JPH11501210A - Dielectrophoresis - Google Patents

Abstract

(57) Abstract: A method and apparatus for concentrating microorganisms, especially bacteria, from a sample material, especially a complex sample material, more particularly a food-derived material, is followed by a desalting means for reducing the conductivity to less than 1000 μS / cm ^2. Consists of using dielectrophoresis. Methods for analyzing microorganisms and producing approved batches of food are also disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION                               Dielectrophoresis Technical field   The present invention concentrates and separates microorganisms from complex sample materials, especially food-derived materials And a method for the same. Target microorganisms suitable for bacteria and yeast It is.Conventional technology   Detection of microorganisms in food is particularly relevant to avoiding food poisoning situations Quantify desired microorganisms in cultures as well as in the food manufacturing and wholesale industries Interest is also growing to do so.   Conventional detection methods use a culture technique that enriches the microorganism of interest and then performs an identification step. For example, EP-A-0 214 340 [Biocon Troll Systems]. This type of method always requires considerable time and effort It takes power. This means that, for example, a sample of food can be corrected before it leaves the factory, for example. Reduced suitability for preparatory activities, such as conducting regulatory tests and placing contaminated batches Let it.   The faster technology underlying is e.g. Rapid Analysis Technology "[P. D. Patel, Blacky] Have been. However, for example, electrical and immunological Is greatly limited by the complex and heterogeneous nature of the food sample . Therefore, a practical method for rapid analysis of microorganisms in food and other complex samples It will be seen that the method has long been awaited in the industry.The present invention   The present inventors have proposed a method using dielectrophoresis. We have developed a practical method for analyzing microorganisms in mixed samples (especially food-derived samples).   Dielectrophoresis is triggered by a non-uniform electric field, especially in polarized uncharged particles This is a phenomenon characterized by movement [GB Patent Application GB 2 071 843 (Po Le)]. This replaces the DC field used in electrophoresis with the AC field. This is the movement that occurs when used.   The movement of microorganisms such as bacteria and yeasts is manipulated using the dielectrophoresis principle It is well known that this can be done [for example, Black Chemistry and -Bioenergetics, Vol. 24, pp. 203-214; Marx et al. (1 994), Microbiology, Vol. 140, pp. 585-591; (1993), Microbios, Vol. 73, pp. 81-86]. Sa It is also well known that various bacterial forms can be similarly controlled [e.g., Archer (1993), Microbios, Vol. 73, pp. 165-172].   In addition, the ability of dielectrophoresis devices to separate mixtures of particles (especially bacteria) This is shown using the differential delay of particles in the sample liquid passing through the moving chamber (EO 93 / 20927 (BTG)). This study uses a changing electric field to isolate specific bacteria. Focuses on achieving separation, but does not include the pH and conductivity of the liquid, and the Wavenumbers were also found to affect delay and thus the order and degree of separation. Have been.   However, published studies on electrophoresis to date have generally been relatively Relating to the use of simple liquid samples, especially with different dielectrophoretic properties Characterization and resolution of bacteria. For example, GB 2 071 In 843A, samples were obtained from pure cultures grown specifically for that purpose. Collected. In each case, dielectrophoresis was used to test "real world" food samples. It was not practical.   The present inventors have proposed that direct and active dielectrophoretic aggregation of microorganisms be induced with high conductivity and high conductivity. 1-20 MHz voltage at 1-10 volts from food and medium broth of electrical conductivity What could not be achieved using the settings, as well as a 1:10 dilution of broth, I figured out not to repair. However, 1: 100 to 1: 100,000 Dilution results in some aggregation using these frequency and voltage parameters It has been found. However, for practical purposes, a dilution of 1: 1000 to 1: 10000 Gives the best results, allowing use up to 15 volts, above which heating Convection is generated by the effect.   Dilution of the sample should be done prior to performing any method that is intended for concentration. Decreasing the concentration is inevitable and is therefore undesirable in principle. this is In particular, samples (such as those that can only be present at low concentrations, at least when bacteria are present) For example, when handling food samples).   This time, the present inventors first started with a composite sample, for example, obtained from food. Can be analyzed by dielectrophoresis without dilution I figured out the way.   Therefore, in a first aspect of the present invention, a food sample characterized by the use of dielectrophoresis The present invention discloses a method for concentrating microorganisms.   