JP2000189150A - Device for measuring number of microorganism and method therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a maintenance-free device for measuring the number of microorganisms capable of rapidly and automatically measuring various samples at high sensitivity. SOLUTION: This device for measuring the number of microorganisms possesses a measuring cell 1 installed with a plurality of electrodes and capable of introducing a microorganism-containing liquid and a measuring vessel 30 connected to the measuring cell 1 through an ion-permeable diaphragm and equipped with a dialysis means to reduce the ion concentration in the measuring cell 1. To the measuring vessel 30, an electrophoresis power circuit 12, a measuring unit 13 for measuring impedance or electric conductivity between the plurality of electrodes, an operation unit 14 for calculating the number of the microorganisms and a control means 15 for controlling the electrophoresis power circuit 12, the dialysis means, the measuring unit 13 and the operation unit 14 are installed. The number of microorganisms is counted by measuring the impedance or electric conductivity by reducing the ion concentration in the measuring cell 1 by the dialysis means when a liquid is introduced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus and a method for measuring the number of microorganisms in a solution.

[0002]

2. Description of the Related Art Conventionally, many techniques such as those described in JP-A-57-50652 have been known as a method for measuring the number of microorganisms in a solution.

[0003] However, in the method of measuring the number of microorganisms according to the conventional technique, a special agent such as an enzyme or a dye is added to a sample solution to cause a biochemical reaction, and the progress or result of the reaction is measured by fluorescence or luminescence. Although the measurement sensitivity is relatively high, specialized knowledge in the field of microorganisms and biochemistry is required, and a dedicated and expensive large measuring device is required, and furthermore, work by a dedicated person is required. However, it was not very common and easy to measure the number of microorganisms.

[0004] Therefore, a simple and simple method that uses only physical means, such as that described in JP-A-59-91900, does not use any chemicals, is small, and can be automatically incorporated into a sample system. A new microbial count detector was proposed,
Since the number of microorganisms cannot be detected unless the number of cells exceeds 10 8 cells / ml (the number of microorganisms is 100 million in 1 ml), its application range has been significantly limited.

[0005]

As described above, in order to increase the measurement sensitivity of the microorganism counting device according to the prior art, it is necessary to use some kind of chemical, use a special measuring device, and operate the device by a dedicated person having specialized knowledge. Was needed. In addition, with a simple device that does not use drugs, measurement can be performed without the need for a dedicated person.However, if the number of microorganisms is not very large, measurement is difficult, and only a low-sensitivity measuring instrument can be obtained. However, there is a problem that there is no simple and maintenance-free means even if it is desired to improve the sensitivity by locally increasing the density.

[0006] In order to solve these problems, the present invention provides a maintenance-free microorganism which can rapidly and automatically measure various samples having high sensitivity and high electrical conductivity without the need for drugs or special equipment. It is an object to provide a number measuring device.

[0007]

In order to achieve the above object, a microorganism counting apparatus according to the present invention comprises: a measuring cell provided with a plurality of electrodes, into which a liquid containing microorganisms can be introduced; Articulated through an ion-permeable membrane,
A measurement container having a dialysis means for reducing the ion concentration in the measurement cell, wherein the measurement container has a migration power supply circuit for dielectrophoretizing microorganisms in the measurement cell; A measuring unit for measuring the impedance or electric conductivity between the electrodes, a calculating unit for calculating the measurement result of the measuring unit to calculate the number of microorganisms, the electrophoresis power supply circuit, the dialysis means, the measuring unit, and Control means for controlling an arithmetic unit is provided, and when the liquid is introduced, the dialysis means reduces the ion concentration in the measurement cell to measure impedance or electric conductivity to calculate the number of microorganisms. I do.

Accordingly, a maintenance-free microbial count measuring method and a microbial count measuring method capable of quickly and automatically measuring various samples having high sensitivity and high electrical conductivity without requiring a medicine or a special device. The purpose is to provide.

[0009]

DETAILED DESCRIPTION OF THE INVENTION The invention described in claim 1 is a measuring cell provided with a plurality of electrodes, into which a liquid containing microorganisms can be introduced, and connected to the measuring cell via an ion-permeable diaphragm. A dialysis unit for reducing the ion concentration in the measurement cell, the measurement container has a migration power supply circuit for dielectrophoresis of the microorganisms in the measurement cell, A measurement unit for measuring impedance or electrical conductivity between the electrodes, a calculation unit for calculating the number of microorganisms by calculating the measurement result of the measurement unit, the electrophoresis power supply circuit, the dialysis unit, and the measurement unit Control means for controlling the arithmetic unit is provided, and when the liquid is introduced, the dialysis means reduces the ion concentration in the measurement cell to measure impedance or electric conductivity to calculate the number of microorganisms. Microorganism counting device characterized by the fact that even if the sample has a high ion concentration and high electrical conductivity, the microorganisms can be efficiently concentrated near the electrodes after the electrical conductivity of the sample is reduced by electrodialysis. Since the number of microorganisms can be measured by electrical means, high-precision and high-sensitivity measurement can be performed without requiring a medicine or a special device.

According to a second aspect of the present invention, the dialysis means comprises a first dialysis cell containing a first dialysis electrode and connected to a measurement cell via a cation-permeable membrane, and a second dialysis cell. A second dialysis cell containing an electrode and connected to the measurement cell via an anion-permeable diaphragm is provided, and a voltage for performing electrodialysis on the first dialysis electrode and the second dialysis electrode is provided. The microorganism counting device according to claim 1, further comprising a dialysis power supply circuit for applying, even if the sample has a high electrical conductivity due to a high ion concentration,
The ion concentration in the measuring cell can be easily reduced by electrical processing, without the need for drugs or special equipment.
Highly accurate and highly sensitive measurement.

According to a third aspect of the present invention, the permeation means includes a dialysis cell connected to the measurement cell via a semipermeable membrane, and the dialysis cell has a higher electrical conductivity than the liquid in the measurement cell. 2. The microorganism counting device according to claim 1, further comprising a dialysis solution supply unit for introducing a liquid having a low electric conductivity, and a simple structure in which a liquid having a low electric conductivity is flowed to efficiently provide ions in the sample. The concentration can be reduced, and the number of microorganisms can be measured with high sensitivity without requiring a drug or a special device.

The invention according to claim 4 is characterized in that the electric conductivity of the microorganism-containing liquid is measured before measuring the number of microorganisms. Since the measurement device is used, the electrical conductivity of the sample can be set to an optimum value in advance before the measurement, so that highly accurate and highly sensitive measurement can be performed.

According to a fifth aspect of the present invention, a microorganism-containing liquid is introduced into a cell having a plurality of electrodes, and an AC voltage is applied between the electrodes to apply a dielectrophoretic force to the microorganisms in the measurement cell. A method for measuring the number of microorganisms by measuring the impedance or electrical conductivity between the electrodes while collecting in the electric field concentrating portion by applying a microbial count, wherein the liquid is deionized before applying the AC voltage. Since the method for measuring the number of microorganisms is characterized by lowering the electrical conductivity of the liquid, even a sample having a high electrical conductivity due to a high ion concentration can perform highly accurate and highly sensitive measurement by deionization. it can.

