Method for extracting living microorganisms contained in sample solution

Abstract

To extract living microorganisms from a sample liquid containing both living and dead microorganisms by dielectrophoresis.SOLUTION: After filling a flow path 113 provided in a migration chamber 10 of a dielectrophoresis device 1 with a migration medium from a second syringe pump 124, a voltage application unit 20 applies an AC voltage to an electrode unit 14 to supply a sample liquid from a first syringe pump 123 to the flow path 113, and a living body in the sample liquid is captured by the electrode unit 14. While maintaining the voltage application to the electrode unit 14, the migration medium is further supplied from the second syringe pump 124 to the flow path 113, the residual liquid in the flow path 113 is replaced by the migration medium by flowing it from a discharge pipe 130 to a waste liquid tank 134, and the voltage application to the electrode unit 14 is released to further supply the migration medium from the second syringe pump 124 to the flow path 113, and the outflow liquid from the flow path 113 is collected in a collector 135 through the discharge pipe 130. The living microorganisms within the effluent liquid are collected in the collector 135 and separated from the dead bodies in the residual liquid that flows into the waste liquid tank 134.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for extracting living microbial organisms contained in a sample liquid, and in particular to a method for extracting living organisms from a sample liquid containing living and dead microbial organisms, using a dielectrophoresis device comprising a migration device equipped with a flow path having an inlet at one end and an outlet at the other end, electrodes arranged between the inlet and outlet in the flow path, and a voltage application unit to the electrodes. [Background technology]

[0002] Drinking water, bath water, and other water for daily use may contain trace amounts of various microorganisms due to various factors such as the water source and environment, and if the content exceeds a certain amount, it may induce infectious diseases. For example, Legionella bacteria, which are often detected in circulating hot spring water and bath water in public baths and lodging facilities, or in circulating cooling water in cooling towers, are acid-resistant microorganisms and are pathogenic bacteria that cause diseases such as Pontiac fever and severe pneumonia (Legionella pneumonia). For bath water such as hot spring water, ordinances stipulate that the regulated value of Legionella bacteria is less than 10 CFU / 100 mL, and annual testing for Legionella bacteria is mandatory. For circulating cooling water in cooling towers, a separate recommended value of less than 100 CFU / 100 mL is set, and similar testing is recommended.

[0003] As a standard test method for determining whether or not the Legionella bacteria contained in bath water or circulating cooling water exceed the regulated value, for example, the culture method specified in Non-Patent Documents 1 and 2 is used. However, since the culture method requires several days for cultivation, it takes a long period of at least about 10 days from the collection of the test water from the test subject to the determination of the result, which is a difficulty in managing the water quality of bath water, circulating cooling water, etc. Therefore, Non-Patent Document 1 recognizes the LC EMA-qPCR method (Non-Patent Documents 3 and 4), which is a rapid test method that can quantitatively detect the nucleic acid of live bacteria of the Legionella genus, as a test method for determining whether or not bath water complies with the water quality standard. In the LC EMA-qPCR method, the test water is filtered and concentrated, and then treated with acid to suppress the habitat of contaminating microorganisms in the test water, and liquid culture (Liquid Culture (LC)) is started to selectively grow the Legionella bacteria. Next, the liquid cultured sample is added with ethidium monoazide (EMA), a selective membrane-permeable dye, and irradiated with light, so that the nucleic acids derived from the contaminating microorganisms whose habitat has been suppressed and the corpses of Legionella bacteria are modified by EMA, and then the sample is subjected to a rapid test method, real-time polymerase chain reaction (qPCR). In this qPCR method, the nucleic acids derived from both the contaminating microorganisms whose habitat has been suppressed and the corpses of Legionella bacteria are modified by EMA and do not become templates for the polymerase chain reaction, so that the nucleic acids derived from live Legionella bacteria are selectively amplified, and the nucleic acids derived from the live bacteria can be selectively detected. With the LC EMA-qPCR method, it is possible to determine whether bath water complies with the water quality standards for Legionella bacteria in two days, which is significantly shorter than the culture method.

[0004] However, the LC EMA-qPCR method is designed to test bath water, which is relatively free of contaminant microorganisms, and the accuracy of the test decreases when testing circulating cooling water, which is highly contaminated with contaminant microorganisms, especially dead Legionella bacteria.

[0005] Meanwhile, Patent Document 1 discloses a method using dielectrophoresis as a method for easily testing the amount of bacteria contained in a sample liquid. This method selectively captures live bacteria contained in the sample liquid by applying a high-frequency AC voltage in a dielectrophoresis device, and quantitatively evaluates the live bacteria in the sample liquid by measuring the impedance change at that time.

[0006] Since dielectrophoresis can selectively capture live bacteria in sample water, it is possible to perform a highly accurate test for live bacteria by separating dead bacteria by dielectrophoresis even in sample water derived from circulating cooling water that is highly contaminated by dead bacteria and applying the captured live bacteria to the LC EMA-qPCR method. However, since the dielectrophoresis device described in Patent Document 1 measures the impedance in a state in which live bacteria are captured, it is difficult to remove the captured live bacteria from the dielectrophoresis device. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] September 19, 2019, Notification from the Director of the Public Health Division, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare: "Testing methods for Legionella bacteria in bath water, etc. in public baths" [Non-Patent Document 2] Japanese Industrial Standards JIS K 0350-50-10:2006, "Testing method for Legionella in industrial water and factory wastewater" [Non-Patent Document 3] Takara Bio Inc., Legionella test live bacteria detection method (LC EMA-qPCR method) [Search date 2023.07.04], Internet<URL:https: / / catalog.takara-bio.co.jp / product / basic_info.php?unitid=U100007673> [Non-Patent Document 4] Takara Bio Inc., Selective detection of DNA derived from live bacteria using the Viable Bacteria Selection system (EMA-PCR method) [Search date 2023.07.04], Internet<URL:https: / / catalog.takara-bio.co.jp / product / basic_info.php?unitid=U100006890#ema-pcr> [Patent documents]

[0008] [Patent Document 1] JP 2007-006858 A Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention utilizes dielectrophoresis to easily extract living microorganisms from a sample liquid containing both living and dead microorganisms. [Means for solving the problem]

[0010] The present invention relates to a method for extracting living organisms from a sample solution containing living organisms and dead organisms of microorganisms, using a dielectrophoresis device including an electrophoretic device having a flow path with an inlet at one end and an outlet at the other end, electrodes arranged in the flow path between the inlet and outlet, and a voltage application unit to the electrodes. The extraction method includes the steps of: supplying the sample solution from the inlet to the flow path while maintaining a voltage applied to the electrodes by the voltage application unit; supplying a migration medium from the inlet to the flow path while maintaining a voltage applied to the electrodes by the voltage application unit after the step 1, and replacing the remaining liquid in the flow path with the migration medium supplied; and supplying a migration medium from the inlet to the flow path while maintaining a voltage applied to the electrodes by the voltage application unit after the step 2, and recovering the outflow liquid from the outlet. Here, the migration medium has an electrical conductivity that allows the electrodes to which a voltage is applied by the voltage application unit to selectively capture living organisms, and the sample solution contains microorganisms in the migration medium.