Concentration itself is associated with exposing the sample to a dielectrophoretic field to aggregate microorganisms. This can be accomplished by removing the remaining sample. Followed by concentrated microorganisms For example, it can be analyzed as described below.   The term "food sample" refers to a culture or suspension in a carrier liquid, for example. Human or animal food or potential food, including samples that have been pretreated by Means any liquid material obtained from the article.   Preferably, the method is performed using a bacterium, such as Salmonella or Listeria sp. Used for sheath analysis. That is, food materials are quickly separated for the presence of bacteria. The use of dielectrophoresis for analysis is important in the food industry at an early stage in manufacturing or distribution. Paving the way for contaminated food to reach end consumers.   This method is, for example, 1000 μS / cm^TwoSamples with higher initial conductivity It is particularly applicable to For this type of sample, Is a method using a sample of 1000 μS / cm^TwoTreated to reduce the conductivity to less than It is further characterized in that dielectrophoresis is performed later. The preferred method of conductivity reduction is desalination. You.   The present inventors have found that the desalting process enables rapid dielectrophoretic separation of microorganisms, It aims to minimize interferences from food particles and the disadvantages associated with sample dilution. stopped.   In a preferred method according to the invention for this aspect, the conductivity is 500 μS / cm^TwoOr Until then, more preferably 1 to 450 μS / cm^TwoUp to, and especially preferably about 100-200 μS / cm^TwoTo reduce.   50-450 μS / cm for practical purposes^TwoConductivity is acceptable. Efficient electricity It is clear that very low conductivity is desirable for electrophoresis, but much higher to achieve Requires intensive effort or a long time, resulting in some loss of sample microorganisms You. Therefore, the desalination technique and the selected conductivity range will balance these factors. Must be. We have taught in GB 2 071 843A Using the present invention with a conductivity much higher than the preferred range of “less than 10 μS / cm” Note the success.   The particular desalting process used should not result in significant bacterial loss from the sample. It is not limited except.   A convenient method realized by the present inventors is a desalting gel [eg, bio Le P-6DG, Biorad Laboratories and PD-10, Pharmacia Dialysis membrane, ion exchange resin, reverse osmosis, ultrafiltration or Includes the use of on-specific balance changes. In addition, simply centrifuge The sample solids were precipitated using, for example, 4000 rev / min for 15 minutes. And then suspending the resulting pellet in, for example, distilled or deionized water. Or just   For example, the method of desalting a sample using a substance such as a gel can vary. Wear. For example, using the exclusion gel method Removing ions directly from solution using ion exchange resins Effectively wash out salts using static, low pressure and countercurrent dialysis. Pressure driven reverse osmosis, microfiltration, countercurrent diffusion or specific ultrafiltration, Diafiltration, linear crossflow or depth filter arrangements can also be used; Or for example electricity Charging systems such as those used in dialysis or ultra-osmosis can also be used.   The gel is used as a coarse filter that is quick and reduces the particulate characteristics of the sample. Use of gel filtration in that it can simplify subsequent dielectrophoresis by acting Is particularly advantageous. Cross-linked dextran gel appears to be particularly effective.   Dialysis cassettes and dialysis chambers are used even by relatively unskilled operators Is advantageous in that it is simple and convenient.   A continuous flow system further simplifies desalination and therefore overall microbial concentration. The contraction process can be simplified.   The advantages resulting from the use of the above desalination step are that all high conductivity (eg> 1000 μS / cm^Two) Also obtained by dielectrophoresis of complex liquid samples (even those of non-food origin) It should be noted that That is, using this kind of method and this method The device also constitutes another aspect of the present invention.   The dielectrophoretic field parameters used are typically 1 to 15 volts. May include frequencies of ２０20 MHz, which use frequencies higher than 20 MHz In that case, it can be increased to 100 volts or more. This is a trial Material conductivity Than prior art dielectrophoresis techniques where the rate does not allow the use of such high voltages. It is advantageous. The remainder of the sample (i.e., the sample without any microorganisms) has been removed After the contamination, the fluid is removed and the carrier liquid is passed through the area where the microorganisms have aggregated. By passing the microorganism, the microorganism itself can be separated by the carrier liquid. Carry The aqueous solution is conveniently prepared with a buffer solution, saline or water, such as deionized or distilled water. is there.   