According to a sixth aspect of the present invention, the deionization treatment is dialysis using a semipermeable membrane.
The microorganism counting method described is a simple method using a semipermeable membrane, but the sample can be efficiently deionized even with a sample with high electrical conductivity due to high ion concentration, with high accuracy and high sensitivity. Measurement.

In the invention described in claim 7, the deionization treatment is electrodialysis using a first dialysis cell and a second dialysis cell, and the microbial count according to claim 5 is provided. Since this method is used, even a sample having a high electric conductivity due to a high ion concentration can be quickly deionized because it is electrically processed, and highly accurate and highly sensitive measurement can be performed in a short time.

Hereinafter, an embodiment of the present invention will be described with reference to FIG.
This will be described with reference to FIG. 8 and (Equation 1) to (Equation 5) described later.

(Embodiment 1) A microorganism counting apparatus according to an embodiment of the present invention will be described in detail with reference to the drawings. FIG. 1 is an overall configuration diagram of an apparatus for measuring the number of microorganisms according to Embodiment 1 of the present invention, FIG. 2 is an explanatory view of an electrode according to Embodiment 1 of the present invention, and FIG. 3 is Embodiment 1 of the present invention.
FIG. 4 is a graph for explaining the relationship between the number of microorganisms, measurement time, and the capacitance C between the electrodes, and FIGS. FIG. 7 is a diagram for explaining a procedure for calculating the inter-electrode capacitance C. FIG. 7 is a diagram for explaining the relationship between force and frequency by dielectrophoresis.
FIG. 3 is a diagram illustrating a flow of a series of microbial count measurement operations in the first embodiment.

In FIG. 1, 1 is a measuring cell, 2 is a measuring electrode composed of a thin film electrode on a substrate, 3 is an anion exchange membrane, 4
Is a cation exchange membrane, 5 is a dialysis electrode A, 6 is a dialysis electrode B,
7 is a dialysis cell A, 8 is a dialysis cell B, 9 is a solenoid valve, 10 is a sample system pipe, 11 is a dialysis power supply circuit, 12 is a migration power supply circuit, 13 is a measurement unit, 14 is a calculation unit, 15 is control means, 1
6 is a display means, and 30 is the whole measurement container. FIG.
In the figure, 17 is a substrate of the measurement electrode, 18 is a thin film electrode on the substrate, and 19 is a gap sandwiched between two poles of the thin film electrode. Reference numeral 20 in FIG. 3 denotes electric lines of force generated by a voltage applied between the thin film electrodes. 2 and 5
5A, reference numeral 50 denotes one pole of the thin-film electrode 18, reference numeral 51 denotes the other pole of the thin-film electrode 18, reference numeral 52 denotes a capacitance constituting an assumed CR parallel equivalent circuit, and reference numeral 53 denotes an assumed CR equivalent circuit. In FIG. 5B, reference numeral 54 denotes a time axis, 55 denotes an axis representing the magnitude of voltage or current, 56 denotes a curve representing a change in current value, and 57 denotes a curve representing a change in voltage value. FIG. 6 is a diagram showing the relationship between the reactance, the resistance, and the phase angle when the impedance is vector-decomposed.

As shown in FIG. 1, the measuring vessel 30 has a dialysis cell A7 and a dialysis cell B8 connected to the measurement cell 1 via an anion exchange membrane 3 and a cation exchange membrane 4, and a solenoid valve 9 is provided. Is closed so that mass transfer between the measurement cell and the two dialysis cells is not performed by any method except through the ion exchange membrane. Other mechanisms may be used as the solenoid valve 9 as long as it can shut off the measurement cell from the sample system. In the first embodiment, the dialysis cell A7
The dialysis cell B8 is also provided with a solenoid valve, in order to prevent current from flowing around the sample system outside the cell when performing electrodialysis, and to make the electric detour distance sufficiently long. When the bypass current is almost negligible, there is no need to provide the bypass current.

As shown in FIGS. 1 and 2, a thin film electrode 18 provided on the measurement electrode 2 is provided in the measurement cell 1 in order to move the microorganisms in the sample liquid to a predetermined position by dielectrophoresis. The two poles 50 and 51 are provided to face each other with a small gap 19 therebetween. In the first embodiment, as shown in FIGS.
Are nested in each other. Each electrode is made of a conductor that is formed in close contact with the measurement electrode substrate 17 by a method such as sputtering, vapor deposition, or plating, and the portion of the substrate where the thin film electrode 18 is in close contact and the substrate surface are exposed. An end line, which is the boundary between the portions, is formed. In the first embodiment, the gaps 19 formed between the end lines of the thin-film electrode by the two poles 50 and 51 are all at the same interval. Since the electric field in the vicinity of the gap 19 becomes strongest by the application of the voltage for dielectrophoresis, the gap 19 becomes the electric field concentration portion in the first embodiment. Although the details will be described later, the microorganisms migrate toward the vicinity of the gap 19 where the electric field is most concentrated.

Here, the microorganism to be detected in the present invention will be described. The microorganisms referred to in the present invention are generally classified as bacteria, fungi, actinomycetes, rickettsia, mycoplasma, viruses, and other microorganisms that are subject to so-called microbiology, as well as small protozoa and protozoa Larvae of living organisms, isolated or cultured animal and plant cells, sperm, blood cells, nucleic acids, proteins and the like in a broad sense. Further, in the present invention, a microorganism existing in a liquid is assumed as a measurement target.

The thickness of the thin film electrode 18 in the first embodiment is about 5 nm. Since the film thickness of 5 nm is very small as compared with an interval of the gap 19 described later of 100 μm and compared with the microorganism to be detected,
Can be considered as an electrode having only a two-dimensional spread that can practically ignore the thickness. Therefore, as described above, the boundary between the portion where the electrode is in close contact with the measurement electrode and the portion where the substrate is exposed on the substrate 17 is expressed as an end line. At this time, the electric field distribution near the gap 19, which is the electric field concentration part, in the measurement electrode 2 used in the first embodiment is as shown in FIG. Since the thickness of the thin film electrode 18 in the first embodiment is as thin as about 5 nm, the electric field is concentrated most in the gap between the end lines of the two poles 50 and 51 as shown in FIG.

The dielectrophoretic phenomenon of the microorganisms that occurs here will now be described. If a more detailed explanation is needed, reference [J. theor. Biol (1972) vol.
37, 1-13.

When a high-frequency AC voltage is applied between the electrodes in the liquid, the action of the AC electric field generated by the application of the AC voltage causes
The microorganisms in the measurement cell 1 are migrated to the most inhomogeneous and non-uniform part due to their dielectric properties, that is, to the electric field concentrated part. Here, the AC voltage is not only a sine wave but also a voltage that changes the direction of flow at a substantially constant cycle, and the average value of the current in both directions is equal.

At this time, assuming that the dipole moment of the microorganism as the dielectric fine particles is μ, the force F has a relationship with the electric field E according to (Equation 1).

[0026]

(Equation 1)

Further, assuming that the relative permittivity of the cytoplasm of the microorganism is ε ₂ , the relative permittivity of the liquid containing the microorganism is ε ₁ , the radius when the microorganism is regarded as a sphere is a, and the circumference is π , And the dielectrophoretic force F can be rewritten as (Equation 2).

