collected at 0 hrs, 20 hrs and immediately after the aqueous two-phase partitioning step. Based on the measured Salmonella and total bacteria concentrations, the target-to-background ratio was calculated at each process time and was used to evaluate the performance of the overall process and two- phase step in particular. Data demonstrating the isolation of target Salmonella from various food matrices via target-to- background ratio are shown in Figure 11. As can be seen by the data in Figure 11 the majority of all of the Salmonella species tested partitioned either into the top phase of composition A or the bottom phase of composition B without regard for the food matrix present. Based on a total of 112 inoculated food experiments the statistics of recovering the seeded Salmonella strain in any of the aforementioned food matrices were: Salmonella Recovered: Two-Phase Two-Phase Sys "A" or Sys "A" Sys "B" Sys "B" Processed Foods 53/55 (96%)W 53/55 (96%)0 ) 53/55 (96%) ( D Raw Foods 55/57 (9 _%) 54/57 f94 57/57 f 100% . 108/112 (96%) 107/112 (96%) 110/112 (98%) It is noteworthy to point out that on several occasions native Salmonella different from the one inoculated, was recovered in the negative control and some inoculated food samples. A sample of coarse black pepper was inoculated with S. newport ATCC (1266) (poly group B) ; several of the isolated suspect colonies were confirmed as poly group A, indicating the potential presence of a native Salmonella strain. The negative control sample of ground turkey was confirmed to have sero group B native Salmonella . The isolates were picked up from both aqueous two-phase systems A and B. Furthermore, several inoculated samples of the same food matrix were confirmed to have sero group B Salmonella (system A) in addition to the inoculated Salmonella strain S. panama ATCC (1250), poly group A/sero group I. The same was true for the ground turkey sample inoculated with S. heidelberg ATCC (1239) which was in poly group A, a sero group B Salmonella was recovered in both systems A and B. It is important to note that the food matrix was completely eliminated the end of the third stage of the counter-current method. It is evident from Example 4 that the present method is able to isolate a broad range of Salmonella strains from a variety of food matrices . It is further evident that the present method is sensitive, being able to isolate Salmonella strains from food, not originally introduced experimentally. EXAMPLE 5 COMPARISON OF THE BIFURCATED SALMONELLA PROCESS WITH FDA'S BAM PROCESS Example 5 demonstrates the the present bifurcated two-phase counter-current method compares favorably with the industry standard for the detection and isolation of Salmonella from contaminated food. Ten experiments were run using raw and processed foods and eight species of Salmonella . The experiments were carried out essentially as described in Example 4 for the two-phase system. The same food matrices and Salmonella strains were analyzed with the BAM method as described in Bacteriological Analytical Manual. 6th Edition supra and the comparative results are given below in Table 6. 7 Dry Milk S. monteridco (1255) 10" 2 Yes Yes 8 Eggs S. typhrimurium (1280) 10" 2 Yes Yes 9 Fresh ground shrimp S. brandenberg (1345) lO -2 No No 10 Fresh ground shrimp S. brandenberg (1345) 10"2 Yes (sys B*) Yes S. typhrimurium (1084) - Yes (sys A*) Yes The bifurcated Salmonella process is comparable to FDA's BAM method, but the former is at least 24 hours faster than the latter. EXAMPLE 6 TSOT.ΑTTON OF LISTERIA FROM FOOD MATRTCF.S The aqueous two-phase system for separation of the Listeria genus from other background organisms from food matrices was developed in a manner similar to that for Salmonella, as described in the previous examples. The optimum aqueous two-phase system is designated LL-P or A 2 P-82, and is described in Table 2 in the MATERIALS AND METHODS section. These compositions were selected after a broad screening of candidate two-phase systems using single-stage partitioning data of pure cultures of several Listeria species and other background bacteria that were known to be potential competitors. The proposed scheme for the isolation of Listeria from food matrices was similar to that of Salmonella, except that a single branch of three-stage, counter¬ current aqueous two-phase system as described in Figure 6 in place of a bifurcated partitioning scheme. As in Example 4, it was also observed that almost all food debris was eliminated by