Sulfur component detection method and device, and microorganism amount determination device and method

Abstract

The objective of the present invention is to provide a sulfur component detection method with which it is possible for a sulfur component such as a sulfur compound in a specimen to be detected easily in a short time using an electrode sensor. The sulfur component detection method according to the present invention is a method for detecting a sulfur component in a specimen 2 using an electrode sensor 1 in which an anode electrode body and a cathode electrode body are formed integrally with an insulator interposed therebetween, the method involving: measuring a galvanic current flowing between both electrode bodies as a result of an electrochemical reaction that occurs between the sulfur component in the specimen 2 and the anode electrode body, in a state in which the specimen 2 is in contact with both electrode bodies; and detecting the sulfur component in the specimen 2 on the basis of the measured current value.

Description

Sulfur component detection method and device, and microbial quantity determination device and method

[0001] The present invention relates to a method and apparatus for detecting sulfur components that detect trace amounts of sulfur compounds in a sample using an electrode sensor, and also to a microbial quantity determination apparatus and method that can detect sulfur components contained in metabolites of microorganisms such as bacteria and quantitatively determine the amount of viable microorganisms.

[0002] Sulfur (element symbol: S) is known to be an essential element for life, and various sulfur compounds play important roles in biological functions in living tissues. Sulfur is also known to be involved in a wide range of redox reactions in living tissues, and attempts have been made to analyze redox reactions in living tissues by detecting sulfur components.

[0003] For example, Patent Document 1 describes measuring the content of methionine-derived sulfur in a biological fluid collected from a subject using an X-ray fluorescence analyzer. Patent Document 2 describes quantitatively analyzing sulfur metabolites using a mass spectrometer, using a solution prepared by adding a treatment liquid to exhaled breath condensate collected from the exhaled breath, in order to diagnose infectious diseases based on sulfur metabolites in the exhaled breath. Patent Document 3 describes detecting sulfur compounds by directly contacting a liquid or paste-like specimen with a detection layer having exposed silver particles and measuring the transmission or reflection spectrum with a spectrometer using a sulfur compound sensor.

[0004] The present inventors have proposed a microbial detection device that detects current changes due to metabolites of microorganisms in a culture medium by measuring the galvanic current flowing between an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, while the electrode sensor is in contact with the culture medium, as disclosed in Patent Document 4. By using such an electrode sensor, it has become possible to detect minute current changes due to metabolites produced by microorganisms in a culture medium, thereby easily and accurately detecting microorganisms.

[0005] Japanese Patent No. 6734489 Japanese Patent Application Laid-Open No. 2022-178150 Republished 2019 / 167567 Japanese Patent No. 6804063

[0006] In Patent Documents 1 to 3, an analytical instrument such as an X-ray fluorescence analyzer is required to detect sulfur compounds, and as described in Patent Document 2, a pretreatment such as adding a treatment liquid must be performed, which poses a problem that it is difficult to perform the detection treatment simply and quickly.

[0007] In particular, when analyzing redox reactions in biological tissues, it is desirable to perform the analysis without affecting the biological reactions as much as possible. However, conventional detection methods require steps such as sample extraction and pretreatment, which have the drawback of easily affecting the biological reactions during these steps.

[0008] SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a method and apparatus for detecting sulfur components that can easily and quickly detect sulfur components such as sulfur compounds in a sample using an electrode sensor.

[0009] The sulfur component detection method according to the present invention is a method for detecting sulfur components in a specimen using an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, and in which, while the specimen is in contact with both electrodes, a galvanic current flowing between the two electrodes due to an electrochemical reaction that occurs between the sulfur components in the specimen and the anode electrode body is measured, and the sulfur components in the specimen are detected based on the measured current value.

[0010] The sulfur component detection device according to the present invention includes an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, and a detection unit that measures a galvanic current that flows between the two electrodes due to an electrochemical reaction that occurs between the sulfur components in the sample and the anode electrode body while at least both electrode bodies of the electrode sensor are in contact with the sample, thereby detecting a change in the current value due to the sulfur components in the sample.

[0011] The microbial quantity determination device according to the present invention is a microbial quantity determination device that determines the microbial quantity by detecting sulfur components in the metabolites of microorganisms, and is equipped with an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, a culture medium for culturing microorganisms, a measurement unit that measures the galvanic current flowing between at least both electrode bodies of the electrode sensor while they are in contact with the culture medium, and an analysis unit that stores the current value measured by the measurement unit and determines the microbial quantity based on time data corresponding to changes in the current value.

[0012] The method for determining the amount of microorganisms according to the present invention is a method for determining the amount of microorganisms by detecting sulfur components in the metabolites of microorganisms, in which an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator is used to bring at least both electrode bodies into contact with a culture medium for culturing microorganisms, measure the galvanic current between the two electrode bodies, and determine the amount of microorganisms based on time data corresponding to changes in the measured current value.

[0013] According to the sulfur component detection method of the present invention, the electrochemical reaction occurring between the sulfur components in the sample and the anode electrode assembly is reflected in the galvanic current flowing between the anode electrode assembly and the cathode electrode assembly, and therefore trace amounts of sulfur components in the sample can be detected based on the measured galvanic current. Furthermore, detection can be performed simply by bringing the electrode sensor into contact with the sample, and no pretreatment or other steps are required, making it possible to detect sulfur components in the sample simply and quickly.

[0014] According to the microbial quantity determination device of the present invention, the amount of viable microorganisms can be determined easily and quickly by measuring the current value generated in the electrode sensor by the sulfur components in the metabolites of the microorganisms while the electrode sensor is in contact with the culture medium in which the microorganisms are cultivated, and determining the amount of microorganisms based on the time data corresponding to the change in the current value.

[0015] 1 is a configuration example of a sulfur component detection device for carrying out a sulfur component detection method according to the present invention; FIG. 2 is a plan view and a partially enlarged cross-sectional view of an electrode sensor; FIG. 3 is a schematic configuration diagram of a microbial quantity determination device according to the present invention; FIG. 4 is a graph showing the time course of current value and hydrogen sulfide (HS) concentration; FIG. 5 is a graph showing the correlation between measured current value and concentration; FIG. 6 is a graph showing the time course of OD value corresponding to the number of bacteria and measured current value; FIG. 7 is a graph showing the measurement results of a detection test using an electrode sensor after the first, first, and second regeneration treatments in the case of regeneration treatment by current application; FIG. 8 is a graph showing the measurement results of a detection test using an electrode sensor after the first, first, and second regeneration treatments in the case of regeneration treatment using thiourea; FIG. 9 is a reduction curve of an anode electrode assembly in a regeneration test; FIG. 10 is a graph showing the measurement results in Example 7; FIG. 11 is a graph showing the correlation between the number of viable bacteria and the time required to reach a predetermined current value in Example 7; FIG. 12 is a graph showing the correlation between the number of viable bacteria and the time required to reach a predetermined current value in Example 7; 10 is a graph showing the correlation between the number of viable bacteria and the maximum current value in Example 7.