[0028]

(Equation 2)

(Equation 2) shows that the force due to dielectrophoresis is a potential gradient,
This indicates that the medium is affected by the difference in the relative dielectric constant between the medium and the microorganism as the dielectric fine particles. Since an AC voltage is applied to the embodiment 1, the dielectrophoretic embodiment, the dielectric constant epsilon has a frequency dependency, for example, when the the epsilon ₁ 'takes into account the response to the AC relative epsilon ₁ , Ε
₁ ′ is a form in which a term using imaginary unit j, electric conductivity σ _{1 of the} medium, and angular frequency ω is added to ε ₁ as in (Equation 3).

[0030]

(Equation 3)

The same applies to ε ₂ , where ε ₂ ′ is a value taking account of the response to an alternating current. Ε ₂ ′ uses imaginary unit j, electric conductivity σ _{2 of the} medium, and angular frequency ω for ε ₂ . Term is added, F is ε ₁ ε ₁ ′, ε ₂ is ε ₂
It is expressed by replacing it with '.

As described above, F also includes a term of the electric conductivity of the medium. Now, how the electric conductivity of the medium acts on the dielectrophoretic force will be specifically described. FIG. 7A is a graph showing the relationship between dielectrophoretic force and frequency using a medium such as water having a low ion concentration and a general value of relative permittivity as a microorganism. FIG.
As is clear from (a), dielectrophoresis acts as an attractive force in a certain frequency range and as a repulsive force in a certain frequency range. In the first embodiment, an attractive force is used to move microorganisms.

FIG. 7 (b) is a graph again obtained in a state where the ion conductivity of water as a medium is increased to increase the electric conductivity. The values of the factors such as the applied voltage are the same as those in FIG. As is clear from FIG. 7 (b), it can be seen that the frequency range showing the attractive force is narrow, and the attractive force itself is weak.

As described above, the force due to dielectrophoresis changes depending on the electric conductivity of the medium, and becomes weaker as the electric conductivity of the medium increases. Therefore, it can be seen that it becomes difficult for the microorganism measuring apparatus using dielectrophoresis to measure microorganisms such as sewage and seawater having high electric conductivity.

In the first embodiment, the measuring electrode 2
The structure in the vicinity of the gap 19 corresponds to the electric field concentration portion, and the electric field is most concentrated in the gap 19. Therefore, the microorganism is most strongly electrophoresed in the gap 19 portion.
The gap 19 shown in FIG. 2 is a portion sandwiched between parallel electrodes, and the distribution of the electric field is uniform in the direction in which the electrodes extend, that is, in the direction perpendicular to the plane of FIG. However, in the direction perpendicular to the substrate surface, an electric field distribution as shown in FIG. 3 occurs, and the electric field concentrates most on the surface connecting the end lines of the electrodes. Microorganisms floating near the gap 19 are attracted to the gap 19 by such an electric field effect generated between the measurement electrodes 2,
Align along the lines of electric force. At this time, the moving state of the microorganisms 24 in the vicinity of the gap 19 depends on the number of microorganisms present in the sample liquid and the interval between the gaps 19, but when the number of microorganisms is sufficiently large, the gap 19 is formed by a chain composed of the microorganisms 24. It becomes even crosslinked. At this time, the microorganisms floating in the vicinity of the gap 19 from the beginning move to the gap 19 immediately, and the microorganisms floating in the place away from the gap 19 reach the gap 19 after a predetermined time elapses according to the distance. Therefore, after a certain time, gap 1
The number of microorganisms collected in a predetermined area near 9 is also proportional to the number of microorganisms in the measurement cell 1. This is, of course, proportional to the number of microorganisms existing in the sample system piping 10.

The thin-film electrode 18 may be made of any material as long as the resistance is not extremely high. However, it is assumed that the thin-film electrode 18 is used in a liquid, particularly in water as in the first embodiment. A metal having a low ionization tendency is desirable. Because a strong electric field is generated between the electrodes during dielectrophoresis,
Electrolysis may occur depending on the frequency of the applied voltage and the concentration of the electrolyte in the water. In the case of an electrode composed of a metal having a high ionization tendency when electrolysis occurs, the electrode is dissolved, and the shape of the electrode is collapsed, and in extreme cases, the electrode is broken. In view of the above,
In the first embodiment, platinum is used as a main material of the electrode.

The substrate 17 of the measuring electrode may be made of any material as long as it can hold the thin film electrode 18 and has a high insulating property, but it is desirable that the dielectric constant is low. That is, it is desirable to use a low dielectric constant substrate such as silica glass or borosilicate glass. The reason why a substrate having a low dielectric constant is more desirable is that the electric field between the thin-film electrodes 18 is distributed in the inside of the substrate, though not shown in detail in FIG. The ratio of the intensity of the electric field inside the substrate to the electric field spreading outside the substrate, ie, the electric field for dielectrophoresis, is determined by the ratio of the dielectric constants. If the substrate has a low dielectric constant, the internal electric field becomes weak. In the first embodiment, a borosilicate glass substrate is used as the low dielectric constant substrate because the smoothness is high, the fine processing is easy, and the dielectric constant is relatively low.

In the first embodiment, the thin-film electrode 18 is formed on the substrate 17 of the measurement electrode by a general method using photolithography. The following briefly describes how to make an electrode by photolithography.

There are many methods for preparing an electrode by photolithography. In the first embodiment, a platinum thin film with chromium as a base is formed by sputtering on the entire surface of one side of a substrate 17 of a measurement electrode made of borosilicate glass. A product that is firmly attached is produced. Then apply a photosensitive resin evenly on this thin film by spin coating,
Prebaking is performed by applying heat to accelerate the curing of the photosensitive resin. A photosensitive resin is exposed by bringing a mask in which an electrode pattern is previously formed into close contact with this. The portion of the photosensitive resin that is shielded from light by the mask does not change, but the portion of the photosensitive resin that is exposed to light softens and is easily peeled off by washing, exposing the platinum-deposited surface. Next, the substrate is etched in a reactive gas atmosphere. At this time, the portion where the platinum is exposed on the substrate is etched and the platinum and the underlying chromium are removed, but the portion where the photosensitive resin remains, that is, the electrode pattern portion is protected by the resin. So it is not affected at all. After the etching is completed, the photosensitive resin is removed by immersing the substrate in a cleaning liquid for removing the cured photosensitive resin. The portion protected by the photosensitive resin, that is, the electrode pattern, remains on the substrate.

In the example of this description, the production method using a mask has been described. Note that a mask is a so-called "mold" composed of a light-shielding thin film on a glass substrate having the same shape as an electrode to be formed, and a light-shielding substance on the glass substrate is removed by an electron beam or the like in accordance with an electrode pattern. Created by: When the number of electrodes to be formed is small, an electrode pattern may be drawn directly on the platinum-deposited substrate by an electron beam. However, when a plurality of same objects are formed, the photolithography described above is generally more efficient.

The method of forming an electrode by photolithography has been described briefly, but this is merely an example.
Numerous other methods for producing the electrode used in the first embodiment are conceivable, and do not prevent the electrode from being produced by a method other than the above method.