Using the preferred method of the invention, samples from undiluted food and media (eg, food) Aggressive dielectrophoresis of the whole broth (culture broth and culture broth) (Eg bacteria) can rapidly enrich the contents or selected microbial component populations You.   In a second aspect, the invention also provides a method for analyzing microorganisms in a food sample, the method comprising: The method includes the step of analyzing the microorganism after concentrating the microorganism as described above.   Preferably, the microorganisms are removed from the field before analysis. To analyze microorganisms The method used is not limited to any particular group of technologies; (E.g., the end of the electrode used to add the field) It can be as simple as direct visual observation using a microscope. this Various techniques are well known to those skilled in the art.   In a third aspect, the invention also provides a method for producing an approved batch of food, the method comprising: A food batch is prepared in the usual manner, a representative sample is taken from this batch, and Analyze microorganisms in a representative sample using the dynamic method, compare the results of the analysis with a standard, Approve batch if analysis meets or exceeds (exceeds) standard It consists of things.   The term "representative sample" may determine overall quality at a statistically significant level Means a sample. The standard used depends on the nature of the food and the microorganism being analyzed. In many cases, the absence of certain microorganisms (eg, Salmonella) I need.   In yet another aspect, the present invention provides^TwoDuplex with higher conductivity It also provides a device for concentrating or separating microorganisms from a combined liquid sample, which device Conductivity of 1000 μS / cm^TwoOr reduce the sample to a lower value , A means for generating a dielectrophoretic field, and Means for passing through the dielectrophoretic field. Equipment preferred for separation purposes Controls the electric field to disappear. And the carrier liquid are passed through a means for generating a dielectrophoretic field and aggregated. Means for collecting the passed microorganisms, and means for collecting the passed carrier liquid.   The means for generating a dielectric field is not limited to any particular device, but Can be passed through the dielectric field holding region in use. Feel The electrodes are conveniently formed using electrodes / field chamber electrodes, which are used for electrophoresis. As is known in the art to preserve the dielectrophoretic field without spawning Away.   Hereinafter, the method and apparatus of the present invention will be described with reference to examples and drawings without limitation. An embodiment will be described. Other embodiments that fall within the scope of the invention will also refer to them. It will be apparent to those skilled in the art.Drawing   Figure 1: Using a microscope to directly observe the number of microorganisms separated from the sample solution 1 is a schematic diagram of the device of the present invention.   Figure 2: Relative permittivity and conductivity of buffered peptone water (BPW) versus dilution factor Rate graph.   Figure 3: Many different bacteria placed in a dielectrophoresis field 4 is a graph showing the effect of BPW dilution on aggregation.   Figure 4: Wrapperport basiliadis enriched broth (RV) versus dilution factor Graph of relative permittivity and conductivity of.   FIG. 5: RV for aggregation of a number of different bacteria placed in a dielectrophoretic field 7 is a graph showing the effect of diluting water.   FIG. 6: Beef and birds fed by centrifugation and resuspension described in Example 2. Graph of dielectric aggregation of natural flora from meat and cod homogenates.   FIG. 7: described in Example 3 for dielectrophoretic aggregation of bacterial cultures in TSB 5 is a graph showing the effect of column desalting.   FIG. 8: Example using dielectrophoresis parameters of 100 KHz and 10 volts Aggregation, conductivity and total viability of flora from beef homogenized products as described in 4. 7 is a graph showing the effect of column desalting on the recovery rate.   FIG. 9: Example using dielectrophoresis parameters of 100 KHz and 10 volts Aggregation, conductivity and total viability of flora from bird meat homogenized material as described in 4. 7 is a graph showing the effect of column desalting on the recovery rate.   Figure 10: 100 KHz and 10 volt dielectrophoresis parameters Surviving flora from cod homogenized material as described in Example 4 using 4 is a graph showing the effect of column desalting on the aggregation, conductivity, and recovery of the column.   FIG. 11: Dialysis cassette of TSB against Salmonella count and conductivity 7 is a graph showing the effect of desalination.   Figure 12: Desalting of dialysis chamber of TSB against Listeria count and conductivity. The graph which shows the effect of.   FIG. 13: Salmonella counts and conductivity for Salmonella counts. Of Tifi suspension 4 is a graph showing the effect of desalting on a PD10 column.   