[0016] Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the embodiments described below are preferred examples for carrying out the present invention, and therefore various technical limitations are imposed thereon. However, the present invention is not limited to these embodiments unless otherwise specified in the following description to the effect that the present invention is limited thereto.

[0017] 1 shows an example of the configuration of a sulfur component detection device for carrying out the sulfur component detection method according to the present invention. The sulfur component detection device includes an electrode sensor 1 having an anode electrode body and a cathode electrode body integrally formed with an insulator interposed therebetween, a liquid or paste-like specimen 2 containing sulfur components, and a detection unit 3 that measures the galvanic current flowing between at least both electrodes of the electrode sensor 1 while in contact with the specimen 2, thereby detecting a current change due to the sulfur components in the specimen 2. In this example, the electrode sensor 1 is set in a container 4, and the specimen 2 is dropped onto the top surface of the electrode sensor 1 so that it adheres to both electrodes, thereby detecting the sulfur components in the specimen 2.

[0018] 2A and 2B are a plan view and a partially enlarged cross-sectional view, respectively, of the electrode sensor 1. In FIG. 2B, a cross section in the left-right direction is shown in the area enclosed by a circle.

[0019] The electrode sensor 1 has an insulator 11 laminated in layers on the upper surface of a rectangular anode electrode body 10, and a cathode electrode body 12 laminated in layers on the upper surface of the insulator 11. Narrow, comb-shaped cutouts are formed in parallel in the central portions of the insulator 11 and the cathode electrode body 12, exposing the surface of the anode electrode body 10. Then, by bringing the specimen 2 into contact with both electrode bodies of the electrode sensor 1 and measuring the galvanic current flowing between the anode electrode body 10 and the cathode electrode body 12 exposed in the cutouts, a change in current due to sulfur components in the specimen 2 can be detected.

[0020] A galvanic current is generated between the two electrode bodies because an oxidation reaction occurs on the electrode surface of the anode electrode body 10 exposed in the cutout, generating electrons, and a reduction reaction occurs on the electrode surface of the comb-shaped electrode portion of the cathode electrode body 12, causing electrons to disappear. Therefore, the electrode bodies are set to a combination of dissimilar metals so that the electrode surface of the cathode electrode body 12 has a more noble potential than the electrode surface of the anode electrode body 10 under environmental conditions in which an electrochemical reaction occurs.

[0021] In the electrode sensor 1, the two electrode bodies are integrally formed in the interdigital electrode portion so as to be opposed to each other via an insulator, and a galvanic current is generated by causing an electrochemical reaction at the contact points of the two electrode bodies with a specimen interposed between them. The output of the generated galvanic current is proportional to the length of the boundary of the contact area with the specimen at the boundary between the two electrode bodies opposed to each other via an insulator, and it is known that the detection sensitivity increases as the distance between the boundary becomes narrower.

[0022] For this reason, in the above example, the electrode sensor is arranged with the anode electrode body and the cathode electrode body stacked with an insulator between them, but is not limited to this configuration. For example, it is also possible to arrange both electrode bodies in parallel on a substrate, form the boundary between the opposing electrode bodies into a comb-like shape, and arrange the formed narrow electrode portions in parallel alternately.

[0023] In addition, such a comb-shaped electrode structure can be formed so as to surround the periphery of a stick-shaped substrate, or the comb-shaped electrode structure can be formed inside a cylindrical substrate to create a compact electrode sensor, and the compact electrode sensor can be inserted into a sample to detect sulfur components.

[0024] The insulator 11 is made of a non-conductive insulating material that electrically insulates the anode electrode body 10 and the cathode electrode body 12. There are no particular limitations on the insulating material, and examples include resin materials and ceramic materials. As shown in the above example, when forming a laminated structure, it can be formed by a printing method using a paste made of an insulating material, similar to the cathode electrode body described below.

[0025] A metal thin film is preferably formed on the electrode surfaces of the anode electrode body 10 and the cathode electrode body 12. The metal thin film is preferably a single composition of gold, platinum, silver, titanium, nickel, chromium, or carbon, or a composition containing one or more of these. Furthermore, the surface resistance of the anode electrode body and the cathode electrode body is preferably low, specifically, preferably set to 0.1 Ω or less. The surface resistance can be measured by connecting measurement terminals to both ends of the center line of the electrode body.

[0026] When silver is used as the metal for the anode electrode body 10, the metal used for the cathode electrode body 12 may be a metal having a composition that results in a more noble potential than silver. Furthermore, when silver is used for the anode electrode body 10, the thickness of the metal thin film is preferably 10 μm or more. By making the thickness 10 μm or more, the electrical resistance value decreases and the current output improves. When the thickness of a silver metal thin film is thinner than 10 μm, problems such as peeling of the thin film are likely to occur during long-term use.

[0027] The anode electrode body 10 can be a silver plate, but can also be configured by forming a metal film made of silver on the substrate surface by wet film formation processing, dry film formation processing, or by sintering a conductive paste. Wet film formation processing includes electroplating, electroless plating, and composite plating, and dry film formation processing includes vapor deposition and sputtering.

[0028] The cathode electrode body 12 has a conductive material containing a metal material at least on the electrode surface, and when a plate-shaped body is used for the anode electrode body 10 serving as the substrate, the cathode electrode body 12 is preferably formed by a printing method using a conductive paste, as shown in Figure 2. By forming the cathode electrode body by such a printing method, the layer thickness is increased to 5 to 100 μm, which reduces the electrical resistance value and enables accurate and stable measurement of the current generated between the two electrode bodies.