Now, the gap 1 in the first embodiment will be described.
9 is set to 100 μm, but the gap 1
The interval 9 is adjusted as necessary because it is affected by the size of the microorganism to be measured. For example, large ones such as yeast and isolated cells need to be wide, and small ones such as rickettsia need to be narrow. The larger the gap 19, the more microorganisms can be concentrated and the wider the dynamic range of the measurement. However, the time required for the measurement is long, and the power required for dielectrophoresis is also large. . Conversely, when the gap 19 is narrowed,
The power and time required for the measurement are reduced, but the dynamic range of the measurement is reduced. For the reasons described above, in the first embodiment,
The interval between the gaps 19 is set to 100 μm, which is 0.2 to 300 μm according to the microorganism to be detected.
It is desirable to adjust appropriately within the range.

Next, the dialysis cells A7 and B8 will be described. The dialysis cell A7 is separated from the measurement cell 1 by the anion exchange membrane 3 and has a dialysis electrode A5 inside. The dialysis cell B8 is separated from the measurement cell 1 by the cation exchange membrane 4 and has a dialysis electrode B6 therein. Each of the two types of ion exchange membranes is a thick membrane having ion exchange ability, and the anion exchange membrane 3 can transmit only anions and the cation exchange membrane 4 can transmit only cations. In the first embodiment, the dialysis electrode A5 and the dialysis electrode B6 are formed by coating a titanium rod-shaped electrode with platinum.

Here, the electrodialysis will be described. When a plurality of electrodes are immersed in an aqueous solution containing ions and a potential difference is applied between the electrodes, an electrochemical reaction occurs at a constant potential difference determined by a solvent and a solute, and ions move in the solution. This phenomenon is commonly known as electrolysis. At this time, it is observed that negatively charged anions move to the anode side and positively charged cations move to the cathode side. If a voltage is applied so that the potential difference between the electrodes does not reverse, for example, a direct current is applied, the moving direction of ions becomes constant.

In the structure of the measuring container 30, the dialysis electrode A5
A case where a direct current voltage is applied between both electrodes as the anode and the dialysis electrode B6 as the cathode will be described. The value of the applied voltage may be any value that is sufficient to cause an oxidation-reduction reaction on the electrode surface.

Electrolysis occurs at the same time as the application of the voltage, and anions in the measurement container 30 move to the anode side and cations move to the cathode side. At this time, since the dialysis electrode A5 is an anode and the anion exchange membrane 3 can transmit anions, the anions in the dialysis cell A7 and the measurement cell 1 can easily move to the anode side. The anions in the dialysis cell B8 separated by the cation exchange membrane cannot remain in the cation exchange membrane 4 because they cannot pass through the cation exchange membrane 4. Similarly, the cations in the dialysis cell B8 and the measurement cell 1 can easily move to the dialysis electrode B6 side, but the dialysis cell A7
The cations therein cannot pass through the anion exchange membrane 3 and remain in the dialysis cell A7.

When the voltage is continuously applied in such a situation, the cations and anions in the measuring cell 1
It moves outside and the ion concentration in the measurement cell 1 decreases. Then, the current flowing through the system gradually decreases as the ion concentration, which is the carrier of the charge in the measurement cell 1, decreases. Thus, the ion concentration in the measurement cell 1 decreases, and the measurement cell 1
The electrical conductivity of the solution inside also decreases.

Now, it is known that microorganisms are generally negatively charged. Therefore, the microorganisms in the measurement cell 1 naturally also try to move to the anode side. However, although microorganisms are small, they are very large particles as compared with ions, and the moving speed in a solution is very slow as compared with ions. Therefore, on the time scale for achieving the purpose of reducing the ion concentration in the measurement cell 1 by the electrodialysis phenomenon as described above, it is almost unnecessary to consider the movement of microorganisms. further,
The pores of the ion exchange membrane through which the ions permeate are very small compared to the microorganisms, and if the microorganisms in the measurement cell 1 try to move to the anode side, they will be caught by the anion exchange membrane. Therefore, the microorganism does not move to the microorganism by electrodialysis, and only the ion concentration in the measurement cell can be reduced.

The dialysis power supply circuit 11 may be of any type as long as it realizes the above-mentioned electrodialysis. In the first embodiment, a DC power supply circuit is used. The dialysis power supply circuit 11 can apply a voltage to the electrodes, and is controlled by the control unit 15. Further, as described later, an arbitrary voltage value can be applied using the dialysis electrode A5 as an anode and the dialysis electrode B6 as a cathode according to the measurement result of the electrical conductivity of the sample performed before the measurement of the number of microorganisms. . In addition,
Hereinafter, the voltage applied by the dialysis power supply circuit is referred to as a voltage for dialysis in order to avoid complicating the description in the future.

The migration power supply circuit 12 supplies an alternating current for causing dielectrophoresis between the measurement electrodes 2. In the first embodiment, as will be described later, an alternating current for causing dielectrophoresis is temporarily interrupted to measure a change in impedance between the electrodes. The migration power supply circuit 12 is controlled by the control means 15 together with the solenoid valve 9 and the like.

The control means 15 comprises a microprocessor (not shown), a memory for storing a preset program, a timer, and the like. The control means 15 opens and closes the solenoid valve 9 according to the program, and connects the dialysis power supply circuit 11 to the electrophoresis circuit. The power supply circuit 12 is controlled to apply a specific DC voltage to the dialysis electrode A5 and the dialysis electrode B6, and to apply an AC voltage having a specific frequency and voltage to the measurement electrode 2. Further, the control unit 15 manages the overall flow of the measurement operation by transmitting and receiving signals to and from the measurement unit 13 and the calculation unit 14 and performing appropriate control.

In the first embodiment, the measuring unit 13 includes a circuit for applying an AC voltage (hereinafter referred to as a voltage for measurement) for checking the impedance of the measuring electrode 2 and a measuring electrode 2 required for checking the impedance. It comprises a voltage applied therebetween, a current flowing through the measuring electrode 2 and a circuit for measuring the phase difference between the voltage and the current. Microorganisms move by dielectrophoresis and are concentrated near the electric field concentration portion. The change in the impedance of the measurement electrode 2 caused by the measurement is measured. In order to check the impedance, the measuring unit 13 measures a voltage value for measurement applied between the measuring electrodes 2, a current flowing through the measuring electrode 2 and a phase difference between the voltage and the current. Hand over to 14.
The calculation unit 14 calculates the impedance according to a procedure described later.

The measuring section 13 may measure the electric conductivity of the sample in addition to the impedance. This is because the electric conductivity is the reciprocal of the impedance, and both of them have substantially the same contents, and the configuration of the measuring unit 13 can use the above-described configuration for checking the impedance as it is.

As described above, the electric conductivity and the impedance are different only in terms of expression, and it is sufficient to add the explanation of measuring one of the impedances. In the case of explanation, it is easier to understand the use of electrical conductivity, which means the ease of passing electricity, than the impedance, which is the electrical resistance of a liquid. Is generally used when explaining, the expression using not only impedance but also electrical conductivity is used to make the explanation of the behavior of ions in a liquid easier to understand.

The impedance or electric conductivity of the sample is measured before measuring the number of microorganisms as described later.
The reason why it is necessary to measure the impedance or electric conductivity of the sample will be described.