[0011] In this extraction method, in step 1, living microorganisms contained in the sample liquid supplied from the introduction part to the flow path are selectively captured by the electrodes, and their corpses remain in the sample liquid. In step 2, the remaining liquid in the flow path is replaced with an electrophoretic medium, thereby removing the corpses in the flow path. In step 3, the voltage applied to the electrodes is removed, causing the living microorganisms captured by the electrodes to detach into the electrophoretic medium. Therefore, the effluent from the discharge part recovered in step 3 contains the living microorganisms that have detached from the electrodes.

[0012] The extraction method according to the present invention further includes, for example, a preparation step prior to step 1, of supplying a loading medium from an introduction part and filling the flow path with the loading medium.

[0013] In one embodiment of the extraction method according to the present invention, the inlet section includes an inlet pipe connected to the flow path, and a first inlet and a second inlet branched from the inlet pipe, and the sample liquid and the electrophoretic medium are supplied from the first inlet and the second inlet, respectively, through the inlet pipe.

[0014] In another embodiment of the extraction method according to the present invention, the inlet section has a first inlet and a second inlet each connected to the flow path, and the first inlet is provided between the second inlet and the electrode, and the sample liquid and the electrophoretic medium are supplied from the first inlet and the second inlet, respectively.

[0015] The extraction method of each of the above-mentioned embodiments may further include, before step 2, step 2A of supplying a migration medium from the second inlet to the flow path while maintaining the voltage application to the electrodes by the voltage application unit, and pumping the sample liquid supplied from the first inlet to the flow path in step 2 toward the electrodes.

[0016] The extraction method including step 2A may further include step 2B, subsequent to step 2A, of supplying a migration medium from the second inlet toward the flow path while maintaining the application of a voltage to the electrodes by the voltage application unit, and sucking at least a portion of the migration medium supplied into the first inlet.

[0017] The microorganisms to be subjected to the extraction method of the present invention are, for example, acid-resistant microorganisms. In this case, the sample solution is, for example, derived from a test water containing acid-resistant microorganisms. This sample solution is prepared by a test water treatment process including: step A, adjusting the pH of the test water to an acidic range in which acid-resistant microorganisms show resistance; step B, passing the test water whose pH has been adjusted in step A through a filter material that has a negative charge in a high pH range including the pH value of the test water adjusted in step A and through which acid-resistant microorganisms can pass; step C, passing the filter material that has undergone step B through an electrophoretic medium A that has a pH in the acidic range in which acid-resistant microorganisms show resistance and has an electrical conductivity that allows an electrode to which a voltage is applied by a voltage application unit to selectively capture a living organism; and step D, passing the filter material that has undergone step C through an electrophoretic medium B that has a pH higher than the isoelectric point of the acid-resistant microorganisms and has an electrical conductivity that allows an electrode to which a voltage is applied by a voltage application unit to selectively capture a living organism, and securing the passed liquid.

[0018] Another aspect of the present invention relates to a method for testing microorganisms contained in a sample liquid. This testing method includes a step of testing the effluent collected in step 3 of the method for extracting living microbial organisms according to the present invention.

[0019] In one embodiment of this testing method, the testing step includes a step of extracting nucleic acid from living microorganisms contained in the effluent, and a step of testing the extracted nucleic acid by a nucleic acid amplification method.

[0020] According to yet another aspect, the present invention relates to a dielectrophoresis device for separating live and dead microorganisms contained in a sample solution in the presence of an electrophoretic medium having an electrical conductivity capable of selectively capturing live microorganisms by dielectrophoresis. The dielectrophoretic device includes a migration device having a flow path having an inlet at one end and an outlet at the other end, an electrode disposed between the inlet and the outlet in the flow path, and a voltage application unit to the electrode. Here, the inlet includes an inlet tube connected to the flow path, and a first inlet for the sample solution and a second inlet for the electrophoretic medium branched off from the inlet tube.

[0021] According to yet another aspect, the present invention relates to a dielectrophoresis device for separating live and dead microorganisms contained in a sample solution in the presence of an electrophoretic medium having an electrical conductivity capable of selectively capturing live microorganisms by dielectrophoresis. The dielectrophoretic device includes a migration device having a flow path having an inlet at one end and an outlet at the other end, an electrode disposed between the inlet and the outlet in the flow path, and a voltage application unit to the electrode. Here, the inlet includes a first inlet for the sample solution and a second inlet for the electrophoretic medium, each of which communicates with the flow path, and the first inlet is provided between the second inlet and the electrode. Effect of the Invention

[0022] According to the method for extracting microbial living organisms of the present invention, the living organisms can be easily extracted by dielectrophoresis from a sample liquid containing microbial living organisms and dead organisms.

[0023] The microorganism testing method of the present invention can improve the accuracy of microorganism testing because the test subject is an effluent recovered by the extraction method of the present invention from a sample liquid containing live and dead microorganisms.

[0024] According to the dielectrophoresis apparatus of the present invention, living organisms can be easily extracted from a sample liquid containing living and dead organisms. [Brief description of the drawings]

[0025] [Figure 1] 1 is a schematic diagram of a dielectrophoresis device according to an embodiment of the present invention. [Diagram 2] FIG. 2 is a schematic plan view of an electrophoretic device used in the dielectrophoresis device. [Diagram 3] Cross-sectional view taken along line III-III in FIG. 2 . [Figure 4] FIG. 13 is a schematic diagram of a dielectrophoresis device according to another embodiment of the present invention. [Diagram 5] FIG. 13 is a schematic plan view of a dielectrophoretic device according to still another embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] A dielectrophoretic device according to an embodiment of the present invention will be described with reference to Figs. 1 to 3. This dielectrophoretic device is for separating living organisms and dead organisms of microorganisms contained in a sample liquid. The microorganisms that can be separated into living organisms and dead organisms by the dielectrophoretic device are organisms in the broad sense or microparticles derived from organisms, including microbiological organisms classified as bacteria, fungi, actinomycetes, rickettsia, mycoplasma, or viruses, as well as small protozoa and protozoa, larvae of organisms, cells isolated from animals and plants, cultured cells of such cells, sperm, blood cells, nucleic acids, proteins, etc., and the living organisms are those that are active, and the dead organisms are those that have irreversibly lost their activity.