FIG. 14: Salmonella counts and conductivity versus count. Of Tifi suspension The graph which shows the effect of Presto (trademark) column desalination.   FIG. 15: S.V. for Salmonella counts and conductivity. Of Tifi suspension The graph which shows the effect of Speedy (trademark) column desalination.   Figure 16: Salmonella counts and conductivity for Salmonella counts. Enteritidis FIG. 5 is a graph showing the effect of Speedy ™ column desalting of a suspension. FIG.Example Dielectrophoresis device:   The devices used in all the examples described below have different diameters and spacings A number of electrodes were provided. Those with a narrow radius of curvature (diameter) are next to the narrowest electrode Increased tangential agglomeration produced the highest field noted. The degree of aggregation It is best for electrodes with a diameter of 1-11 μm and a spacing of 30-135 μm, It was minimal at a diameter of 2-43 [mu] m and a spacing of 500 [mu] m. Microbial aggregation Observation was performed using the apparatus shown in FIG. 1, where the image analyzer (1) was a camera ( Dielectrophoresis fee in microelectrode array (4) obtained from microscope (3) via 2) Accepts an image of the field, which is then the switching matrix of each electrode in the row ( 5) was supplied with a control signal via a function generator (6).Example 1 Effect of Dilution on Dielectrophoretic Sample:   2 and 3 show the conductivity and dielectrophoretic characteristics of buffered peptone water (BPW). 9 shows the effect of dilution on gender. 4 and 5 show the wrapper port basiliage 5 shows the corresponding results for the RV medium.Example 2: Reduction of conductivity of food pre-enriched samples by centrifugation and resuspension and Dielectrophoresis:   Add a beef, chicken or cod sample (10 g) to a 90 mL volume of sterile BPW, Digest for 1 minute [Colworth digester, A. J. Stewart Limited], Then, the cells were cultured at 37 ° C. for 18 hours. After the culture, add 1 mL of each homogenized product. Centrifuge at 4000 rev / min for 15 minutes and pellet in distilled water. Resuspended. Dielectrophoretic aggregation of the natural flora in suspension was measured at 10 KHz, 100 It was measured at KHz, 1 MHz and 10 MHz. The results are shown in FIG.Example 3: Reduction of conductivity and dielectrophoresis of bacterial cultures by using desalting columns:   About 10⁷-10⁸Soy broth (TSB) containing microbes / mL An aliquot (1 mL) of the bacterial culture in Commercially available desalting columns (Bio-gel, P6DG, Biorad La) (Voratories). Collect the eluate in 100 μL fractions For each fraction, the dielectrophoretic aggregation of bacteria in each fraction was determined at 100 KHz and 10 vol. Measured at The results are shown in FIG.Example 4: Reduction and induction of conductivity of food pre-enrichment samples by using desalting columns Electrophoresis:   Add beef, chicken and cod samples (10 g) to a 90 mL volume of sterile BPW, Incubated at 37 ° C. for 18 hours. After incubation, 1 Aliquots of the mL enriched broth were transferred to 2 mL hydrated desalting gels (Bio-Gel, P-6D). G, biospin) through the column containing 100 μL of the eluate. Collected in action. Each fraction was analyzed for conductivity, total viable count (trypton Soy agar: using the spread plate method in TSA)), and 100 KHz And analyzed for dielectrophoretic aggregation at 10 volts. The results are shown in 8th, 9th and Figures 10 and 10 show the results.Example 5: Reduction of conductivity of bacterial cultures and food pre-enriched samples: desalting column, permeability Comparison of analysis cassette and dialysis room: Method   S. aureus is resistant to streptomycin and nalidixic acid. Chifimurium (55698) and S.M. Enterobacteria (p48120) strain at TSB Cultured overnight. 2.5 mL of the culture was subjected to desalting gel (PD10, Pharmacia). It was applied to a column (1.5 cm × 1 cm) and eluted with distilled water. 0.5 Collect aliquots of the eluate and use a small conductivity meter [Horiba Limited]. Was used to measure the conductivity. The Salmonella in each fraction was counted, Serially dilute with a 1/4 concentration Ringer's solution, streptomycin and Nariji The measurement was performed by applying a 0.1 mL surface coating to TSA containing cusic acid. 37 each plate Cultured at 24 ° C. for 24 hours.   In addition, the desalting column was replaced with a cocoon inoculated with two antibiotic resistant strains of Salmonella. Evaluated using pre-enriched cultures of nuts and poultry. 25g of each food sample The charge was added to 225 mL of BPW. The pre-enrichment culture was then added to S. aureus. Entericity Or S. 10 of any overnight culture of Typhimurium^-Five1 from diluent 00 μL was inoculated. A sample (0.5 mL) of the cultured pre-enrichment is 1 cm deep Column and transfer each fraction for conductivity measurement and Salmonella counting. Collected as above.   S on dialysis cassette [Slide-A-Riser, Pierce & Wallina] . Fill 1 mL of TSB inoculated with Typhimurium and place in 500 mL of distilled water. Dialysis was performed at room temperature (RT) and at 4 ° C. with stirring. 100 μL ants Coats were removed at predetermined time intervals for conductivity measurements. as mentioned above Salmonella was counted before and after dialysis.   