[0029] Furthermore, even when both electrodes are formed on an insulating substrate, they can be formed by a printing method using a conductive paste. Examples of printing methods using a conductive paste include relief printing, lithographic printing, intaglio printing, stencil printing, electrostatic printing, inkjet printing, and laser printing. The electrode sensor shown in Figure 2 can be produced by screen printing, which is a stencil printing method, but other printing methods may also be used, and are not particularly limited.

[0030] In the electrode sensor 1, a galvanic current flows due to an electrochemical reaction that occurs between the anode electrode body 10 and the cathode electrode body 12 due to trace amounts of sulfur contained in the specimen 2 that contacts the two electrodes. For example, in the case of hydrogen sulfide, a sulfur compound contained in the metabolites of microorganisms, the following reaction is thought to occur, causing a change in the current that flows between the two electrodes. <Specimen> HS → HS - +H + <Cathode electrode body> O2 + 2H2O + 4e - →4OH - <Anode electrode body> 2Ag + H2S → Ag2S + 2H + +2e - 2Ag+HS - →AgS+H + +2e - 2Ag+S 2- →AgS + 2e -

[0031] In the above reaction, silver ions (Ag) are produced in the anode electrode body as the oxidation reaction progresses. + occurs at the interface with the sample, causing a current to flow, but silver ions Ag + are the sulfur components in the sample, hydrogen sulfide H2S and hydrogen sulfide ions HS - and sulfide ions S 2- Immediately after generation, these react with each other to form silver sulfide Ag2 S. The formed silver sulfide Ag2 S is insoluble in water, so it deposits on the surface of the anode electrode body.

[0032] The deposition of silver sulfide (AgS) results in the formation of silver ions (Ag) at the interface between the anode electrode body and the specimen. + disappears, but the oxidation reaction progresses and silver ions Ag + Therefore, silver ions Ag are supplied between the anode electrode body and the sulfur component in the sample. + A series of electrochemical reactions will proceed, consisting of the supply of hydrogen sulfide and the deposition of silver sulfide Ag2S. Therefore, the change in the concentration of hydrogen sulfide in the sample will cause the silver ion Ag + It is thought that the amount of generated current changes, causing the current to change.

[0033] Furthermore, when measuring metabolites produced by microorganisms, the environmental conditions remain the same and no pretreatment using reagents or the like is required, so measurements can be taken in their natural state without damaging the microorganisms, and the state of the microorganisms can be grasped in real time.

[0034] Figure 6, which will be described later, shows the correlation between the growth process of live bacteria and changes in current. It can be seen that the current value increases during the logarithmic growth phase, when live bacteria rapidly increase, and reaches its maximum value during the deceleration phase before moving to the stationary phase, when the growth rate of live bacteria decreases. This shows that the growth process of live bacteria is reflected in the current value in real time.

[0035] Similarly, in the case of biological tissues other than microorganisms, when detecting sulfur compounds in biological reactions or products resulting from biological reactions, it becomes possible to easily detect them without changing environmental conditions, etc.

[0036] When the surface of the anode electrode body on which silver sulfide Ag2S is deposited is covered with silver sulfide Ag2S, silver ions Ag + Therefore, it is preferable to perform a regeneration process for the electrode sensor to remove silver sulfide Ag2S as needed.

[0037] A method for regenerating an electrode sensor involves removing the silver sulfide (AgS), a sulfur compound, formed on the exposed surface of the anode electrode assembly through a reduction reaction. Specifically, the silver sulfide (AgS) formed on the exposed surface can be removed by a reduction reaction similar to that of a cathodic reduction method, which occurs when an electric current is applied to the anode electrode assembly in an electrolyte. The electrolyte may be any liquid that can be electrically conducted, and is not particularly limited, but an example of such an electrolyte is a potassium chloride aqueous solution. A known electrochemical reaction measuring device, such as a galvanostat, can be used as a regeneration device for applying an electric current, and the anode electrode assembly can be used as the working electrode.

[0038] Furthermore, silver sulfide Ag2S formed on the exposed surface can be removed by contacting at least the exposed surface of the anode electrode body with a sulfuric acid solution containing thiourea. The sulfuric acid solution containing thiourea may be set to a thiourea concentration of 0.1 to 0.5 M (molar), and silver sulfide Ag2S can be easily removed by dropping the solution onto the exposed surface and leaving it for a predetermined period of time.

[0039] By removing silver sulfide (Ag2S) from the exposed surface of the anode electrode body, silver ions (Ag) are generated by an oxidation reaction at the interface with the specimen. + is generated, making it possible to detect sulfur components.

[0040] The detection unit 3 constantly measures the galvanic current generated between the anode electrode body 10 and the cathode electrode body 12 using a resistanceless ammeter connected between the two electrodes. The measurable current range is preferably set to 0.1 nA to 1000 nA.

[0041] The detection unit 3 can be powered by a battery such as a cell or by an AC adapter from a commercial power source, thereby making the device smaller and more portable. The measurement unit 12 can also be equipped with a function for measuring on multiple channels, and can measure current changes of multiple analytes in parallel.

[0042] The current measurement is preferably carried out at short time intervals in order to measure minute current changes, and specifically, the time intervals should be set to 0.1 to 60 seconds. The measured current values ​​are stored, and the presence or absence of sulfur components in the sample and their transition can be detected and analyzed based on the changes in the current values.

[0043] The substance to be detected is not particularly limited as long as it contains a sulfur component. For example, inorganic compounds include hydrogen sulfide, sodium sulfide, sulfurous acid, sulfur monoxide, sulfur dioxide, sulfur trioxide, disulfur monoxide, and dithionous acid. Organic compounds include cysteine, propanethiol, ethanedithiol, thiophenol, glutathione, and 4-mercaptobenzoic acid. Of the organic compounds, organic compounds having a thiol group are preferred.

[0044] The specimen to be detected is preferably in a paste or liquid form, and may be prepared by dissolving the substance to be detected in a solvent, etc. Examples of specimens include body fluids such as blood, urine, and sweat, biological tissues such as brain and liver, and foods such as milk and meat.

[0045] When a microbial metabolite is used as a specimen, the medium in which the microorganism is cultured can be used as the specimen. The medium is not particularly limited as long as it can culture a wide range of microorganisms, and known media such as liquid media, solid media, gel media, and membrane media can be used. The microorganism may be any type of living microorganism that produces metabolites, including bacteria, actinomycetes, fungi, etc. For example, it is also possible to determine the causative bacteria of food poisoning or infectious diseases.