[0027] In FIG. 1, a dielectrophoresis device 1 mainly includes a migration device 10 and a voltage application unit 20.

[0028] As shown in Figs. 1 to 3, the electrophoretic device 10 is formed in a rectangular flat plate shape, and mainly includes a main body 11, an inlet 12, an outlet 13, and an electrode section 14. The main body 11 is made of a material that has electrical insulation and is not easily deteriorated even when various solvents or living organisms are attached thereto, such as glass or resin, and is a laminate of a substrate 110, a cover 111, and a spacer 112 disposed between the substrate 110 and the cover 111. The main body 11 is also formed with a flow path 113 extending in the longitudinal direction at the center in the width direction, partitioned by the spacer 112 between the substrate 110 and the cover 111. The volume of the flow path 113 is not particularly limited as long as it is suitable for dielectrophoresis, but is, for example, about 50 to 100 μL.

[0029] The inlet 12 is provided at one end of the longitudinal direction of the flow path 113, is connected to the flow path 113, and protrudes from the cover 111. The tip of the inlet 12 is open, and an inlet tube 120 extends from the opening. The inlet tube 120 branches into a first inlet tube 121 (an example of a first inlet) connected to a first syringe pump 123 for supplying a sample liquid, and a second inlet tube 122 (an example of a second inlet) connected to a second syringe pump 124 for supplying an electrophoretic medium. The first syringe pump 123 and the second syringe pump 124 are capable of discharging stored liquid during normal operation, and of sucking liquid from the outside during reverse operation, and are capable of discharging or sucking liquid at a predetermined flow rate.

[0030] The discharge part 13 is provided at the other end of the flow path 113 in the longitudinal direction, is connected to the flow path 113, and protrudes from the cover 111. The tip of the discharge part 13 is open, and a discharge pipe 130 extends from the opening. The discharge pipe 130 branches into a waste liquid pipe 131 and a recovery pipe 132, and a three-way valve 133 is provided at the branch point between the waste liquid pipe 131 and the recovery pipe 132. The three-way valve 133 is used to switch the discharge pipe 130 to either the waste liquid pipe 131 or the recovery pipe 132. The waste liquid pipe 131 and the recovery pipe 132 are connected to a waste liquid tank 134 and a recovery device 135, respectively.

[0031] The electrode section 14 is formed in the flow channel 113 between the inlet section 12 and the outlet section 13, and is composed of two electrodes, a first electrode 141 and a second electrode 142, which are thin-film electrodes arranged on the substrate 110. The first electrode 141 and the second electrode 142 are both formed in a comb shape, and are arranged so that the comb teeth are parallel to the longitudinal direction of the flow channel 113 and are nested together. The first electrode 141 has a first terminal 143 extending to one end of the body 10 in the width direction, and the second electrode 142 has a second terminal 144 extending to the other end of the body 10 in the width direction. The first terminal 143 and the second terminal 144 are both formed in a thin-film shape on the substrate 110, and their tips are exposed at the cutouts 114 and 115 of the cover 111 and the spacer 112, respectively.

[0032] The voltage application section 20 is for applying an AC voltage, the frequency and voltage amplitude of which can be controlled, to the electrode section 14 , and is connected to the first terminal 143 and the second terminal 144 .

[0033] Next, a method for separating live microorganisms from dead ones using the dielectrophoresis device 1 and extracting the live microorganisms will be described. (Initial setting) First, with the voltage application unit 20 in a stopped state, the three-way valve 133 is operated so that the discharge pipe 130 communicates with the waste liquid pipe 131, and the dielectrophoresis device 1 is set to an initial state. In this initial state, the sample liquid is stored in the first syringe pump 123, and the migration medium is stored in the second syringe pump 124. The first syringe pump 123 and the second syringe pump 124 are set to a stopped state.

[0034] The sample liquid stored in the first syringe pump 123 is, for example, a dispersion of target microorganisms in physiological saline or a buffer solution. As the buffer solution, for example, an inorganic buffer solution such as hydrochloric acid-potassium chloride buffer solution or phosphate buffer solution, an organic buffer solution such as citrate buffer solution or acetate buffer solution, or a mixed buffer solution of inorganic and organic systems such as glycine-hydrochloric acid buffer solution is used. On the other hand, the migration medium stored in the second syringe pump 124 is physiological saline or the same buffer solution as that used in the sample liquid. When physiological saline is used in the sample liquid, it is preferable to use physiological saline as the migration medium. When a buffer solution is used in the sample liquid, it is preferable to use the same buffer solution as the sample liquid as the migration medium.

[0035] When a buffer solution is used as the sample liquid and the migration medium, the buffer solution has an electrical conductivity that allows the electrode unit 14 to selectively capture living organisms of microorganisms when a voltage is applied by the voltage application unit 20. A buffer solution having such an electrical conductivity can be prepared by controlling the electrolyte concentration, for example, by diluting a required buffer solution with deionized water. It is preferable to adjust the electrical conductivity of the buffer solution used as the migration medium and the buffer solution used in the sample liquid to be substantially the same.

[0036] (Process 1) In the dielectrophoresis device 1 set to the initial state, the voltage application unit 20 is operated to apply an AC voltage to the electrode unit 14. Here, the frequency of the AC voltage applied to the electrode unit 14 is adjusted so that a positive dielectrophoretic force acts only on the living organisms of the living organisms and the dead organisms of the microorganisms contained in the sample liquid. Then, while maintaining the state in which the AC voltage is applied to the electrode unit 14, the first syringe pump 123 is operated for a predetermined time, and the sample liquid is supplied from the first syringe pump 123 through the first introduction tube 121 and the introduction tube 120 from the introduction unit 12 into the flow path 113, and the flow path 113 is filled with the sample liquid. The living organisms of the microorganisms contained in the sample liquid filled in the flow path 113 migrate due to the action of the dielectrophoretic force, and are selectively captured by the first electrode 141 and the second electrode 142 of the electrode unit 14. On the other hand, the dielectrophoretic force does not act on the dead organisms of the microorganisms, and the dead organisms are in a state of floating in the sample liquid.