the end of the third stage with the Listeria two-phase system. Several raw and processed foods where Listeria was expected to be present, were tested. These included dry milk, salami, ricotta cheese, ground port, bacon, brie cheese, and sour cream. The foods were inoculated at very low levels (approximately 1.0 x 10 ~2 and 1.0 x 10 _1 cells/ml) with various Listeria species following the protocol outlined in Example 4. After a 24 hr. the inoculated and negative control samples in the shaker/incubator (300 rpm; vigorous agitation) at 30°C, one sample from the Listeria enrichment broth was transferred into the first stage of the aqueous two- phase system. Once the counter-current partitioning was done, 10 μl of the targeted top-phase (system LL-P) was streaked onto Modified Oxford Agar (MOXA) plates. The plates were incubated at 30°C for 24 hrs. Suspect Listeria colonies were differentiated from background based on their metallic gray color and on block/bacon one surrounding the colony, which was the result of aesculin hydrolysis. In addition to isolating Listeria onto selective, differentiation agar (MOXA) , the concentration of Listeria and the total background microorganisms was determined at the end of the enrichment/growth step (t=24 hrs.) and after the aqueous two-phase step, by dilution/enumeration on BHI and MOXA plates. Evaluation of the target-to-background ratio was also used, together with the streak plate results, to evaluate the isolation of inoculated Listeria species and the utility of the aqueous two-phase partitioning step. All food matrices were inoculated with Listeria in triplicate flasks. A summary of the results is given below in Table 7: (Resting other growth conditions, i.e., silica gel or alumina instead of charcoal EXAMPLE 7 PARTITIONING BEHAVIOR OF YEARS AND ALGAE Example 7 demonstrates that organisms other than bacteria may be isolated and detected according to the present method. The partitioning characteristics of several yeast and algae species in the aqueous two-phase systems A*, B* and A 2 P82 (single stage) were examined. The yeast and algae species were: Yeasts Saccharonyces cerevisiae (2341) ATCC Candida tropicalis (750) ATCC Algae Navicula pelliculosa (brown algae) Chorella pyrenoidosa (green algae) Peridinium sp . (gold-colored algae) Anacystis nidulans (cyanobacteria) Calithamnion sp . (red algae) The partitioning behavior of yeasts, was determined by conducting mass balances as per Example 1) . Enumeration in each phase was done by decimal dilutions and plating onto BHI agar. Taking advantage of the colors that algae exhibit, their partitioning characteristics were qualitatively determined by visually examining the algae migration either at the top or bottom phases, or at the interface, and was also photographed. Data for demonstrating the partitioning behavior of the yeast and algae are summarized below in Table 8: TABLE 8 Partitioning Behavior of Algae in Aqueous Two-Phase Systems A*, B* and A2P82 Based on the data above Applicant believes that definitive separation of yeasts and algae could be obtained by applying the instant method using the bifurcated or single stage two-phase system.

Claims: CLAIMS. 1. A method for the separation of a target micro¬ organism contained within a complex matrix, comprising the steps of: i) homogenizing said complex matrix and incubating said complex matrix in the presence of a pre- enrichment media to resuscitate and grow said target microorganism whereby the number of said target microorganisms in said complex matrix is enhanced; ii) emulsifying a sample of said enhanced complex matrix of step i) in at least one aqueous polymeric two-phase separation system; and iii) allowing said emulsified system to separate into a top phase and a bottom phase, whereby a preponderance of said target microorganisms from the sample is found in either said top phase or said bottom phase. 2. The method of Claim 1 wherein said target microorganism is a bacterium. 3. The method of Claim 2 wherein said bacterium is a pathogenic bacterium. . The method of Claim 3 wherein said pathogenic bacterium is Salmonella, Listeria, Klebsiella, E. coli, staphylococcus or Lactobacillus . 5. The method of Claim 1 wherein said complex matrix is food. 6. The method of Claim 5 wherein said food is ground beef, ground poultry, ground pork, eggs, dry milk, whole milk cocoa, salad dressing, spices or cheese products. 