[0046] By removing microorganisms from their living environment and adding them to a culture medium to prepare a sample, sulfur compounds contained in the microbial metabolites can be easily detected. The sample can be used as is, allowing for detection without stressing the microorganisms. Furthermore, the microorganisms may be concentrated as needed, allowing for simple operations. It is preferable to select a combination of the medium and the metal material used for the electrode body so that the current flowing between the two electrodes is as stable and low as possible when the two electrode bodies are in contact with a culture medium free of microorganisms.

[0047] 3 is a schematic diagram of a microbial quantity determination device according to an embodiment of the present invention. The microbial quantity determination device 100 includes an electrode sensor 51 in which an anode electrode body and a cathode electrode body are integrally formed with an insulator interposed therebetween, a culture medium 52 in which microorganisms can be cultured, a measurement unit 53 that measures the galvanic current flowing between the electrodes of the electrode sensor 51 while both electrodes are in contact with the culture medium 52, and an analysis unit 54 that determines the microbial quantity based on time data corresponding to changes in the measured current value.

[0048] In this example, the electrode sensor 51 can have the same structure as that shown in Fig. 2. A galvanic current flows between the anode electrode body and the cathode electrode body of the electrode sensor 51 due to an electrochemical reaction that occurs between sulfur components such as hydrogen sulfide in metabolites produced by the microorganisms cultured in the culture medium 52 and the anode electrode body of the electrode sensor 51.

[0049] In the electrochemical reaction occurring in the electrode sensor 51, a change in the concentration of sulfur components such as hydrogen sulfide causes a change in the current, and the amount of surviving microorganisms can be determined based on the time data corresponding to the change in the current value.

[0050] Furthermore, since an increase or decrease in the galvanic current value occurs only when microorganisms are present in the culture medium, it is also possible to determine whether the microorganisms in the culture medium are alive or dead by observing the change in the galvanic current. Furthermore, when measuring metabolites produced by microorganisms, the environmental conditions are left unchanged and no reagents are used, so measurements can be taken in their natural state without damaging the microorganisms, and the state of the microorganisms can be grasped in real time.

[0051] The electrode sensor 51 and culture medium 52 are placed in a storage container 50. The storage container 50 is configured by attaching a rectangular cover plate 55 and a bottom plate 56 to the upper and lower openings of a cylindrical body 57, and the electrode sensor 51 is placed on the upper surface of the bottom plate 56. An inlet is formed in the center of the cover plate 55, and a plug 58 is inserted into the inlet to close it in a manner that allows ventilation.

[0052] The culture medium 52 is introduced into the container 50 by removing the stopper 58 and dropping it through the opening. The introduced culture medium 52 is placed on the upper surface of the electrode sensor 51 and comes into contact with the anode electrode body and cathode electrode body of the electrode sensor 51. After the culture medium 52 is introduced, the opening of the cover plate 55 is closed with the stopper 58, thereby preventing contamination from outside.

[0053] The medium 52 is not particularly limited as long as it can cultivate a wide range of microorganisms, and known media such as liquid media, solid media, gel media, and membrane media can be used.

[0054] Bacterial media include meat broth, meat gelatin, nitrate, milk, cornmeal, MR-VP, tomato juice, TSI, SIM, glucose broth, and YM.

[0055] Examples of fungal media include oatmeal medium, malt agar, malt, soil extract, PGY medium, Fowell acetate medium (medium for yeast spore formation), V-8 juice medium, yeast extract, fermentation test medium, yeast assimilative medium, acidogenic medium, hyperosmotic medium, starch-producing medium, and arbutin decomposition medium.

[0056] Examples of media for algae include AF6 medium, C medium, URO medium, VT medium, marine algae medium, ESM medium, f / 2 medium, IMR medium, MNK medium, and Daigo artificial seawater medium.

[0057] In addition to the above media, other media used for research include complete medium for bacteria, Lennox medium (L medium), LB medium, minimal medium for Escherichia coli (Davis medium), minimal salts medium for Escherichia coli (MS), Tris-glucose medium (TG medium), EMB sugar indicator medium, minimal medium for Bacillus subtilis (Spizizen minimal medium), minimal salts medium for Bacillus subtilis, complete medium for yeast (YPAD), minimal medium for yeast, complete medium for Neurospora crassa, minimal medium for Neurospora crassa (Vogel medium N), complete medium for Aspergillus (ANA medium), and Czapek-Dox medium.

[0058] The amount of microorganisms can be easily determined by removing a specimen from an environment where quantitative determination of microorganisms is required and adding it to the culture medium 52. The specimen can be used as is, or the microorganisms can be concentrated as necessary, making the process simple. Furthermore, it is preferable to select a combination of the medium and the metal material used for the electrode body so that the current flowing between the two electrodes is as stable and low as possible when the two electrode bodies are in contact with a culture medium that does not contain microorganisms.

[0059] The microorganisms to be determined may be any type of living microorganism that produces metabolites, including bacteria, actinomycetes, fungi, etc. For example, bacteria that cause food poisoning or infectious diseases can also be determined.

[0060] Specifically, Staphylococcus genus (e.g., Staphylococcus aureus such as methicillin-resistant Staphylococcus aureus (MRSA)), Campylobacter genus (e.g., Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter sputum, etc.), Salmonella genus (e.g., Salmonella typhi, Salmonella paratyphi A, Salmonella enteritidis, Salmonella typhimurium, Salmonella cholera suis, Salmonella dublin, etc.), Escherichia genus (e.g., enterohemorrhagic Escherichia coli (O111, O157, etc.)), Vibrio genus (e.g., Vibrio cholerae, Vibrio parahaemolyticus, etc.), Legionella genus (e.g., Legionella pneumophila, etc.), Shige Examples of bacteria include bacteria of the genus S. pneumoniae (e.g., Shigella sonnei, Shigella dysenteriae, Shigella flexneri, Shigella boydii, etc.), bacteria of the genus Clostridium (e.g., Clostridium botulinum, etc.), bacteria of the genus Listeria (e.g., Listeria monocytogenes, etc.), bacteria of the genus Mycobacterium (e.g., Mycobacterium tuberculosis, etc.), bacteria of the genus Cryptosporidium (e.g., Cryptosporidium parvum, etc.), bacteria of the genus Coxiella (e.g., Coxiella burnetii, etc.), bacteria of the genus Francisella (e.g., Francisella tularensis, etc.), bacteria of the genus Bacillus (e.g., Bacillus cereus, Bacillus anthracis, etc.), bacteria of the genus Yersinia (e.g., Yersinia pestis, etc.), and the like.