[0037] (Process 2) Next, the three-way valve 133 is maintained in the initial state, and the second syringe pump 124 is operated for a predetermined time while maintaining the state in which an AC voltage is applied to the electrode unit 14, and the migration medium is supplied from the second syringe pump 124 through the second inlet pipe 122 and the inlet pipe 120 from the inlet part 12 into the flow channel 113. As a result, the residual liquid originating from the sample liquid in which the corpses of microorganisms are suspended, present in the flow channel 113, overflows from the outlet part 13 and is forced out into the outlet pipe 130, and is discarded into the waste liquid tank 134 through the waste liquid pipe 131. As a result, the residual liquid in the flow channel 113 is replaced by the newly supplied migration medium. Here, it is preferable to adjust the operation time of the second syringe pump 124 and control the supply amount of the migration medium to the flow channel 113 so that the residual liquid in the outlet pipe 130 is replaced by the migration medium.

[0038] (Step 3) Next, the three-way valve 133 is operated so that the discharge pipe 130 is connected to the recovery pipe 132, and the voltage application unit 20 is stopped to release the AC voltage application to the electrode unit 14. As a result, the microbial living organisms captured by the electrode unit 14 are liberated from the first electrode 141 and the second electrode 142 into the migration medium in the flow path 113. Then, the second syringe pump 124 is operated for a predetermined time to further supply the migration medium from the introduction unit 12 to the flow path 113 through the second introduction pipe 122 and the introduction pipe 120. The migration medium in which the liberated living organisms are suspended is forced out of the discharge unit 13 into the discharge pipe 130 and is recovered in the recovery device 135 through the recovery pipe 132 as an outflow from the discharge unit 13.

[0039] Through the above steps 1 to 3, the microorganisms contained in the sample liquid stored in the first syringe pump 123 are separated into dead microorganisms discarded in the waste liquid tank 134 and living organisms collected in the collector 135, and as a result, the living organisms in the sample liquid are extracted in a dispersed state in the electrophoretic medium.

[0040] It is preferable that the flow path 113 of the electrophoretic device 10 used in the above-mentioned extraction method is filled with the migration medium in its entirety in advance. Therefore, it is preferable that the electrophoretic device 1 is used in which the migration medium is filled in the flow path 113 in advance as the electrophoretic device 1, or that a preparation step is performed for filling the flow path 113 of the electrophoretic device 10 with the migration medium before step 1 in carrying out the above-mentioned extraction method. In the preparation step, for example, after setting the dielectrophoretic device 1 to an initial state, the second syringe pump 124 is operated for a predetermined time to supply the migration medium from the second syringe pump 124 through the second introduction pipe 122 and the introduction pipe 120 from the introduction part 12 into the flow path 113. Then, the migration medium overflowing from the discharge part 13 is discharged from the discharge pipe 130 through the waste liquid pipe 131 to the waste liquid tank 134.

[0041] The above extraction method can also be modified to include the following steps: (Process 2A) Before executing step 2, the three-way valve 133 is maintained in the initial state, and the second syringe pump 124 is operated for a predetermined time while maintaining the state in which an AC voltage is applied to the electrode unit 14, and the migration medium is supplied from the second syringe pump 124 through the second inlet pipe 122 and the inlet pipe 120 from the inlet part 12 into the flow path 113. By supplying the migration medium, the sample liquid remaining in the flow path 113 between the inlet part 12 and the electrode unit 14 is pressure-transferred to the electrode unit 14, so that living microorganisms contained in the sample liquid supplied to the flow path 113 in step 2 are easily captured by the electrode unit 14.

[0042] The amount of electrophoretic medium supplied in this step is sufficient to pump the sample liquid retained between the introduction part 12 and the electrode part 14 to the electrode part 14, and therefore can be set to a small or trace amount compared to the amount of electrophoretic medium supplied in step 2.

[0043] (Process 2B) In the extraction method including step 2A, the second syringe pump 124 is operated for a predetermined time while maintaining the state in which an AC voltage is applied to the electrode unit 14 following step 2A, to supply the migration medium toward the flow path 113. At the same time, the first syringe pump 123 may be operated in the reverse direction for a predetermined time, thereby sucking at least a part of the migration medium supplied toward the flow path 113 in this step into the first introduction tube 121. This makes it possible to prevent the sample liquid remaining in the first introduction tube 121 after step 2 from leaking into the flow path 113, and to prevent the living organisms of the microorganisms extracted in step 3 from being contaminated by the corpses of the microorganisms contained in the sample liquid.

[0044] In the dielectrophoresis device 1 according to the above-described embodiment, the electrophoretic device 10 can be modified, for example, as shown in Fig. 4. In Fig. 4, the inlet section 12 of the electrophoretic device 10 includes a first inlet 12a and a second inlet 12b, each of which is individually connected to a flow channel 113. The first inlet 12a is provided between the second inlet 12b and the electrode section 14 in the flow channel 113, and is connected to a first syringe pump 123 for supplying a sample liquid through a first communication channel 123a. The second inlet 12b is connected to a second syringe pump 124 for supplying an electrophoretic medium through a second communication channel 124a.

[0045] This modified form of the dielectrophoresis device 1 can carry out the above-mentioned extraction method by supplying a sample liquid from the first syringe pump 123 to the flow path 113 through the first connecting path 123a and the first inlet 12a, and supplying a migration medium from the second syringe pump 124 to the flow path 113 through the second connecting path 124a and the second inlet 12b.

[0046] In the dielectrophoresis device 1 according to the above-described embodiment, the discharge pipe 130 extending from the discharge unit 13 of the electrophoresis device 10 may receive the discharged liquid from the discharge unit 13 by moving its tip to communicate with a waste liquid tank 134 or a collector 135 without the waste liquid pipe 131, the recovery pipe 132, and the three-way valve 133. For example, as shown in Fig. 5, the discharge pipe 130 is attached to the discharge unit 13 so as to be rotatable about the discharge unit 13 between a state shown by a solid line and a state shown by a dashed line in the figure, and by appropriately rotating the discharge pipe 130, the discharged liquid from the discharge unit 13 can be received in the waste liquid tank 134 or the collector 135.

[0047] The dielectrophoresis device 1 according to each of the above-mentioned embodiments is typically equipped with a control device that stores an operating program for executing the above-mentioned steps, and performs the above-mentioned extraction method by sending operating commands from this control device to each part, such as the voltage application unit 20, the first syringe pump 123, the second syringe pump 124, and the three-way valve 133.

[0048] Typical examples of microorganisms from which living organisms can be extracted by the above-mentioned extraction method are acid-tolerant microorganisms, such as bacteria, molds, and yeasts, such as Legionella or Salmonella. Acidophilic microorganisms are also included in the category of acid-tolerant microorganisms.