As described above, the dielectrophoretic force increases and decreases depending on the electric conductivity of the microorganism-containing liquid, and tends to decrease as the electric conductivity increases (the impedance decreases). Therefore, in order to move microorganisms efficiently and measure the number of microorganisms accurately in a short time, it is most important to deionize the sample solution and reduce the electrical conductivity of the sample before measuring the number of microorganisms. Become. Therefore, the first embodiment is described using electrodialysis as the deionization process. By this electrodialysis, the electrical conductivity of the sample can be reduced to a certain value or less. In the configuration of the first embodiment, the electric conductivity of the sample is set to 100 μS /
cm, and the microorganisms can be moved at a sufficient moving speed.

By measuring the impedance or electric conductivity of the sample liquid after performing the electrodialysis for a predetermined time, if the above-mentioned value is satisfied, it is possible to immediately proceed to the step of measuring the number of microorganisms. Furthermore, it was confirmed that the conditions were not satisfied for the sample whose electrical conductivity did not sufficiently decrease within a predetermined time due to the high initial ion concentration, and the electrodialysis was performed again to perform the predetermined electrical conductivity. Rate can be reduced. Therefore, even a sample having high electric conductivity such as sewage or seawater can be adjusted to a sample satisfying certain conditions, and can be used for measuring the number of microorganisms.

In the first embodiment, the measuring section 13 is controlled by the control means 15 and can smoothly advance a series of measuring operations in cooperation with each other in accordance with a preset program.

The operation unit 14 is composed of a microprocessor, a memory, and the like (not shown). The details will be described later, but the impedance of the measurement electrode 2 is analyzed based on the result measured by the measurement unit 13, and the electrostatic capacitance between the measurement electrodes 2 is measured. Calculate the capacity. Then, the number of microorganisms contained in the sample system pipe 10 is finally calculated by storing the calculation result in the memory 14 as necessary, reading out the data stored in advance and comparing the data, and displaying the result. For example, a display is made on the display 16. The calculation unit 14 also performs calculation of impedance or electrical conductivity measurement of the sample solution performed prior to measurement of the number of microorganisms, compares the calculation result with a value stored in advance, and performs electrodialysis again on the sample. Determine if it is needed.

This microprocessor can be shared by the control means 15 and the arithmetic section 14. Also, the arithmetic unit 14 is controlled by the control unit 15 similarly to the measuring unit 13, so that a series of measuring operations can be smoothly performed in cooperation with each other according to a preset program.

The display means 16 digitally displays the calculated number of microorganisms as the number of microorganisms per 1 mL of the sample. The display on the display means 16 is the final output of the microorganism counting device in the first embodiment. In the first embodiment, the user can directly know the measured number of microorganisms as the number of microorganisms per 1 mL of the sample. The display method may be used. Furthermore, there is no need for the user to directly know the number of microorganisms, such as controlling the sterilization device by checking the number of microorganisms in the sample or controlling the culture conditions such as temperature. In the case where it is sufficient that the number of microorganisms is clear to perform the control, it is needless to say that control corresponding to the number of microorganisms is performed as it is, and no display means is particularly required.

Hereinafter, a series of flows from the introduction of the sample to the concentration, measurement, and washing of the microorganisms in the measurement cell 1 will be described with reference to the flowchart of FIG. In the initial state, the solenoid valve 9 for shutting off the sample system pipe 10 and the measurement cell 1 is in an open state, and the liquid in the sample system pipe 10 freely passes through the measurement cell 1, the dialysis cell A7 and the dialysis cell B8. I have.
At a predetermined timing, when a measurement operation set in advance by a program is started, the control means 15 closes the solenoid valve 9, shuts off the above-mentioned three cells from the sample system pipe 10,
A sample is introduced by constructing a closed system (step 1).

At this time, as already described, when the current bypassing the outside of the cell during the electrodialysis can be neglected, the solenoid valves of the dialysis cell A7 and the dialysis cell B8 do not need to be particularly provided. In this case, the ions that have moved to the outside of the measurement cell 1 by electrodialysis are diffused as they are, and the ions can be prevented from reentering the inside of the measurement cell 1 due to the concentration gradient even after the voltage for dialysis is cut off. Further, it is preferable that the dialysis cell A7 and the dialysis cell B8 are not provided with an electromagnetic valve, and the liquid in the dialysis cell A7 and the dialysis cell B8 is freely flown so that the moved ions are immediately flowed away.

Next, the control means 15 controls the dialysis power supply circuit 11
To apply a DC voltage of 5 V between the dialysis electrodes A and B to start electrodialysis (step 2). The progress of electrodialysis is as described above. When the electrodialysis for a predetermined time is completed, the control unit 15 sends a signal to the measuring unit 13 to measure the impedance or electric conductivity of the sample solution in the measuring cell 1 (Step 3). A measuring unit 13 for measuring the impedance or electric conductivity of the sample liquid in the measuring cell 1;
Applies an alternating current having a frequency of 1 kHz and a voltage of 1 V to the measuring electrode 2, measures a current value flowing at that time, and sends the measured current value to the arithmetic unit 14. The calculation unit calculates the impedance or the electrical conductivity from the measured value and compares it with a value stored in advance to check whether the impedance or the electrical conductivity of the sample has risen or decreased sufficiently to measure the number of microorganisms. (Step 4). In the first embodiment, the electric conductivity of the sample is 1
The condition for starting the measurement of the number of microorganisms is set to be not more than 00 μS / cm. Naturally, electric conductivity 1
It goes without saying that the condition determination may be performed by performing the calculation of the impedance corresponding to 00 μS / cm.

When the impedance or electric conductivity of the sample satisfies the above-mentioned condition, the process immediately proceeds to the measurement of the number of microorganisms (step 5). When the condition is not satisfied, electrodialysis of the sample is performed again (step 5). Step 2). When the electrodialysis is completed and the electric conductivity satisfies a predetermined condition, the process proceeds to measurement of the number of microorganisms (step 5).

The control means 15 sends a signal to start measurement of the number of microorganisms to the measurement section 13 to instruct the measurement section 13 to start measurement. Upon receiving the measurement start command, the measurement unit 13 immediately applies a sine wave AC voltage having a frequency of 100 kHz and a voltage of 1 V between the measurement electrodes 2 as a voltage for the measurement, and the current value and the phase difference between the voltage and the current at that time. Is measured. Here, the applied AC frequency and voltage for measurement may be optimally selected at the time of calibration with a sample containing a microorganism whose concentration is known in advance, as described later. It is not necessary to be bound by the values exemplified in the above.
Further, at least two electrodes are required for the measurement electrode 2, but three or more electrodes can be provided depending on the measurement purpose to perform measurement.

The measurement result is sent to the arithmetic section 14. Based on the measurement results obtained, the arithmetic unit 14 determines the impedance between the measurement electrodes 2 and the static circuit when the equivalent circuit assumed between the measurement electrodes 2 is regarded as a CR parallel circuit composed of a resistance and a capacitance, which will be described later. Calculate the capacitance value. The impedance can be obtained by dividing the applied voltage and the current. Although the calculation of the capacitance is a little complicated, the impedance is expressed on the complex plane using the value representing the difference between the phase of the voltage and the current for measurement as the angular difference of the angular frequency (hereinafter referred to as the phase angle). It can be calculated by expressing it in polar coordinates and analyzing it. The impedance is Z, the capacitance is C, the reactance is x, and the resistance is r, which will be described in detail with reference to FIGS. 5 and 6 and equations (4) to (8).