[0061] In particular, preferred target microorganisms for the purpose of food hygiene inspection include methicillin-resistant Staphylococcus aureus (MRSA), Campylobacter bacteria (e.g., Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter sputum, etc.), Salmonella bacteria (e.g., Salmonella typhi, Salmonella paratyphi A, Salmonella enteritidis, Salmonella typhimurium, Salmonella cholera suis, Salmonella dublin, etc.), enterohemorrhagic Escherichia coli (O111, O157, etc.), Vibrio bacteria (e.g., Vibrio cholerae, Vibrio parahaemolyticus, etc.), Legionella bacteria (e.g., Legionella pneumophila, etc.), Bacillus cereus (e.g., Bacillus cereus), and the like.

[0062] The measurement unit 53 constantly measures the galvanic current generated between the anode electrode body and the cathode electrode body using a resistanceless ammeter connected between the anode electrode body and the cathode electrode body of the electrode sensor 51. The measured current value can then be output to the analysis unit 54. The measurable current range is preferably set to 0.1 nA to 1000 nA.

[0063] The measuring unit 53 can be made smaller and more portable by being powered by a battery such as a cell or by an AC adapter from a commercial power source. The measuring unit 53 can also be provided with a function for measuring on multiple channels, and can measure current changes of multiple analytes in parallel.

[0064] The analysis unit 54 performs a storage process for storing the current value output from the measurement unit 53 at predetermined time intervals and a determination process for determining the amount of microorganisms based on time data corresponding to changes in the stored current value. For example, software for performing the storage process and determination process can be installed in an information processing device such as a personal computer to realize such processing by the analysis unit 54.

[0065] The storage process is preferably performed at short time intervals to measure minute current changes, and specifically, the time interval should be set to 0.1 to 60 seconds. The current value is then stored together with the time data elapsed since the start of measurement.

[0066] In the analysis process, the stored changes in current value are analyzed, and the current change that correlates with the amount of surviving microorganisms is identified, and the time data at that point is obtained, thereby making it possible to determine the amount of microorganisms.

[0067] As shown in Figure 6 (described later), if the measurement start point is the start point of contact between the microorganisms and the culture medium, the current value flowing through the electrode sensor 51 shows almost no increase immediately after the start of measurement. However, over time, the current value increases in accordance with the increase in metabolites from the surviving microorganisms added to the culture medium. After reaching a peak, the current value gradually decreases. As described above, this change in current value is due to the metabolites produced by the microorganisms, and therefore is thought to correlate with the amount of surviving microorganisms. Therefore, it is possible to identify current changes that correlate with the amount of microbial life within the change in current value, and determine the amount of surviving microorganisms from the time data at the identified point in time.

[0068] In the example of the measurement results shown in Figure 6, the amount of microorganisms can be determined based on the time data from the start of contact with the medium to the time when the current value reaches a predetermined value during the period when the current value corresponding to the logarithmic growth phase increases and reaches its maximum value. Alternatively, the amount of microorganisms may be determined based on the time data from the start of contact with the medium to the time when the current value changes the most during the period when the current value increases and reaches its maximum value. The time when the current value changes the most can be identified, for example, as the time when the slope of the graph showing the change in current value becomes maximum.

[0069] During the period from when the current value increases until it reaches its maximum value, the rate at which the current value increases increases as the amount of surviving microorganisms increases, so the time at which the current value reaches a predetermined value is correlated with the amount of microorganisms. Furthermore, since the time at which the rate of increase in the current value reaches its maximum is also correlated with the amount of microorganisms, the amount of microorganisms can be determined based on time data having such a correlation. Furthermore, in this determination process, the amount of microorganisms can be determined early within the period from when the current value increases until it reaches its maximum value, making it possible to perform the determination process efficiently.

[0070] It is also possible to combine multiple pieces of time data having such correlations to make a comprehensive judgment. Also, by analyzing the correlation between the amount of microorganisms and time data in advance depending on the combination of culture medium and microorganism, the judgment process can be performed more accurately and efficiently based on the analysis results.

[0071] For the measurement results, an approximation curve may be calculated using a known method such as the least squares method to remove noise, and a judgment process may be performed based on the time data calculated from the approximation curve. In addition, noise removal processes such as the moving average method and low-pass filtering may be performed.

[0072] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

[0073] <About the Electrode Sensor> An electrode sensor with the structure shown in Figure 2 was manufactured. A commercially available iron substrate (64 mm long x 64 mm wide x 0.8 mm thick) was used as the anode electrode body, and a silver metal thin film was formed on one exposed surface of the substrate by wet film formation. In the wet film formation process, as a pretreatment, the substrate was immersed in an alkaline aqueous solution for 1 minute, then washed with deionized water, degreased with acetone, and dried indoors. Next, the substrate was immersed in a commercially available plating solution containing silver cyanide and potassium cyanide, and a current of 120 C was applied to the substrate at 20°C. As a posttreatment, the plated substrate was washed several times with ion-exchanged water and then dried indoors. The thickness of the silver metal thin film formed on the substrate surface was measured to be 12 μm using a micrometer (manufactured by Mitutoyo Corporation).

[0074] Next, a resin layer was formed on the surface of the metal thin film formed on the substrate. The resin layer was formed by screen printing a resin paste (manufactured by Henkel AbleStick Japan Co., Ltd.) made of epoxy resin material into a comb-like shape and then heat-curing it at 150°C for 1 hour in a nitrogen atmosphere. The resin layer had a thickness of 20 μm.

[0075] Next, a conductive layer serving as a cathode electrode was formed on the resin layer. A conductive material containing a carbon material was used for the conductive layer. A conductive paste (manufactured by Tamura Corporation) was screen-printed in a comb shape so as to overlap the resin layer, and then heat-cured in a nitrogen atmosphere at 130°C for 1 hour. The conductive layer had a thickness of 20 μm.

[0076] The obtained electrode sensor has an electrode structure in which a cathode electrode body made of a conductive layer is laminated on the surface of an anode electrode body on which a silver metal thin film is formed, with an insulator made of a resin layer interposed between them, and 1 mm wide notches are arranged in a comb-like pattern at 1 mm intervals in a circular area with a radius of 35 mm in the center of the cathode electrode body, with no resin layer formed in the notched areas and the silver metal thin film being exposed.