Double-sided dialysis room [Spin Biodialyzer, SialoMed Inc. A 0.1 μm or 0.45 μm polycarbonate film, Mo Fill 1 mL of TSB inoculated with Nocytogene and add 500 mL of distilled water. The mixture was dialyzed at room temperature with stirring. Count Listeria before and after dialysis, which is 1 PARCAM agar [Oxoid] diluted in series with 1/4 concentration Ringer's solution 0.1 mL of the appropriate dilution was applied to the surface. result   Using a gel for PD10 desalting, 9.6 × 10⁷cfu / mL S.P. Entericity And 1.2 × 10⁸cfu / mL S.P. TSB culture containing typhimurium The conductivity of the nutrients is> 2,000 to 84 μS / cm respectively^TwoUp to and 117μ S / cm^TwoDropped within 5 minutes in the second fraction from the column. Hula Action 2 Enteritidis and S. aureus. Recovering of Chifimurium The rates were 100% and 17%, respectively.   Coconut inoculated with two serotypes of Salmonella and For chicken pre-enriched cultures, the conductivity of the enrichment is> 2,000 μS / cm before desalting^Two 10 to 14 μS / cm in fraction 2 after desalting^TwoDown to. 5 . 5 × 10^Fivecfu / mL S.P. Coconuts and birds containing typhimurium For the meat pre-enrichment, the recovery after desalination was 6% and 10%, respectively. 2 . 1 × 10^FiveS. For coconut and chicken pre-enriched products containing enteritidis Recoveries were 57% and 20%, respectively.   At room temperature, the slide-A-riser dialysis cassette increases the conductivity of TSB after 1 hour. > 2,000 to 530 μS / cm^Two155 μS / cm until and after 6 hours^TwoMa Reduced by. log_TenSalmonella count was 8.6 from the initial 8.6 to 9 after 6 hours . Increased slightly to zero. At 4 ° C, the desalination rate decreased slightly, and the final conductivity decreased slightly. It became higher (Fig. 11). Spin Bio Dializer is Slide-A-La A similar proportion of desalting was given (Figure 12), but the final conductivity was much lower. (36 μS / cm^Two). 0.1% of desalting rate and final conductivity There was no difference between the membrane and the 0.45 μm membrane. Listeria count is the initial 8 . It decreased slightly from 6 to 8.1 after 6 hours.   Thus, desalting columns enable the ionic concentration of food enrichment to be positively dielectrophoretically Level (<500 μS / cm^Two) Is effective as a means of rapidly reducing You. Salmonella recovery after passing through the column varied with serotype. Dialysis machine Set-up and dialysis rooms are an easy and convenient way to desalinate food reserves. But with a slide-A-riser and spin biodialyzer The rate of SB desalting was much slower than when using a gel column system. Magnetic stirring Dialysis chambers with built-in magnets for use with the machine are larger than dialysis cassettes A significant conductivity reduction.Example 6: Reduction of conductivity of food pre-enriched samples using a dialysis cassette of 3 mL capacity : Method   A chicken and coconut sample (25 g) was weighed into a 225 mL BPW And incubated overnight at 37 ° C. After incubation, enriched culture In TSB in TSB. 1 mL of a 1/1000 dilution of an overnight culture of Typhimurium Seeded. Transfer a sample (1 mL) of the enriched culture to a dialysis cassette and add 1 L of distilled water. And dialyzed. Aliquot of enriched culture (100 μL) Is dialyzed at hourly intervals for conductivity measurement using a card-type conductivity meter [Horiba] Removed from cassette. The Salmonella count was measured before and after dialysis, A series of suspensions at Maxim Recovery Diluent (MRD) Make a 0-fold dilution and prepare xylose-lysine-desoxycholate agar (X LD) by spraying 0.1 mL on the surface. result   The cassette has a conductivity of chicken and coconut-enriched cultures> 2000 μS / cm^Two To 260 μS / cm^TwoAfter one hour at room temperature was reduced. Then, desalination If the conductivity of the chicken and coconut enrichment was 160 μS / c each after 2 hours m^TwoAnd 179 μS / cm^TwoTo about 100% after 6 hours. μS / cm^TwoReached. S. in dialyzed chicken and coconut enrichments H The number of fimurium did not change significantly, but after 6 hours at room temperature, respectively. Increased at 0.6 and 0.3 log cycles.   Therefore, these cassettes can be used to rapidly produce food-enriched cultures prior to dielectrophoresis. For example, 2-3 hours) to provide a simple and convenient way of desalting, There is no loss.Example 7: Conductivity of food pre-enriched samples using 15 mL dialysis cassettes Reduction and dielectrophoresis: Method   Enriched culture of minced beef, chicken, skim milk powder and coconut Products were prepared and used as described in Example 6 for S.p. Chifimurium (S596 8) The antibiotic resistant strain was inoculated. Large capacity cassette (10,000) MW Enrichment medium for each 15mL in the 15mL capacity of Pierce & Wallina Nourishment was filled. These cultures were transferred to 3 L of distilled water at 1 hour intervals. Dialysis was performed. Aliquots of 100 μL of the suspension were given at regular time intervals prior to dialysis. Post-conductivity measurement and streptomycin (1 mg / mL) and Nariji Xylose-ricin agar containing xylic acid (50 μg / mL) (XLNS) Removed for salmonella count. After completion of dialysis, desalted enrichment culture One part of the product was transferred to a dielectrophoresis cell, Observed for dielectrophoretic separation of the whole microbiota in SMP at 0 MHz 20 V Was. result   Chicken, minced beef (MB), skimmed mill using 15 mL dialysis cassette Conductivity of coconut powder (SMP) and coconut enrichment> 2000 μS before dialysis / Cm^Two520, 650, 580 and 240 μS / c respectively 2 hours after m^TwoTo 240; 740, 310 and 143 μS respectively after 3 hours / Cm^TwoAnd after 5 hours, 104, 210, 143 and 104 respectively. And 76μS / cm^TwoReached. Salmonella counts after 5 hours of dialysis are chicken Slight increase in enrichment (0.1 log cycle), MB, SMP and coco Slightly reduced in nut enrichment (0.5, 0.3 and 0.9 lo respectively) g cycle). After dialysis, positive electrophoresis of the SMP enrichment was favorably shown.   