[0068]

(Equation 4)

[0069]

(Equation 5)

[0070]

(Equation 6)

[0071]

(Equation 7)

[0072]

(Equation 8)

(Equation 4) is an equation representing the combined impedance of the CR parallel equivalent circuit, (Equation 5) is an equation representing the resistance of the CR parallel equivalent circuit, (Equation 6) is an equation representing the reactance of the CR parallel equivalent circuit, 7) is an equation representing the resistance value of the CR parallel equivalent circuit, and (Equation 8) is an equation representing the capacitance value of the CR parallel equivalent circuit.

FIG. 5A shows an electrical state between the electrodes in an equivalent circuit. Water containing microorganisms is present between the electrodes 50 and 51. Before the microorganisms move to the gap between the electrodes by dielectrophoresis, the water is formed by the capacitance C52 formed as a dielectric between the electrodes and the water. It is considered that the electric conduction resistance R53 connects the electrodes 50 and 51 in parallel. Further, even after the microorganisms move by dielectrophoresis, the microorganisms behave as dielectric fine particles as described later, so that even if the absolute values of the capacitance C52 and the resistance R53 change, the connection form of the equivalent circuit does not change. be able to. Hereinafter, this equivalent circuit is called a CR parallel circuit.

It is generally known that when an AC voltage is applied to such a CR parallel circuit, a phase difference as shown in FIG. 5B appears between the current 57 flowing through the circuit and the applied voltage 56. . When the frequency of the voltage to which the phase difference is applied is expressed in polar coordinates on a complex plane using the angle difference θ when the frequency is expressed by the angular frequency ω, the voltage, current, and phase angle are shown in FIG.
There is a relationship shown in

The impedance Z is obtained by dividing the measured applied voltage and current, and corresponds to the absolute value of the vector shown in FIG. At this time, the impedance Z is Z = r + jx
(J is an imaginary unit), and the resistance r is represented by C = Rsin θ shown in FIG.
The resistance component x and the reactance x of the combined impedance of the R parallel circuit are related to the reciprocal of the capacitive component of the same circuit as x = Zcos θ.

On the other hand, the combined impedance of the CR equivalent circuit of FIG. 5A is expressed by (Equation 4), and (Equation 4) is obtained by calculating Z = r +
By decomposing into a resistance r and a reactance x from the relation of jx, (Equation 5) and (Equation 6) are obtained. When (Equation 5) and (Equation 6) are simultaneously transformed, (Equation 7) and (Equation 8) are obtained. (Equation 7) and (Equation 8) show the voltage value for measurement, the current value at that time, r, x, calculated from the measured value of the phase angle of voltage and current.
By substituting ω, the resistance R53 and the capacitance C52 can be known.

Although it is very complicated to explain in this way,
The operation unit 14 includes a microprocessor (not shown), and a series of operations is completed in an instant.

The calculation unit 14 stores the calculated value of the capacitance C as an initial value in the memory, and sends a signal to the control means 15 that the measurement of the initial value has been completed. Hereinafter, the control unit 15, the measurement unit 13, and the calculation unit 14 appropriately exchange signals as needed, and perform a smooth operation according to a preset program.

If the calculation corresponding to the measured value is performed in advance and stored in the form of a conversion table and stored in a memory, the calculation is not performed every time the measurement is performed, but the conversion into microorganisms can be performed simply by referring to the conversion table. Can also. That is, the measurement is performed after the microorganisms are concentrated by dielectrophoresis for a preset time, and the voltage value for the measurement, the current value at that time, and the phase difference between the voltage and the current are measured. If the conversion table is referred to, it means that the previously calculated number of microorganisms is written therein. With such a configuration, it is possible to provide a microorganism count measuring apparatus having a simpler structure without providing the arithmetic unit 14. In this case, the control means 15 includes the dialysis power supply circuit 11 and
It controls the migration power supply circuit 12 and the measurement unit 13 and accesses the conversion table.

Next, the control means 15 controls the electrophoretic power supply circuit 12 to control the peak voltage 1 between the measuring electrodes 2 at a frequency of 1 MHz.
A sine wave AC voltage of 00 V (hereinafter referred to as a voltage for electrophoresis) is applied. The frequency of the alternating current applied at this time can be arbitrarily selected within a frequency range in which dielectrophoresis occurs. However, if the frequency is too low, undesired electrolysis occurs between the measurement electrodes 2 and, conversely, too much. A high frequency complicates the power supply circuit. Therefore, in the first embodiment, dielectrophoresis can be sufficiently generated, and the electrophoresis power supply circuit 12 can be relatively simple.
Hz is selected.

At this time, depending on the type of microorganism to be measured, the voltage for measurement and the voltage for electrophoresis may use the same frequency instead of two different frequencies. The AC frequency to be applied for the most efficient migration of any microorganism and the AC frequency to be applied for the most efficient measurement are not necessarily the same. Will be the same. In this case, the migration and measurement can be performed at a single frequency, so that the migration power supply circuit 12 can be simplified. In addition, the measurement can be performed continuously while performing the migration. Precise results can be obtained.

After a lapse of a predetermined time, the control means 15 once interrupts the AC voltage applied between the measuring electrodes 2 to perform a measuring operation. The measurement unit 13 and the calculation unit 14 measure the impedance between the measurement electrodes 2 and calculate the capacitance between the measurement electrodes 2 in cooperation with the above-described method, and store the obtained value in the memory. After the measurement is completed, the control means 15 again applies an AC voltage for dielectrophoresis.

Thereafter, the control means 15 and the measuring section 13 repeat the electrophoresis and the measurement in cooperation with each other at preset time intervals, and the measuring section 13 stores the calculated capacitance in the memory each time. Thus, the gap 19 between microorganisms by dielectrophoresis was
By repeating the movement to the vicinity and the measurement of the impedance of the measurement electrode 2, it is possible to examine the time change of the capacitance between the measurement electrodes 2.

After the start of the application of the AC voltage for dielectrophoresis, the impedance of the measuring electrode 2 is measured a predetermined number of times in advance, and the measuring unit 13 detects the capacitance C at a plurality of times stored in the memory. From the calculation result of the above, as shown in FIG.
Is calculated, and the number of microorganisms in the sample system pipe 10 is calculated according to a conversion formula described later.

The reason why the number of microorganisms can be calculated by measuring the gradient of the time change of the capacitance is as follows. Microorganisms are cell walls that are ion-rich and have relatively large electric conductivity.
It is composed of phospholipids and is surrounded by a cell membrane with small electric conductivity, and can be regarded as fine dielectric particles. The dielectric constant of microorganisms viewed as dielectric fine particles has a large value as compared with a general liquid, and also as compared with water having a high dielectric constant as a liquid. Therefore, as the number of microorganisms moving to the gap 19 by dielectrophoresis increases, the apparent dielectric constant near the gap 19 increases. It is a well-known fact that the capacitance C changes when the dielectric constant of the medium is changed while the electrode conditions are fixed. Therefore, if the change in the dielectric constant between the measurement electrodes 2 is measured through the change in the capacitance C between the measurement electrodes 2, the value is the number of microorganisms that have moved to the vicinity of the gap 19, and thus the number of microorganisms existing in the sample system piping 10. Can be obtained. FIG. 4 shows an example of such a time change of the capacitance C. As can be seen from FIG. 4, it can be seen that the gradient (gradient) of the time change of the capacitance C at the initial stage of measurement also increases in accordance with the number of microorganisms, similarly to the time change of the capacitance C. Capacitance C
When calculating the number of microorganisms with the time change of the time, it is more accurate to measure after passing the transient state, so it takes a long time, but the number of microorganisms depends on the slope (gradient) of the time change of the capacitance at the initial stage of measurement. Is characterized in that the number of microorganisms can be calculated in a relatively short time.