[0077] <Detection Device> The detection device used was assembled as shown in Figure 1. In a sterile chamber, an electrode sensor was placed on the top surface of a resin bottom plate, with the cathode electrode assembly facing up. A glass cylinder (inner diameter 42 mm, height 32 mm) was placed on the cathode electrode assembly via a silicone packing material. A resin cover plate was placed to cover the upper opening of the cylinder, and the four corners of the bottom plate and cover plate were fastened with fasteners consisting of bolts and nuts to ensure close contact between the components, thereby assembling the storage container. An opening was formed in the center of the cover plate for introducing the sample. Each part of the assembled storage container, including the container, was sterilized in an autoclave at 121°C for 15 minutes and placed in a constant temperature bath at 37°C.

[0078] One end of a conductive wire was connected to each of the anode and cathode electrodes of the electrode sensor at the portion exposed to the outside of the container, and the other end was connected to a zero-resistance ammeter (manufactured by Shrinks Co., Ltd.) to measure the current between the two electrodes. Detection processing was performed using an information processing device that stores the measurement results and analyzes the transition of the current value.

[0079] <Culture solution used for sample preparation> LB broth was used as a medium for culturing microorganisms. The LB broth was prepared by adding 1 L of pure water to tryptone (10.0 g; manufactured by BD Biomedicals), yeast extract (5.0 g; manufactured by BD Biosciences), and NaCl (5.0 g; manufactured by Wako), stirring the mixture, and then sterilizing it with high-pressure steam at 121°C for 15 minutes using an autoclave.

[0080] Example 1 A detection test was carried out using test solutions containing various sulfur compounds as specimens.

[0081] <About the test solution> Sulfur compounds, sodium sulfide (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), cysteine ​​(manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), cystine (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), methionine (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), propanethiol (manufactured by Wako Pure Chemical Industries, Ltd.), and ethanedithiol (manufactured by Wako Pure Chemical Industries, Ltd.) were added to LB culture medium to a predetermined concentration to prepare a test solution. The added concentrations were set to 0.312 mM (millimolar) for sodium sulfide, 0.825 mM for cysteine, 4.16 mM for cystine, 6.70 mM for methionine, 13.1 mM for propanethiol, and 10.6 mM for ethanedithiol. In addition, a test solution was prepared by dissolving sodium sulfide in degassed pure water. The added concentration was set to 0.312 mM. For comparison, a test solution containing only LB culture medium without any sulfur compound added was also prepared.

[0082] <About the detection test> After dropping 10 ml of each test solution onto the top surface of the cathode electrode of the electrode sensor through the opening of the container of the detection device, the current flowing between the anode electrode and the cathode electrode was measured. During the measurement, the opening of the container was set in an open state.

[0083] The stable current values ​​100 minutes after the test solution was added were 180 nA (nanoamperes) for sodium sulfide, 215 nA for cysteine, below the detection limit (10 nA) for cystine, below the detection limit for methionine, 104 nA for propanethiol, and 272 nA for ethanedithiol. Furthermore, the sodium sulfide test solution containing pure water had a current of 620 nA, while the test solution containing only LB culture medium had a current below the detection limit.

[0084] <Measurement Results> The measurement results showed that different current values ​​were measured depending on the type of sulfur compound. The current value per measured concentration was highest for sodium sulfide, followed by cysteine, ethanediol, and propanethiol. No sulfur was detected in cystine or methionine.

[0085] In the case of sodium sulfide, hydrogen sulfide ions HS are present in the test solution. - or sulfur ions S 2- It is believed that a sulfur compound having a thiol group is produced. In addition, since a current value is measured for a sulfur compound having a thiol group, it is believed that a sulfur compound having a thiol group is detected.

[0086] In the test solution for sodium sulfide, the current value was higher when it was added to pure water than when it was added to the LB culture solution. This is because hydrogen sulfide ions (HS), which are sulfur components in the test solution, - or sulfur ions S 2- It is presumed that the concentration of sulfur components in the LB culture solution is lower than that in pure water because the sulfur components are adsorbed to proteins contained in the LB culture solution.

[0087] Example 2 A detection test was carried out using a bacterial solution containing live bacteria as a sample.

[0088] <Preparation of bacterial suspension> Commercially available Escherichia coli (E. coli DH5α) was used for the bacterial suspension. E. coli was cultured on a plate medium, and independent colonies were picked up and cultured in LB culture medium, followed by centrifugation. The resulting precipitated bacterial cells were washed with LB culture medium to remove any metabolites produced, and then resuspended in LB culture medium. The bacterial count in the bacterial suspension was determined by measuring the optical density (OD) at 600 nm of the bacterial suspension using a spectrophotometer (manufactured by Hitachi High-Tech Corporation), and when OD = 0.1, the bacterial count was 107 cfu / ml equivalent, and the number of bacteria is 10 6 The culture was diluted with LB culture medium to a concentration of cfu / ml.

[0089] <Detection test> After dropping 10 ml of bacterial solution onto the top surface of the cathode electrode of the electrode sensor through the opening of the container of the detection device, the current flowing between the anode electrode and the cathode electrode was continuously measured at 0.5 second intervals. During the measurement, the opening of the container was set to be open.

[0090] <Quantitative Analysis of Hydrogen Sulfide (HS) Concentration in Bacterial Solution> In parallel with the measurement of the current value, the hydrogen sulfide (HS) concentration in the bacterial solution was quantitatively analyzed. 0.5 mL samples were taken from the bacterial solution at appropriate times during the measurement, and quantitative analysis of the samples was performed using an ion chromatograph (ICS-3000 electrochemical detector, manufactured by Thermo Fisher Scientific K.K.) using the calibration curve method.

[0091] <Measurement Results> Figure 4 shows the time course of the current value and hydrogen sulfide HS concentration, with the horizontal axis representing time (minutes), the left vertical axis representing current value (nA), and the right vertical axis representing concentration (µM (micromolar)). Figure 5 is a graph showing the correlation between the measured current value and concentration, with the horizontal axis representing concentration (µM) and the vertical axis representing current value (nA).

[0092] It is known that E. coli releases trace amounts of hydrogen sulfide (HS) during proliferation through metabolic decomposition of cysteine, etc. As shown in Figure 4, quantitative analysis confirmed that a small amount of hydrogen sulfide (HS) was released into the LB culture medium as E. coli proliferated, and an accompanying increase in current value was confirmed.