[0049] When the sample liquid to be used in the above-mentioned extraction method is highly contaminated by dead bacteria of acid-resistant microorganisms and various contaminating microorganisms, for example, when the sample liquid is sample water collected from circulating water such as circulating hot spring water or bath water in a public bath or accommodation facility, or circulating cooling water in a cooling tower for testing Legionella genus bacteria, when the target live acid-resistant microorganism is extracted from such a sample liquid, the target live acid-resistant microorganism is likely to be mixed with dead bodies or contaminating microorganisms. Therefore, when the target live acid-resistant microorganism is extracted from the sample water collected from circulating water highly contaminated by dead bodies or contaminating microorganisms, it is preferable to pretreat the test water and use the pretreated liquid obtained thereby as the sample liquid.

[0050] In the pretreatment of the test water, the pH of the test water is adjusted to an acidic range in which the acid-tolerant microorganism to be tested exhibits resistance, preferably to a pH lower than the isoelectric point of the acid-tolerant microorganism to be tested (Step A). ​​For example, when the acid-tolerant microorganism to be tested is a bacterium of the genus Legionella (it is known that the isoelectric point of general bacteria is generally 4 to 5, and the isoelectric point of bacteria of the genus Legionella is also in this range), the pH of the test water in this step is preferably adjusted to a range of 2 to 6, particularly 3.0 to 4.5.

[0051] The pH of the test water can be adjusted using an acidic agent capable of lowering the pH of the test water. Examples of the agent include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, and hydrofluoric acid, and organic acids such as citric acid, acetic acid, formic acid, and oxalic acid. Two or more of these agents may be used in combination.

[0052] By adjusting the pH in this process, among the various microorganisms contained in the test water, acid-tolerant microorganisms can live stably in a state of having a positive charge, while the survival of contaminating microorganisms other than the acid-tolerant microorganisms is suppressed in the acidic environment of the test water.

[0053] Next, the test water whose pH has been adjusted in step A is passed through the filter material (step B). The filter material used in this step has a negative charge in a high pH range that includes the pH value of the test water adjusted in step A. Such a filter material can be formed by using a material that is negatively charged in a high pH range that includes the pH value of the test water adjusted in step A. Examples of such materials include glass fiber, glass wool, and mixed fibers thereof, quartz wool, and cation exchange resin. Of these, it is preferable to use glass fiber, glass wool, or mixed fibers thereof.

[0054] The filter material is not limited in shape as long as it is formed using the above-mentioned material so that the test water can pass through it. For example, the material may be formed into a filter paper shape or the like, or the material may be filled in a water passage through which the test water passes in this step. However, since it is necessary to elute acid-resistant microorganisms from the filter material in step D described below, the filter material must be designed so that the acid-resistant microorganisms to be tested can pass through it. For example, when the acid-resistant microorganisms to be tested are Legionella bacteria and a filter paper-shaped filter material is used, it is preferable to set the auxiliary particle size of the filter material to 0.2 μm or more, particularly 0.4 μm or more.

[0055] When the test water, the pH of which has been adjusted in step A, is passed through the filter in this step, the filter becomes negatively charged by being exposed to the pH environment of the test water. In contrast, the acid-resistant microorganisms and their corpses in the test water that pass through the filter become positively charged, and are attracted to the filter by electrostatic attraction and captured by the filter. As a result, the acid-resistant microorganisms and their corpses in the test water (hereinafter sometimes referred to as acid-resistant microorganisms, etc.) are separated from the test water onto the filter, and the test water containing some of the impurity microorganisms and their corpses passes through the filter.

[0056] After the completion of this step and before proceeding to the next step, if necessary, the filter media that has captured the acid-resistant microorganisms and the like may be subjected to a heat treatment. The purpose of the heat treatment is to enhance the effect of inhibiting the habitation of some of the contaminating microorganisms captured in the filter media together with the acid-resistant microorganisms to be tested.

[0057] In the heat treatment of the filter medium, the filter medium is heated under the heat resistance conditions of the acid-resistant microorganism to be tested. For example, when the acid-resistant microorganism to be tested is a bacterium of the genus Legionella, the heating temperature of the filter medium is preferably set to 40°C to lower than the heat resistance temperature of the bacterium of the genus Legionella, particularly lower than 60°C.

[0058] Next, a buffer solution is supplied to the filter and passed through it (step C), whereby the test water remaining in the filter together with the acid-resistant microorganisms etc. is replaced with the buffer solution.

[0059] The buffer solution used here (hereinafter referred to as the "first buffer solution") is one that can be used as an electrophoretic medium, and has a pH adjusted to an acidic region in which the acid-resistant microorganism to be tested shows resistance, preferably the pH is adjusted to a range from the pH of the test water adjusted in step A to the isoelectric point of the acid-resistant microorganism to be tested, and has an electrical conductivity that allows the acid-resistant microorganism to be selectively captured when the acid-resistant microorganism to be tested is subjected to dielectrophoresis. As such a first buffer solution, various types of electrophoretic media similar to those stored in the second syringe pump 124 can be used as long as the pH and electrical conductivity are adjusted as described above. The electrical conductivity of the first buffer solution can be adjusted by controlling the electrolyte concentration, for example, by diluting the required buffer solution with deionized water. The electrical conductivity of the first buffer solution is preferably adjusted to be substantially the same as that of the second buffer solution described below.

[0060] When the acid-tolerant microorganism to be tested is a bacterium of the genus Legionella, the lower limit of the acidic range in which it shows resistance is pH 2, so that, for example, when the pH of the test water is set to 3.5 in step A, it is preferable to use a first buffer solution whose pH is adjusted to within a range of about 3.5 to 5.0 and whose electrical conductivity is adjusted to 100 μS / cm or less, particularly 25 μS / cm or less. In this example, the lower limit of the electrical conductivity is not particularly limited, but it is usually preferable to set it to 1 μS / cm or more, particularly 15 μS / cm or more.

[0061] Next, a buffer solution different from the first buffer solution is supplied to the filter, and the buffer solution that has passed through the filter is secured as a passing liquid (step D). The buffer solution used here (hereinafter referred to as "second buffer solution") is one that can be used as an electrophoretic medium, and is adjusted to a pH higher than the isoelectric point of the acid-resistant microorganism to be tested, and has an electrical conductivity that allows the acid-resistant microorganism to be selectively captured when the acid-resistant microorganism to be tested is applied to dielectrophoresis. As such a second buffer solution, various types of electrophoretic medium similar to that stored in the second syringe pump 124 can be used as long as the pH and electrical conductivity are adjusted as described above. The electrical conductivity of the second buffer solution can be adjusted by controlling the electrolyte concentration, for example, by diluting the required buffer solution with deionized water, just like the first buffer solution.