In order to correlate the change in the capacitance C with the number of microorganisms in the sample system pipe 10, a conversion formula between the capacitance C and the number of microorganisms is required. This conversion equation measures a calibration sample with a clear number of microorganisms in advance using the measurement system of the microorganism number measurement device described in the first embodiment, and shows a correlation between the number of microorganisms and the capacitance C at that time. And a function representing a curve obtained by regression analysis of variations. This conversion formula is stored in the memory of the calculation unit 14, and when a sample whose number of microorganisms is unknown is measured, the capacitance C within a predetermined time is measured.
By substituting the value of the change, the number of microorganisms in the sample system pipe 10 can be calculated.

Here, the sample of the first embodiment is assumed to be a single microorganism system such as a culture solution of yeast, etc. Unless otherwise, a similar conversion equation can be calculated in advance and measured.

As described above, after calculating the number of microorganisms,
After a lapse of a predetermined time programmed in advance, the arithmetic unit 14 sends a notification of the end of the measurement to the control unit 15. In response, the control means 15 stops the energization of the measuring electrode 2 and opens the solenoid valve 9 to start cleaning (step 6). Microorganisms that have gathered near the gap 19 are washed away by the liquid in the sample system piping 10 that flows in by opening the solenoid valve 9, and a series of measurement operations is completed.

As described above, in the first embodiment, since the impedance or electric conductivity of the sample solution is reduced by electrodialysis prior to the measurement of the number of microorganisms, accurate measurement can be performed even on a sample having a high ion concentration. It is possible to provide a maintenance-free microbial count measuring apparatus which has high measurement sensitivity, is capable of automatic measurement, and has a simple structure in a short time.

(Embodiment 2) A microorganism counting apparatus according to another embodiment of the present invention will be described in detail with reference to the drawings. In the second embodiment, since there is a portion that overlaps with the microbial count measuring device of the first embodiment, a detailed description will be given of a portion different from the first embodiment.

FIG. 8 is an overall configuration diagram of a microorganism counting device according to the second embodiment of the present invention, and FIG. 10 is a diagram illustrating a flow of a series of microorganism counting operations according to the second embodiment of the present invention. A graph for explaining the relationship between the number of microorganisms in the sample, the measurement time, and the capacitance C between the electrodes is the same as that in FIG. 4, and a detailed description thereof will be omitted in the first embodiment.

In FIG. 8, reference numeral 40 denotes an ion-permeable semipermeable membrane; 41, an inlet for a low electrical conductivity liquid; 42, an outlet for a low electrical conductivity liquid; 43, a dialysis cell according to the second embodiment; Is a measurement container in Embodiment 2, and 45 is a dialysis solution supply unit.

The most different point of the second embodiment from the first embodiment is the structure of the measuring container 44. The measurement container 44 includes the measurement cell 1 and the dialysis cell 43. The measurement cell 1 has a measurement electrode 2 inside similarly to the first embodiment, and is connected to a sample system pipe 10 via an electromagnetic valve 9. Further, the measurement cell 1 and the dialysis cell 43 are connected via an ion-permeable semipermeable membrane 40, so that ions can be freely transmitted but larger particles such as microorganisms cannot be transmitted.

The dialysis cell 43 has an inflow port 41 and an outflow port 42, and completely covers the measurement cell 1. The dialysate supply unit 45 is composed of a liquid supply source, a control valve, and the like, and is controlled by the control unit 15 to supply a required amount of a liquid having a lower ion concentration than the sample liquid, that is, a low electric conductivity liquid, through the inlet 41. can do. The low electric conductivity liquid means that the ion concentration is sufficiently lower than that of the liquid flowing through the sample piping 10 used as a sample for measurement, as will be described later in detail. The inside of the dialysis cell 43 is filled with the low electric conductivity liquid, and the measurement cell is immersed therein.

Since the configurations of the measuring electrode 2, the electrophoresis power supply circuit 12, the measuring unit 13, the calculating unit 14, the control unit 15, the display unit 16 and the like are the same except that the operation for electrodialysis is not performed. The description is omitted from the description of the first embodiment.

The basic concept of measurement and calculation performed in order to know the number of microorganisms in the second embodiment is the same as in the first embodiment. That is, the capacitance C of the electrode, which changes as the microorganism moves to the vicinity of the gap due to dielectrophoresis,
Is converted to the number of microorganisms. The flow of a series of these measurements will be described below with reference to FIG. 10. The second embodiment differs from the first embodiment in that the ionic concentration of the sample liquid is reduced prior to the measurement of the number of microorganisms, that is, the electric conductivity. The part of the dialysis operation for realizing the reduction (increase in impedance) will be described in detail, and the part overlapping with the first embodiment will be omitted from the description of the first embodiment.

When the sample liquid is introduced into the measuring cell 1 (step 11), the control means 15 waits for a predetermined time until the ion concentration in the measuring cell 1, that is, the electric conductivity is reduced by the dialysis action. Since the wall surface of the measurement cell 1 is in contact with the low-conductivity liquid filling the inside of the dialysis cell 43 via the semipermeable membrane 40, ions in the sample liquid fill the inside of the dialysis cell 43 according to the concentration gradient. It diffuses and moves into the electric conductivity liquid. As a result, the ion concentration in the measurement cell 1 decreases, and the electrical conductivity of the sample liquid decreases (the impedance increases) (step 12).

After the elapse of a predetermined time, the control means 15
3 to measure the impedance or electrical conductivity of the sample liquid in the measurement cell 1 (step 13). If the impedance or electric conductivity of the sample does not satisfy the conditions for measuring the number of microorganisms (step 14), the sample is dialyzed again (step 12). Since the electric conductivity and the impedance are substantially the same,
It is the same as in the first embodiment that the condition determination may be performed up to the calculation of the impedance. However, in order to judge the behavior in the liquid, the electric conductivity is easier to understand, so the second embodiment uses the electric conductivity.

The movement of the ions in the measurement cell to the low electric conductivity liquid is caused by the concentration gradient. Therefore, the larger the difference between the ion concentrations of the sample liquid and the low electric conductivity liquid, the faster the movement. Therefore, immediately after the introduction of the sample liquid, the electric conductivity sharply decreases, and the electric conductivity becomes slower as the difference between the concentration of ions transferred to the low electric conductivity liquid and the concentration of ions in the sample liquid decreases over time. It is. Eventually, when the ion concentration in the dialysis cell 43 and the ion concentration in the measurement cell 1 become the same, the diffusion reaches an equilibrium and the electrical conductivity of the sample liquid in the measurement cell 1 does not further decrease. That is, the final point of the electric conductivity of the sample liquid in the measurement cell 1 is determined by the initial value of the ion concentration of the low electric conductivity liquid introduced into the dialysis cell 43. Therefore, when the electrical conductivity of the sample liquid does not satisfy the conditions for measuring the number of microorganisms at the end of the first dialysis, the control means 15 controls the dialysate supply unit 45 to control the inside of the dialysis cell 43. The liquid is replaced with a new liquid having a low electric conductivity, and a second dialysis is performed (step 15).