[0093] In Figure 5, a high positive correlation (correlation coefficient R = 0.993) was confirmed between the hydrogen sulfide (HS) concentration in the LB culture solution and the current value of the electrode sensor, and a linear equation (y = 530x - 1.70) was obtained that quantitatively shows the correlation between the two.

[0094] Furthermore, it was shown that the electrode sensor can detect trace concentrations of hydrogen sulfide (HS) ranging from 0.022 μM to 0.70 μM. Based on the obtained linear relationship, trace amounts of hydrogen sulfide (HS) in a sample can be detected from the current value of the electrode sensor, and the detection limit based on the linear relationship is a trace concentration of 0.003 μM.

[0095] The above detection test results confirmed that even in a solution such as LB culture medium, which contains many impurities such as proteins and amino acids, trace amounts of hydrogen sulfide (HS) present in the solution can be directly detected without pretreatment such as pH adjustment or extraction.

[0096] Example 3 A detection test was carried out by changing the thickness of the metal thin film of the anode electrode body of the electrode sensor.

[0097] <Regarding the detection test> Four types of electrode sensors, each with a silver metal thin film formed on the surface of the substrate as the anode electrode body having a thickness of 5, 8, 10, or 15 μm, were manufactured in the same manner as in Example 1. For comparison, an electrode sensor manufactured in the same manner as in Example 1 was prepared, using a silver plate as the substrate but without forming a metal thin film.

[0098] Using the same medium and microorganisms as in Example 2, 8 A sample of cfu / ml was prepared, and the current value was measured by the same detection test as in Example 2.

[0099] <Regarding the Measurement Results> From the obtained measurement results, the relationship between the silver film thickness and the maximum current value was examined. When the film thickness was 5 μm, the maximum current value was 350 nA, and when it was 8 μm, the maximum current value was 400 nA. In contrast, when the film thickness was 10 μm, the maximum current value was 610 nA, when the film thickness was 12 μm in Example 2, the maximum current value was 650 nA, and when the film thickness was 15 μm, the maximum current value was 650 nA when a silver plate was used.

[0100] From these measurement results, it was confirmed that by forming the silver film thickness of the anode electrode body to 10 μm or more, the maximum current value can be increased and stable measurement results can be obtained.

[0101] Example 4 A test was carried out to confirm the effect of the detection test on live bacteria in a sample.

[0102] <Confirmation Test> Using the electrode sensor manufactured in the same manner as in Example 1, a detection test was carried out on a sample containing live bacteria in the same manner as in Example 2. The number of live bacteria was 10 5 A bacterial solution adjusted to cfu / ml was used.

[0103] Three milliliters of the bacterial solution on the electrode sensor in the container was sampled every 30 minutes, and the OD value at 600 nm of the sampled bacterial solution was measured using a spectrophotometer in the same manner as in Example 2. Meanwhile, a bacterial solution similar to the sample used in the detection test was allowed to stand in a commercially available plastic container at 37°C, and the OD value at 600 nm of the bacterial solution was measured every 30 minutes in the same manner.

[0104] <Measurement Results> The measurement results are shown in Figure 6. In Figure 6, the horizontal axis represents time (minutes), the left vertical axis represents OD values, and the right vertical axis represents current values ​​(n). Comparing the time transitions of the OD values ​​corresponding to the number of bacteria and the measured current values, it was confirmed that the proliferation of bacteria did not coincide with the increase in current value, and that the current value began to increase approximately 100 minutes after the bacteria began to proliferate, and that the bacteria continued to proliferate while the current value was increasing.

[0105] Furthermore, the changes in OD values ​​of the bacterial solution on the electrode sensor and the bacterial solution in the plastic container were consistent, suggesting that the growth rates of both were the same. Specifically, the doubling time of E. coli was calculated from the slope of the change in OD value over the logarithmic growth period of 60 to 210 minutes, and the doubling time was 27 minutes for both. In other words, it was confirmed that the growth of live bacteria was not hindered at all when in contact with the electrode sensor.

[0106] Example 5 A regeneration test was carried out on the electrode sensor used in the detection test.

[0107] <Regarding the regeneration test> The same detection test as in Example 2 was carried out with the number of bacteria set at 10 7 The detection test was carried out using a bacterial solution containing cfu / ml of bacteria. After the detection test, the electrode sensor had turned brown due to the formation of silver sulfide on the exposed silver-plated surface of the anode electrode body.

[0108] A regeneration test was conducted using a used electrode sensor as a test specimen, using a potentiogalvanostat (SI1287, manufactured by Toyo Corporation) with a reduction reaction similar to that of the cathodic reduction method. The test specimen was immersed in a 0.1 N potassium chloride (KCl) electrolyte, and a current of -125 μA was applied for 1,000 seconds using a three-electrode method in which the anode electrode of the test specimen was used as the working electrode, SCE as the reference electrode, and platinum as the counter electrode.

[0109] In addition, a test solution was prepared by dissolving thiourea at a concentration of 0.5 molar in a 0.01 N aqueous sulfuric acid solution, and a regeneration test using thiourea was carried out. Using a used electrode sensor as a test specimen, 6 ml of the test solution was dropped onto the discolored surface of the test specimen and left at room temperature for 1 minute.

[0110] After the regeneration test, the electrode sensor was washed with running water and dried indoors. Next, the same detection test as the above-mentioned detection test was performed using the electrode sensor that had undergone the first regeneration treatment. Then, the electrode sensor used in the second detection test was subjected to a second regeneration treatment, and the second detection test was performed in the same manner as the above-mentioned detection test.

[0111] <Measurement Results> Figure 7 shows the measurement results of a detection test using an electrode sensor in the case of a regeneration treatment by current application, the first time, after the first regeneration treatment, and after the second regeneration treatment. In Figure 7, the horizontal axis represents time (minutes) and the vertical axis represents current value (μA), showing the change in current value over time. The maximum current value was 284 nA in the first regeneration treatment, 273 nA in the first regeneration treatment, and 242 nA in the second regeneration treatment.

[0112] 8 shows the measurement results of the detection test using the electrode sensor in the case of the regeneration treatment using thiourea, the first regeneration treatment, and the second regeneration treatment. Similar to FIG. 6, FIG. 8 shows the time transition of the current value. The maximum current value was 305 nA in the first regeneration treatment, 284 nA in the first regeneration treatment, and 251 nA in the second regeneration treatment.