Therefore, these cassettes can be used in relatively large volumes of food-enriched culture prior to dielectrophoresis. It provides a convenient method for rapid desalination of substances, and there is no loss of bacterial counts during the desalination process. this The results show that, for example, a continuous flow dialysis system as commercially available It suggests that there is a reasonable expectation that the law can be used to advantage.Example 8: In conductivity of bacterial cultures and food pre-enrichment samples using dialysis room Reduction and dielectrophoresis: Method   S. C. typhimurium was cultured overnight in TSB at 37 ° C. and serially Make a 10-fold dilution of XLD and apply 0.1 mL of the appropriate dilution to XLD on the surface. Counted by smearing. The XLD plate was cultured at 37 ° C. for 24 hours. Then 10^-3Measure the conductivity of the dilution and aliquot one part of this dilution to 0.45 μm or Is a spin biodialyzer room equipped with a 0.6 μm polycarbonate film Transferred to [Sialomed Inc.]. The culture dilution is mixed with a magnetic stirrer for 5 Dialyze against 1 L of distilled water over time and introduce 100 μL of sample at hourly intervals. Removed for power measurement. Salmonella at the end of dialysis as described in Example 7. Was counted. This experiment was repeated with L. monocytogene using only the 0.45 μm membrane. Restored. The dilution is spread on PARCAM agar and incubated at 30 ° C. for 72 hours. Listeria were counted.   The antibiotic-enriched S. aureus prepared in Example 6 was added to the food-enriched culture. Contact Chifimurium Biodiamond each sample of the enriched culture Transferred to a riser and dialyzed against 1 L volume of distilled water. Start of dialysis, and 30 Conductivity measurements were taken after 1 minute, 1 hour, 2 hours and 3 hours. Salmonella Was counted on XLNS agar before and after dialysis as described in Example 7.   After completion of the dialysis, a sample of the desalted enriched culture was transferred to a dielectrophoresis cell, Inspection was performed as described above. result   Spin biodialyzer equipped with 0.45μm polycarbonate membrane S. The conductivity of the suspension of Typhimurium is> 2000 μS / cm^TwoFrom 470, 1 04, 62 and 39 μS / cm^Two0.6μm polycarbonate > 2000 to 490, 152, 72 and 40 μS / cm for membranes^TwoUntil Decreased after 1 hour, 2 hours, 3 hours and 4 hours, respectively. Salmonet La counts showed slight fluctuations during dialysis, with 0.45 μm membrane and Shows an overall reduction of about 0.7 and 0.5 log cycles per 0.6 μm membrane Was. L. Similar results were obtained with the suspension of monocytogenes, There was no significant overall change in Listeria counts.   Chicken, SMP and coconut-enriched cultures all <100 μS / cm^TwoReached a conductivity of. MB <300 μS / cm within this time^TwoReached. Food Dielectrophoretic aggregation of the whole microbiota from a material-enriched culture Suitably performed after desalting in Isa.   Thus, these dialysis chambers rapidly desalinate food-enriched cultures prior to dielectrophoresis It provided a convenient method and there was no evidence of bacterial loss during the desalting process. 0.45μ No significant difference was observed between the ratios using the m and 0.6 μm films.Example 8: Bacterial culture using dextran and polyacrylamide desalting columns Decrease in conductivity of objects: Method   Cross-linked dextran [Presto (trademark), 5 mL, Pierce & Warlinna And PD10, Pharmacia] and beaded polyacrylamide P.D. (trademark), 5 mL, Pierce & Warlinner Co.] Culture suspensions and food enriched cultures were desalted using two columns. These columns Is first washed with 3 mL of a 95% ethanol solution and then with distilled water. Low effluent conductivity (About 20μS / cm^Two) Until destabilization. 1 part of effluent (1 mL ) Were retained for sterilization checks. Splebutomycin and nalidixic acid S. of resistance to resistance. Typhimurium strain (S5698) in TSB at 37 ° C overnight Cultured. Salmonella was then serially diluted 10-fold in MRD and Example 7 Counted by surface smear as in. Then 10^-71 part of diluent (1.5m L) into a column (3 mL of 10 per PD10 column).^-3Dilution) and distilled water Was replaced. Ten 200 μL fractions (1 mL per PD10 column) Was collected in an Eppendorf tube and its conductivity was measured. Each fraction (100 μL) portion with MRD and Salmonella counts as above Was measured. result   The PD10 desalting column has a conductivity of Salmonella suspension of> 2 in the first fraction. 