[0062] In this step, when the second buffer solution is passed through the filter, the acid-resistant microorganisms captured on the filter are negatively charged by being exposed to the pH environment of the second buffer solution, and are repelled by the filter, which is also negatively charged. As a result, the acid-resistant microorganisms are detached from the filter and transferred to the second buffer solution, and pass through the filter together with the second buffer solution. Here, since the test water remaining on the filter together with the acid-resistant microorganisms has been replaced by the first buffer solution in the previous step C, the electrical conductivity of the passing liquid obtained by passing the second buffer solution through the filter is maintained at an electrical conductivity that allows selective capture of the acid-resistant microorganisms to be tested by dielectrophoresis.

[0063] In this step, the greater the total amount of the second buffer solution passed through the filter, the higher the elution rate of the acid-tolerant microorganisms captured on the filter (i.e., the recovery rate of the acid-tolerant microorganisms captured on the filter). On the other hand, when a second buffer solution containing added proteins, sugars, alcohols, or surfactants is used, the elution rate of the acid-tolerant microorganisms from the filter can be promoted, and the elution rate of the acid-tolerant microorganisms can be increased while reducing the total amount of the second buffer solution passed through the filter.

[0064] In the second buffer solution containing the above-mentioned additives such as proteins, the concentration of each additive is preferably adjusted to be within the range described below. If the concentration is lower than this range, it may be difficult to obtain the effect of the additive in promoting the elution of acid-resistant microorganisms, etc. If the concentration is higher than this range, the reliability of the test results for acid-resistant microorganisms may be impaired by the influence of the additive.

[0065] Examples of proteins that can be added to the second buffer solution include beef extract, bovine serum albumin, and bovine gamma globulin. Two or more kinds of proteins may be used in combination. The concentration of the protein added to the second buffer solution is usually preferably set to 0.1 to 5.0 W / V%, more preferably set to 1.0 to 3.0 W / V%.

[0066] The sugars that can be added to the second buffer solution are not particularly limited, and specific examples include heptosaccharide ketoses such as sedoheptulose and coriose, hexose aldoses such as allose, talose, gulose, glucose, altrose, mannose, galactose and idose, hexose ketoses such as psicose, fructose, sorbose and tagatose, pentose aldoses such as ribose, lyxose, xylose, arabinose and apiose, pentose ketoses such as ribulose and xylulose, tetraose aldoses such as erythrose and threose, tetraose ketoses such as erythrulose, triose aldoses such as glyceraldehyde, and triose ketoses such as dihydroxyacetone. Two or more sugars may be used in combination.

[0067] The concentration of the saccharide added in the second buffer solution is usually set preferably to 0.1 to 5.0 W / V %, and more preferably to 0.5 to 3.0 W / V %.

[0068] The alcohols that can be added to the second buffer solution are not particularly limited, but specific examples include monohydric lower alcohols such as methanol, ethanol, and isopropanol, dihydric alcohols such as ethylene glycol, and trihydric alcohols such as glycerin. Two or more types of alcohols may be used in combination. The concentration of the alcohols added to the second buffer solution is usually preferably set to 5.0 to 15.0 W / V%, more preferably set to 8.0 to 10.0 W / V%.

[0069] The surfactant that can be added to the second buffer solution is not particularly limited, and soap or various synthetic surfactants can be used. Specific examples of usable synthetic surfactants include anionic surfactants such as carboxylic acid type, sulfonic acid type, sulfate ester type, and phosphate ester type, cationic surfactants such as quaternary ammonium salt type, alkylamine salt type, and pyridine derivative type, amphoteric surfactants such as alkyl betaine type, fatty acid amidopropyl betaine type, alkyl imidazole type, amino acid type, and amine oxide type, and nonionic surfactants such as ester type, ether type, ester ether type, alkanolamide type, alkyl glycoside, and higher alcohol. Two or more types of surfactants may be used in combination.

[0070] The concentration of the surfactant added in the second buffer solution is usually set preferably to 0.01 to 5.0 W / V %, and more preferably to 0.1 to 1.0 W / V %.

[0071] The above-mentioned additives may be used in combination of two or more kinds, but it is particularly preferable to select alcohols or surfactants.

[0072] In the pretreatment of the test water by the above steps, the filter material used in step B may be treated with a second buffer solution before step B. Specifically, the filter material may be treated with the second buffer solution by immersing the filter material in the second buffer solution or by passing the second buffer solution through the filter material. If the filter material is treated with the second buffer solution, the electrostatic attraction force that attracts acid-resistant microorganisms and the like to the filter material in step B is alleviated, and therefore, it becomes easier to increase the elution rate of acid-resistant microorganisms and the like captured by the filter material when the second buffer solution is passed through in step D.

[0073] When carrying out the above-mentioned extraction method, since the second buffer contains the target acid-resistant microorganisms and the like, the passed-through liquid secured in the above pretreatment step D can be stored as it is as a sample liquid in the first syringe pump 123. In this case, the migration medium stored in the second syringe pump 124 is preferably the second buffer solution.

[0074] In the test of microorganisms contained in the sample liquid, the effluent collected in the collector 135 by the above-mentioned extraction method can be used as a test sample. The method of testing the effluent can be selected according to the microorganism to be tested. For example, when the microorganism is a bacterium such as Legionella, various rapid test methods such as nucleic acid amplification methods such as PCR (polymerase chain reaction), qPCR, or LAMP (loop-mediated isothermal amplification), antibody methods such as immunochromatography, or ATP measurement can be used. In addition, ELISA, which is one of the immunoassay methods, or a general culture method for testing various microorganisms may be used. Among these test methods, it is preferable to adopt the nucleic acid amplification method because the test results can be obtained quickly and the test accuracy is high.

[0075] In this test, the effluent obtained by the above-mentioned extraction method is the subject of testing, so false positives and false negatives due to the influence of dead microorganisms or contaminating microorganisms contained in the test water or sample water are less likely to occur. For example, if the microorganism being tested is Legionella, there is a high degree of agreement with the results obtained by the culture method used to determine the water quality standards, and highly reliable test results can be obtained.