7. The method of Claim 1 wherein at step i) said target microorganism is Salmonella and said pre- enrichment broth is BBSM. 8. The method of Claim 7 further wherein at step ii) said sample is emulsified in two distinct aqueous polymeric two-phase systems, a first system and a second system, wherein said first system comprises 6.5% w/w dextran T500 and 6.5% w/w polyethylene glycol 3400; and said second system comprises 5.0% w/w dextran T500 and 3.5% w/w polyethylene glycol 8000. 9. The method of Claim 8 further comprising after step iii) : iv) withdrawing an aliquot of the top phase of said first system which contains a preponderance of said target microorganism and emulsifying said aliquot in a fresh first aqueous polymeric two-phase system; while additionally withdrawing an aliquot of the bottom phase of said second system which contains a preponderance of said target organism and emulsifying said aliquot of said second system in a fresh second aqueous polymeric two-phase system; and v) repeating step iii) . 10. The method of Claim 9 further comprising sequencially repeating steps iv) and v) one or more times. 11. The method of Claim 1 wherein said bacterium is Listeria and said pre-enrichment broth is LBHI. 12. The method of Claim 11 further wherein at step ii) said sample is emulsified in an aqueous polymeric two-phase system comprising 3% w/w dextran T500, 1.5% w/w polyethylene glycol 8000 and 6.0% w/w polyethylene glycol 2000. 13. The method of Claim 12 further comprising after step iii) : iv) withdrawing an aliquot of the top phase of said aqueous polymeric two-phase separation system which contains a preponderance of said target microorganism and emulsifying said aliquot in a fresh aqueous polymeric two-phase system of step ii) ; and v) repeating step iii) . 14. The method of Claim 13 further comprising sequencially repeating steps iv) and v) one or more times. 15. A method for the separation and detection of a target microorganism contained within a complex matrix, comprising the steps of: a) homogenizing said complex matrix and incubating said complex matrix in the presence of a pre- enrichment broth to resuscitate and grow said target microorganism whereby the number of said target microorganisms in said complex matrix is enhanced; b) emulsifying a sample of said enhanced complex matrix of step a) in at least one aqueous polymeric two-phase separation system; c) allowing said emulsified system to separate into a top phase and a bottom phase, whereby a preponderance of said target microorganism from the sample is found in either said top phase or said bottom phase; and d) removing an aliquot from that phase of step c) containing a preponderance of said target microorganism and detecting the presence of said target microorganism within the aliquot. 16. The method of Claim 15 wherein at step d) said detection is accomplished by inoculating said aliquot onto a solid selective growth media, and incubating said solid selective growth media whereby isolated target microorganisms which grow on said media are detected visually. 17. The method of Claim 16 wherein at step a) said target microorganism is Salmonella and said pre-enrichment broth is BBSM; and wherein at step b) two distinct aqueous polymeric two-phase separation systems are used; the first system comprising 6.5% w/w dextran T500 and 6.5% w/w dextran T500 and 6.5% w/w polyethylene glycol 3400, and the second system comprising 5.0% w/w dextran T500 and 3.5% w/w polyethylene glycol 8000; and wherein steps b) and c) are sequencially performed three times, wherein during the second and third times a sample withdrawn from that phase of step c) containing the preponderance of Salmonella is used as the sample in step b) ; and wherein at step d) said solid selective growth media is HE or XLD media. 18. The method of Claim 16 wherein at step a) said target microorganism is Listeria and said pre-enrichment broth is LBHI; and wherein at step b) said aqueous polymeric two- phase separation system comprises 3% w/w dextran T500, 1.5% w/w polyethylene glycol 8000 and 6.0% w/w polyethylene glycol 2000; and wherein steps b) and c) are sequencially performed three times, wherein during the second and third times a sample withdrawn from that phase at step c) containing the preponderance of Listeria- is used as the sample in step b) ; and wherein at step d) said solid selective growth media is modified MOXA.

Inventors: KOPATSIS ALEXANDER DEMETRIOS, KOPATSIS, ALEXANDER, DEMETRIOS
Applicants: DU PONT, E.I. DU PONT DE NEMOURS AND COMPANY
Application year: 1995
Main classification: C12Q1
IPC codes: C12Q1