000 to 166 μS / cm^TwoReduced to Then the conductivity is fraction 430 μS / cm at 2^TwoUp to> 2000 μS in fractions 3-6 / Cm^TwoAnd then decreased again in fractions 7 to 10 (FIG. 13). L. Similar results for monocytogene suspension Was obtained, and the conductivity was 210 μS / cm in the first fraction.^TwoTo hula 760 μS / cm in section 2^TwoAnd further in fractions 3-6 > 2000 μS / cm^TwoAnd then decrease again in fractions 7-10 Was. Maximum recovery of Salmonella and Listeria was obtained in fraction 2 ( 2.5% and 8.5% respectively).   FIG. 14 shows a duplicate experiment using a Presto ™ dextran desalting column. With S. 1 shows the average conductivity and recovery of typhimurium. These columns are monkeys Average conductivity of Monera suspension> 2000 μS / cm before treatment^TwoFraction from 18 μS / cm at 1^TwoReduced to Conductivity in subsequent fractions Gradually increases to 169 μS / cm in the tenth fraction.^TwoReached. Salmo Maximum recovery of Nera was obtained in fraction 8 (69%).   FIGS. 15 and 16 show Speedy ™ polyacrylamide desalinated water. Figure 3 shows the results of a similar desalination experiment using ram. S. In the case of Chifimurium (15th Figure), conductivity> 2000 to 980 μS / cm in fraction 1^TwoDown to And then in fraction 2 at 1088 μS / cm^TwoUp to a hula > 2000 μS / cm in sections 8, 9 and 10^TwoI went back up. S. Ente In the case of Rigitis (FIG. 16), the conductivity was from 2000 to 720 in fraction 2. μS / cm^TwoTo> 2000 μS / in fractions 8, 9 and 10 cm^TwoI went back up. The maximum salmonella recovery was obtained in fraction 10 and From 40% of C. typhimurium (FIG. 15); 64% of enteritidis (16th (Figure). The desalting process took about 5 minutes.   As shown in Examples 3, 4, and 5, desalting columns desalinate food-enriched cultures. Give a very quick way. However, bacterial recovery changed. press G dextran column appeared to be superior in this regard.

[Procedure for Amendment] Article 184-8, Paragraph 1 of the Patent Act [Date of Submission] March 12, 1997 [Content of Amendment] Claims 1. A method for concentrating microorganisms in a human or animal food sample having an initial conductivity greater than 1000 μS / cm ^{2, comprising} : (a) treating the sample to reduce conductivity; A method for concentrating microorganisms, comprising concentrating microorganisms. 2. The method of claim 1 wherein the conductivity is reduced to 500 μS / cm ² or less. 3. 3. The method according to claim ² , wherein the conductivity is reduced to 10-450 [mu] S / cm < ² >. 4. The method according to any one of claims 1 to 3, wherein the food sample is in the form of a culture medium. 5. A method for concentrating microorganisms in a composite liquid sample other than a food sample having an initial conductivity greater than 1000 μS / cm ^{2, comprising} : (a) treating the sample to reduce the conductivity to 10-450 μS / cm ² ; (B) A method for concentrating microorganisms, which comprises concentrating microorganisms using dielectrophoresis. 6. The method according to any one of claims 1 to 5, wherein the conductivity is reduced to 100 to 200 µS / cm ² . 7. 7. The method according to claim 1, wherein the conductivity is reduced by desalination. 8. 8. The method according to claim 7, wherein the desalting is carried out by using a desalting gel, a dialysis membrane sized to retain microorganisms, an ion exchange resin, reverse osmosis, ultrafiltration or an ion-specific balance change. 9. 9. The method according to claim 8, wherein the desalting is performed by using a desalting gel. 10. The method according to claim 9, wherein the desalting gel is a cross-linked dextran gel. 11. 9. The method according to claim 8, wherein the desalting is performed by using a dialysis cassette. 12. The method according to claim 11, wherein the desalting is performed by using a dialysis room. 13. The method according to claim 12, wherein the sample is desalted by a continuous dialysis method. 14． A method for analyzing microorganisms in a sample, comprising the method for enriching microorganisms in a sample according to any one of claims 1 to 13, and further comprising an analysis step. 15. 15. The method according to claim 14, wherein the microorganism is removed from the dielectrophoretic field prior to the analyzing step. 16. A batch of food is manufactured in a conventional manner, a representative sample is taken from the batch, and the microorganisms in the representative sample are analyzed using the method of claim 14 or claim 15, A method of manufacturing an approved batch of food comprising comparing the results of the analysis to a standard and, if the analysis meets or exceeds the standard, approving the batch. 17． 17. The method according to any one of the preceding claims, wherein the parameters of the dielectrophoretic field used comprise a frequency of 1 to 20 MHz at 1 to 15 volts. 18. 18. The method according to any one of the preceding claims, wherein the parameters of the dielectrophoretic field used include frequencies higher than 20 MHz at 1 to 100 volts. 19. An apparatus for separating microorganisms from a food sample having a conductivity higher than 1000 μS / cm ^2, a desalting means for processing the sample to reduce the conductivity to a value of 1000 μS / cm ² or less, and a dielectrophoresis field And means for passing the processed sample through the dielectrophoretic field. 