[0076] In the test using the nucleic acid amplification method, nucleic acid DNA or RNA is extracted from the living organisms of microorganisms contained in the recovery liquid (step X), and in the case of RNA, a reverse transcription reaction is applied, and then the nucleic acid is subjected to the nucleic acid amplification method (step Y). In this case, it is preferable to treat the living organisms of microorganisms contained in the recovery liquid with a selective cell membrane permeable dye such as ethidium monoazide (EMA) or propidium monoazide (PMA) (step Z). When treated with the selective cell membrane permeable dye, the nucleic acid derived from the corpses of the microorganisms contaminating the living organisms of the microorganisms extracted from the sample liquid is modified by the selective membrane permeable dye, and the nucleic acid derived from the corpses is prevented from being amplified in step Y. Therefore, in step Y, the results due to the amplification of the nucleic acid of the corpses are less likely to be reflected, and more reliable test results can be obtained.

[0077] When the microorganism to be tested is Legionella and immunochromatography is used as the test method, immunochromatographic test paper for testing Legionella may be used. Commercially available immunochromatographic test paper for testing Legionella can be used. For example, "Check Legionella" (product name of SA Scientific, Inc.) or "Q-Line Kyokuto Legionella" (product name of Kyokuto Pharmaceutical Industries Co., Ltd.), which are reagents for detecting Legionella pneumophila serotype 1 antigen (LPS) in urine, can be used. EXAMPLES

[0078] [Example] The number of Legionella bacteria contained in the circulating cooling water collected from the cooling tower was examined according to the following procedure. Here, the circulating cooling water collected from the cooling tower was concentrated 50 times to prepare the test water. 1.5 mL of this test water was added with 1.5 mL of 0.2 M acetate buffer (pH 3.6) to adjust the pH to 3.8, and the water was allowed to stand for 5 minutes (step A). ​​Then, the entire amount (3.0 mL) of the test water after standing for 5 minutes in step A was passed through a syringe filter (made of glass fiber by Techno Lab S.C. Co., Ltd. / diameter 13 mm, pore size 1.00 μm) that had been treated by passing 1.0 mL of a commercially available dielectrophoresis buffer (trade name "Elesta Buffer" by AFI Technology Co., Ltd.) with a pH of 7 and an electric conductivity of 20 μS / cm (step B).

[0079] 3.0 mL of diluted acetate buffer (pH 4.2, electrical conductivity 20 μS / cm) was passed through the syringe filter that had been subjected to step B (step C), and then 4.0 mL of the dielectrophoresis buffer used in step B was passed through, and the passed dielectrophoresis buffer was collected. The collected dielectrophoresis buffer was then centrifuged at 8,000 g for 5 minutes at 4° C., and 2.5 mL of the supernatant was discarded, thereby concentrating the volume to 1.5 mL, the same as that of the test water used in step A (step D).

[0080] The dielectrophoresis buffer concentrated in step D was used as a sample liquid, and live bacteria of the genus Legionella were extracted from the sample liquid using a dielectrophoresis device 1 according to the embodiment shown in Fig. 1. The dielectrophoresis device 1 used had a flow path 113 with a volume of 60 µL as an electrophoresis device 10, stored the sample liquid in a first syringe pump 123, and stored the dielectrophoresis buffer used in step B in a second syringe pump 124. The AC voltage applied to the electrode unit 14 by the voltage application unit 20 was set to 6 MHz and 22 V.

[0081] In the extraction operation, after setting the dielectrophoresis device 1 to an initial state, the second syringe pump 124 was operated to supply 0.5 mL of the dielectrophoresis buffer solution to the flow path 113 at a flow rate of 500 μL / min, and the dielectrophoresis buffer solution overflowing from the discharge unit 13 was discarded in the waste liquid tank 134 (preparation step). Next, the voltage application unit 20 was operated to apply an AC voltage to the electrode unit 14, and then the first syringe pump 123 was operated to supply 1 mL of the sample liquid to the flow path 113 at a flow rate of 50 μL / min (step 1). Then, while continuing to apply a voltage to the electrode unit 14, the second syringe pump 124 was operated to supply 0.1 mL of the dielectrophoresis buffer solution to the flow path 113 at a flow rate of 50 μL / min (step 2A). Following this, while maintaining the application of AC voltage to the electrode portion 14, the second syringe pump 124 was operated for 0.5 minutes at a flow rate of 100 μL / min to supply the dielectrophoresis buffer toward the flow path 113, and at the same time, the first syringe pump 123 was operated in the reverse direction for 0.5 minutes at a flow rate of 100 μL / min to suck the dielectrophoresis buffer from the second syringe pump 124 into the first inlet tube 121 (step 2B).

[0082] Next, while maintaining the state in which an AC voltage was applied to the electrode unit 14, the second syringe pump 124 was operated to supply 4 mL of the dielectrophoresis buffer solution to the flow path 113 at a flow rate of 100 μL / min, and the dielectrophoresis buffer solution overflowing from the discharge unit 13 was discarded in the waste liquid tank 134 (step 2). Then, the three-way valve 133 was operated so that the discharge pipe 130 was connected to the recovery pipe 132, and the voltage application to the electrode unit 14 was stopped. Thereafter, the second syringe pump 124 was operated to supply 1 mL of the dielectrophoresis buffer solution to the flow path 113 at a flow rate of 500 μL / min, and the outflow from the discharge unit 13 was recovered in the recovery vessel 135 (step 3).

[0083] Next, the effluent was added with ethidium monoazide, a selective membrane-permeable dye, and irradiated with light. Nucleic acid (DNA) was extracted from the effluent using a method that uses a lysis buffer, and the copy number was quantified by testing the extracted nucleic acid with a real-time PCR testing device (product name "Thermal Cycler Dice Real Time System III" by Takara Bio Inc.). The copy number was then converted into the number of Legionella bacteria using the LC EMA-qPCR method, a rapid testing method approved for testing Legionella bacteria in bath water.

[0084] The Legionella count was examined for three types of sample water (sample water 1 to 3) that were collected and prepared from three different types of circulating cooling water, using the above-mentioned process, and the results are shown in Table 1. It took about four hours from the time the collection of the circulating cooling water was completed until the Legionella count was determined.

[0085] [Comparative Example] According to the culture method of Non-Patent Document 2, the number of Legionella bacteria contained in the circulating cooling water collected from the cooling tower was examined. Here, the circulating cooling water collected from the cooling tower was concentrated 50 times to prepare test water. Then, 1.0 mL of hydrochloric acid-potassium chloride buffer (pH 2.2) was added to 1.0 mL of this test water and allowed to stand for 5 minutes. The test water thus treated was applied to GVPC medium (manufactured by Nissui Pharmaceutical Co., Ltd.), which is a selective isolation medium for Legionella bacteria, and cultured at 36°C for 7 days. Among the colonies formed on the GVPC medium, those thought to be Legionella bacteria were picked up on BCYEα agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and sheep blood agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), which is an L-cysteine-free medium, and cultured at 36°C for 5 to 7 days to determine the number of Legionella bacteria.