Of course, if the volume of the dialysis cell 43 is made larger than that of the measurement cell 1, the ion concentration in the measurement cell 1 finally reached by diffusion can be further increased without replacing the liquid in the dialysis cell 43. descend. However,
This is not very desirable because the device becomes large.

A better method for further reducing the electrical conductivity of the sample liquid than the method described above is the dialysis cell 43.
The low electric conductivity liquid in the inside is continuously supplied from the dialysate supply unit 45, and the ions diffused from the measurement cell 1 are discharged from the outlet 42.
Is to wash away from With such a configuration, the sample liquid is always in contact with the cleanest low-conductivity liquid through the semipermeable membrane 40, and dialysis is performed while always maintaining the maximum ion concentration difference. Will be able to With such a configuration, in principle, the ion concentration of the sample liquid in the measurement cell 1 can be reduced until the electric conductivity becomes the same as the low electric conductivity liquid supplied to the dialysis cell 43. Although such a configuration is somewhat complicated in terms of mechanism, it is a desirable method when performing more precise measurement or when measuring microorganisms in a sample having extremely high electrical conductivity. However, if the electrical conductivity of the sample liquid is reduced at an extremely extreme speed, that is, if the ion concentration is reduced, microorganisms in the sample liquid to be measured may be destroyed by a rapid change in osmotic pressure. Caution must be taken. In the case where the measurement target is a soft one with only a cell membrane, such as an isolated animal cell, it is necessary to reduce the dialysis speed.

When the electric conductivity in the measurement cell 1 satisfies the condition for measuring the number of microorganisms, the control unit 15 starts measuring the number of microorganisms (step 16). The procedure of measurement of the microorganisms and the subsequent washing are the same as those in the first embodiment, and the explanation is omitted for the first embodiment (step 17).

As described above, dialysis of a sample liquid in a clean liquid using a semi-permeable membrane lowers the ion concentration in the sample liquid, lowers the electrical conductivity of the sample liquid, and efficiently performs dielectrophoresis. As a result, it is possible to provide a highly accurate and maintenance-free microorganism counting apparatus with a simple structure.

[0105]

According to the present invention, a maintenance-free microorganism counting apparatus which can quickly and automatically measure a sample having high sensitivity and high electric conductivity without requiring a medicine or a special device. Can be provided.

[Brief description of the drawings]

FIG. 1 is an overall configuration diagram of a microorganism counting apparatus according to Embodiment 1 of the present invention.

FIG. 2 is an explanatory diagram of an electrode according to Embodiment 1 of the present invention.

FIG. 3 is a diagram illustrating a cross section of an electrode and a state of an electric field between the electrodes according to the first embodiment of the present invention.

FIG. 4 is a graph for explaining the relationship among the number of microorganisms, measurement time, and capacitance C between electrodes.

FIG. 5 is a diagram for explaining a method of calculating capacitance between electrodes;

FIG. 6 is another diagram for explaining a method of calculating capacitance between electrodes;

FIG. 7 is a diagram for explaining the relationship between force and frequency by dielectrophoresis.

FIG. 8 is an overall configuration diagram of a microorganism counting apparatus according to Embodiment 2 of the present invention.

FIG. 9 is a diagram illustrating a flow of a series of operations for measuring the number of microorganisms according to the first embodiment of the present invention.

FIG. 10 is a view for explaining a flow of a series of microorganism count measurement operations in Embodiment 2 of the present invention.

[Explanation of symbols]

 Reference Signs List 1 measurement cell 2 measurement electrode 3 anion exchange membrane 4 cation exchange membrane 5 dialysis electrode A 6 dialysis electrode B 7 dialysis cell A 8 dialysis cell B 9 solenoid valve 10 sample piping 11 dialysis power supply circuit 12 migration power supply circuit 13 measuring unit 14 arithmetic unit 15 control means 16 display means 17 substrate of measurement electrode 18 thin film electrode 19 gap 20 line of electric force 30 measurement container 40 semipermeable membrane 41 inflow port 42 outflow port 43 dialysis cell 44 measurement container 45 dialysate supply unit 50, 51 Pole 52 Capacitance 53 Resistance 54 Time axis 55 Axis representing magnitude of voltage or current 56 Curve representing change in current value 57 Curve representing change in voltage value

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 27/447 G01N 33/483 E 33/483 27/26 301A F-term (Reference) 2G045 AA28 CB21 FA34 FB05 GC16 GC22 GC30 HA02 JA01 JA04 JA07 2G060 AA06 AD08 AE40 AF06 AF08 AG08 JA10 4B029 AA07 BB01 CC01 FA10 FA11 4B063 QA01 QQ05 QS31 QS39 QX04

Claims (7)

[Claims] 1. A measuring cell provided with a plurality of electrodes, into which a liquid containing microorganisms can be introduced, and connected to the measuring cell via an ion-permeable diaphragm to reduce the ion concentration in the measuring cell. A dialysis unit, wherein the measurement container includes: a migration power supply circuit for performing dielectrophoresis of microorganisms in the measurement cell; and measuring impedance or electrical conductivity between the plurality of electrodes. A measuring unit for calculating the number of microorganisms by calculating a measurement result of the measuring unit; and a control unit for controlling the electrophoresis power supply circuit, the dialysis unit, the measuring unit, and the calculating unit, When the liquid is introduced, the dialysis means reduces the ion concentration in the measurement cell, measures impedance or electric conductivity, and calculates the number of microorganisms. . 2. A dialysis means comprising: a first dialysis cell containing a first dialysis electrode and connected to a measurement cell via a cation-permeable diaphragm; and a second dialysis cell containing an anion-permeable electrode. A second dialysis cell connected to the measurement cell via a diaphragm; and a dialysis power supply circuit for applying a voltage for performing electrodialysis to the first dialysis electrode and the second dialysis electrode. The microorganism counting device according to claim 1, wherein: 3. A dialysis solution, wherein the permeation means includes a dialysis cell connected to the measurement cell via a semipermeable membrane, and a liquid having a lower electric conductivity than the liquid in the measurement cell is introduced into the dialysis cell. The microorganism counting device according to claim 1, further comprising a supply unit. 4. The method according to claim 1, wherein the electrical conductivity of the microorganism-containing liquid is measured before the measurement of the number of microorganisms.
The microorganism counting device according to any one of claims 1 to 3. 5. A microorganism-containing liquid is introduced into a cell having a plurality of electrodes, and an alternating voltage is applied between the electrodes to exert a dielectrophoretic force on the microorganisms in the measurement cell and collect the microorganisms in an electric field concentration portion. A method for measuring the number of microorganisms by measuring impedance or electric conductivity between electrodes together with the method of measuring the number of microorganisms, wherein the liquid is deionized before applying the AC voltage to reduce the electric conductivity of the liquid. A method for measuring the number of microorganisms. 6. The method according to claim 5, wherein the deionization treatment is dialysis using a semipermeable membrane. 7. The method according to claim 5, wherein the deionization treatment is electrodialysis using the first dialysis cell and the second dialysis cell.