20. Means for controlling the disappearance of the dielectrophoretic field, means for collecting the aggregated microorganisms by passing the carrier liquid through the means for generating the dielectrophoretic field, and means for collecting the carrier liquid thus passed Apparatus according to claim 19. 21. The method or apparatus according to any one of claims 1 to 20, wherein the microorganism is a bacterium.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), OA (BF, BJ, CF, CG , CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, U G), UA (AZ, BY, KG, KZ, MD, RU, TJ , TM), AM, AT, AU, BB, BG, BR, BY , CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, K P, KR, KZ, LK, LR, LT, LU, LV, MD , MG, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, T M, TT, UA, UG, US, UZ, VN (72) Inventor Pinbury, David             Sally Kay Tay 22.UK             7. Earl Wy, Leather Head, Land             Charles Road, Leather Headed Hood             Research Association, Rapid             Methods sexual section (no address)

Claims (1)

[Claims] 1. A method of concentrating microorganisms in a food sample, including the use of dielectrophoresis. 2. The method of claim 1 wherein the sample is treated to reduce its conductivity prior to dielectrophoresis. 3. 3. A method according to claim 1 or claim ^{2 wherein the} sample has an initial conductivity greater than 1000 S / cm2. 4. 4. A method according to claim 2 or claim 3 wherein the conductivity is reduced to 500 [mu] S / cm < ² > or less. 5. 5. The method of claim 4, wherein the conductivity is reduced to 10-450 [mu] S / cm < ² >. 6. The method according to claim 4 or 5, wherein the conductivity is reduced to 100 to 200 µS / cm ² . 7. 7. The method according to claim ² , wherein the conductivity of the sample is reduced by precipitating its solids by centrifugation and resuspending the pellet in a liquid having a conductivity of 1000 .mu.S / cm.sup.2 or less. The method according to any one of the preceding claims. 8. The method of claim 7, wherein the sample pellet is resuspended in distilled or deionized water. 9. The method according to any one of claims 2 to 6, wherein the conductivity is reduced by desalination. 10. 10. The method according to claim 9, wherein the desalting is performed by using a desalting gel, a dialysis membrane sized to retain microorganisms, an ion exchange resin, reverse osmosis, ultrafiltration or an ion-specific balance change. 11. The method according to claim 10, wherein the desalting is performed by using a desalting gel. 12. The method according to claim 11, wherein the desalting gel is a cross-linked dextran gel. 13. The method according to claim 10, wherein the desalting is performed by using a dialysis cassette. 14． The method according to claim 10, wherein the desalting is performed by using a dialysis room. 15. The method according to claim 10, wherein the sample is desalted by a continuous dialysis method. 16. A method for analyzing microorganisms in a sample, comprising the method for enriching microorganisms in a sample according to any one of claims 1 to 15 and further comprising an analysis step. 17． 17. The method according to claim 16, wherein the microorganism is removed from the dielectrophoretic field prior to the analyzing step. 18. A batch of food is manufactured in a conventional manner, a representative sample is taken from the batch, and the microorganisms in the representative sample are analyzed using the method of claim 16 or claim 17 to determine the microorganisms. A method of manufacturing a food product in an approved batch, comprising comparing the results of the analysis with a standard and, if the analysis meets or exceeds the standard, approving the batch. 19. 19. The method according to any one of the preceding claims, wherein the parameters of the dielectrophoretic field used comprise a frequency of 1 to 20 MHz at 1 to 15 volts. 20. 19. The method according to any one of the preceding claims, wherein the parameters of the dielectrophoretic field used include frequencies higher than 20 MHz at 1 to 100 volts. 21. An apparatus for separating microorganisms from a food sample having a conductivity higher than 1000 μS / cm ^2, a desalting means for processing the sample to reduce the conductivity to a value of 1000 μS / cm ² or less, and a dielectrophoresis field And means for passing the processed sample through the dielectrophoretic field. 22. Means for controlling the disappearance of the dielectrophoretic field, means for collecting the aggregated microorganisms by passing the carrier liquid through the means for generating the dielectrophoretic field, and means for collecting the carrier liquid thus passed An apparatus according to claim 21. 23. 23. The method or device according to any one of claims 1 to 22, wherein the food sample is in the form of a culture medium. 24. The method or apparatus according to any one of claims 1 to 23, wherein the microorganism is a bacterium. 25. The method according to any one of claims 9 to 15, or 16, or 17, wherein the sample to be used is a complex liquid sample other than a food sample.