[0086] The results of examining the number of Legionella bacteria using the above-mentioned process for three types of sample water (sample water 1 to 3) that were collected from the same three types of circulating cooling water as in the Examples, are shown in Table 1. According to Table 1, the converted bacterial count value of the Examples is in the same order (number of digits) as the bacterial count of the Comparative Example, and the results of the Examples have the same reliability as the results of the Comparative Example.

[0087] [Table 1] [Explanation of symbols]

[0088] 1. Dielectrophoresis device 10 Electrophoresis device 20 Voltage application section 12 Introduction 12a First entrance 12b Second entrance 13 Discharge section 14 Electrode section 113 Flow Path 120 Introductory tube 121 1st introduction pipe 122 2nd introduction pipe

Claims

1. A method for extracting living organisms from a sample solution containing living organisms and corpses of microorganisms, using a dielectrophoresis device including an electrophoretic device having a flow path with an inlet at one end and an outlet at the other end, an electrode disposed between the inlet and the outlet in the flow path, and a voltage application unit to the electrode, the method comprising: a step 1 of supplying the sample liquid from the inlet to the flow channel while maintaining the voltage application to the electrodes by the voltage application unit; a step 2, after the step 1, of supplying a migration medium from the inlet to the flow channel in a state where the voltage application unit maintains the voltage applied to the electrodes, and replacing the remaining liquid in the flow channel with the migration medium thus supplied; a step 3 of further supplying the electrophoretic medium from the inlet to the flow channel while the voltage application unit is released from the voltage application unit after the step 2, and recovering an effluent from the outlet; Including, the electrophoretic medium has an electrical conductivity that allows the electrodes to selectively capture the living body when a voltage is applied by the voltage application unit; The sample solution contains the microorganism in the electrophoretic medium. A method for extracting living microbial organisms contained in a sample liquid.

2. 2. The method for extracting living microorganisms contained in a sample liquid according to claim 1, further comprising, before step 1, a preparation step of supplying the migration medium from the inlet and filling the flow path with the migration medium.

3. 2. The method for extracting living microbial organisms contained in a sample liquid according to claim 1, wherein the inlet section comprises an inlet pipe connected to the flow path, and a first inlet and a second inlet branched from the inlet pipe, and the sample liquid and the electrophoretic medium are supplied from the first inlet and the second inlet through the inlet pipe, respectively.

4. 2. The method for extracting living microbial organisms contained in a sample liquid according to claim 1, wherein the introduction section includes a first inlet and a second inlet each connected to the flow path, the first inlet being provided between the second inlet and the electrode, and the sample liquid and the electrophoretic medium are supplied from the first inlet and the second inlet, respectively.

5. 5. The method for extracting living microorganisms contained in a sample liquid according to claim 3 or 4, further comprising, before step 2, step 2A of supplying the electrophoretic medium to the flow path from a second inlet while maintaining the voltage application to the electrode by the voltage application unit, and pressure-feeding the sample liquid supplied to the flow path from the first inlet in step 1 toward the electrode.

6. 6. The method for extracting living microbial organisms contained in a sample solution according to claim 5, further comprising, subsequent to step 2A, step 2B of supplying the migration medium from a second inlet toward the flow path while maintaining application of a voltage to the electrodes by the voltage application unit, and aspirating at least a portion of the migration medium supplied to the first inlet.

7. 2. The method for extracting living microbial organisms contained in a sample solution according to claim 1, wherein the microorganisms are acid-resistant microorganisms.

8. The sample liquid is derived from a test water containing the acid-resistant microorganism, A step A of adjusting the pH of the test water to an acidic range in which the acid-tolerant microorganisms are resistant; a step B of passing the test water, the pH of which has been adjusted in the step A, through a filter material that has a negative charge in a high pH range including the pH value of the test water adjusted in the step A and through which the acid-tolerant microorganisms can pass; A step C of passing the filter material that has been subjected to the step B through an electrophoretic medium A having a pH in an acidic range to which the acid-resistant microorganisms are resistant and an electrical conductivity that allows the electrodes to selectively capture the living organism when a voltage is applied by the voltage application unit; A step D of passing an electrophoretic medium B, which has a pH higher than the isoelectric point of the acid-resistant microorganism and has an electrical conductivity that allows the electrode to selectively capture the living organism when a voltage is applied by the voltage application unit, through the filtering material that has been subjected to the step C, and collecting the passing liquid; 8. The method for extracting living microorganisms contained in a sample liquid according to claim 7, wherein the sample liquid is prepared by a water test treatment process comprising the steps of:

9. The method for extracting microbial organisms according to claim 1 further comprises a step of inspecting the effluent collected in step 3. A method for testing microorganisms contained in a sample liquid.

10. The inspection step includes: extracting nucleic acids from microbial organisms contained in the effluent; testing the extracted nucleic acid by a nucleic acid amplification method; Including, The method for testing microorganisms contained in a sample liquid according to claim 9.

11. 1. A dielectrophoresis device for separating living organisms and dead organisms of microorganisms contained in a sample liquid in the presence of an electrophoretic medium having an electrical conductivity capable of selectively capturing the living organisms by dielectrophoresis, comprising: a migration device having a flow path with an inlet at one end and a outlet at the other end; an electrode disposed in the flow path between the inlet and the outlet; A voltage application unit for applying a voltage to the electrode; Equipped with the inlet section includes an inlet pipe connected to the flow path, and a first inlet for the sample liquid and a second inlet for the migration medium branched from the inlet pipe; Dielectrophoresis apparatus.

12. 1. A dielectrophoresis device for separating living organisms and dead organisms of microorganisms contained in a sample liquid in the presence of an electrophoretic medium having an electrical conductivity capable of selectively capturing the living organisms by dielectrophoresis, comprising: a migration device having a flow path with an inlet at one end and a outlet at the other end; an electrode disposed in the flow path between the inlet and the outlet; A voltage application unit for applying a voltage to the electrode; Equipped with the inlet section includes a first inlet for the sample liquid and a second inlet for the electrophoretic medium, each of which communicates with the flow channel, and the first inlet is provided between the second inlet and the electrode; Dielectrophoresis apparatus.

Images