[0113] It was visually confirmed that the discolored surface of the anode electrode body of the electrode sensor changed from brown to white after the regeneration treatment. It is believed that the silver sulfide precipitated on the surface was removed by the regeneration treatment using a reduction reaction. Regardless of the regeneration treatment performed using either reduction reaction, the electrode sensor after the first regeneration treatment obtained detection results equivalent to those of the initial electrode sensor. Therefore, it was confirmed that detection tests using electrode sensors that had undergone the first regeneration treatment were possible.

[0114] In the case of the electrode sensor that underwent the second regeneration process, the current value increased as in the first process, but the maximum current value decreased by about 15%. However, it was confirmed that it was still possible to detect the presence or absence of sulfur components.

[0115] [Example 6] The same detection test as in Example 2 was carried out with the number of bacteria being 10 8 The detection test was carried out using a bacterial solution containing cfu / ml of bacteria. After the detection test, the electrode sensor had turned brown due to the formation of silver sulfide on the exposed silver-plated surface of the anode electrode body.

[0116] A regeneration test using a reduction reaction was performed on the discolored anode electrode assembly and an unused anode electrode assembly in the same manner as in Example 5, and the potential of the anode electrode assembly was measured. Figure 9 shows the reduction curve of the anode electrode assembly in the regeneration test. In Figure 9, the vertical axis represents the potential (mV vs. SHE) based on the standard hydrogen electrode (SHE), and the horizontal axis represents time (seconds).

[0117] <Measurement Results> The electrode potential of the electrode surface of the discolored anode electrode assembly decreased from Ecorr (natural potential) 40 mV, remained constant at approximately -530 mV for approximately 380 seconds, and then rapidly decreased again. The disappearance of the coloration on the electrode surface was confirmed visually when the electrode potential immediately after the decrease was approximately -756 mV. In contrast, with an unused anode electrode assembly, the electrode potential decreased to approximately -1000 mV immediately after the start of measurement and then remained approximately constant.

[0118] Therefore, when a used anode electrode body is regenerated by a reduction reaction using an electrolyte, the completion of the regeneration process can be confirmed when the electrode potential decreases after remaining at a constant potential of −530 mV.

[0119] Example 7 The amount of microorganisms was determined using the same electrode sensor as in Example 1.

[0120] <Regarding the Determination Device> The determination device used was assembled as shown in Figure 3. In a sterile chamber, an electrode sensor was placed on the top surface of a resin bottom plate, with the cathode electrode assembly facing up, and a glass cylinder (inner diameter 42 mm, height 32 mm) was placed on the cathode electrode assembly via a silicone packing material. A resin cover plate was placed to cover the upper opening of the cylinder, and the four corners of the bottom plate and cover plate were fastened with fasteners consisting of bolts and nuts so that each component was in close contact with each other, thereby assembling the storage container. A silicone stopper was inserted into the opening in the center of the cover plate to removably seal it. Each component, including the assembled storage container, was sterilized in an autoclave at 121°C for 15 minutes and placed in a constant temperature bath at 37°C.

[0121] One end of a conductive wire was connected to each of the anode and cathode electrodes of the electrode sensor at the portion exposed outside the container, and the other end was connected to a zero-resistance ammeter to measure the current between the two electrodes. The zero-resistance ammeter was connected to an information processing device consisting of a personal computer and set up to output the measurement results. A program for storing the measurement results and a program for processing and judging the stored measurement results were installed in the information processing device in advance.

[0122] <Regarding the Medium> A medium having the following composition was used: Tryptone: 10.0 g (manufactured by BD Biomedicals) Yeast Extract: 5.0 g (manufactured by BD Biosciences) NaCl: 5.0 g (manufactured by Wako) The medium was stirred with 1000 milliliters of distilled water, and sterilized with high-pressure steam at 121°C for 15 minutes using an autoclave.

[0123] <Preparation of Microorganisms> Commercially available Escherichia coli (E. coli DH5α) was used as the microorganism to be added to the medium. E. coli was cultured on a plate medium, and independent colonies were picked up and cultured in LB culture medium, followed by centrifugation. The precipitated bacterial cells were washed with LB culture medium to remove generated metabolites, and then resuspended in LB culture medium. The viable cell count of the bacterial solution was determined by measuring the optical density (OD) at 600 nm of the bacterial solution using a spectrophotometer, and the bacterial count at OD = 0.1 was 10 7 The LB culture medium was diluted to a predetermined number of bacteria, with the standard being equivalent to 1, 10, and 10 cfu / ml. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 Ten samples of cfu / ml were prepared.

[0124] <Measurement Method> After dropping 10 ml of each sample onto the top surface of the electrode sensor in the container, the current flowing between the anode and cathode electrodes was sampled at 0.5 second intervals at a temperature of 37° C. and measured continuously for 1000 minutes. The measurement results for each sample are shown in Figure 10.

[0125] <Determination method> In Fig. 10, the vertical axis represents current value (nA) and the horizontal axis represents time (minutes). The time at which the current value starts to rise and the time at which the current value reaches the maximum value change depending on the number of viable bacteria, and it can be seen that there is a correlation between the number of viable bacteria and the change in current value.

[0126] First, the correlation between the number of viable bacteria in the sample and the time data from the start of contact with the medium to the time when the current value reaches a predetermined value during the period when the current value increases and reaches its maximum was analyzed. Next, the correlation between the time data from the start of contact with the medium to the time when the current value changes the most during the period when the current value increases and reaches its maximum was analyzed. For the time when the current value changes the most, the difference between the current values ​​sampled during the target period was calculated, and the time data showing the maximum value was used.

[0127] 11A and 11B are graphs showing the time from the start of contact with the medium until the current value reaches a predetermined value, which is set to 10, 20, 30, 50, 100, 200, 300 nA, and the maximum current value, respectively. The vertical axis represents the time (minutes) until each predetermined value is reached, and the horizontal axis represents the number of viable bacteria (cfu / milliliter).

[0128] 11A and 11B clearly show that there is a positive correlation between the viable cell count and the time data until a predetermined current value is reached. Furthermore, there is also a positive correlation with the time data until the maximum current value is reached, indicating that during the period from when the current value increases to when it reaches its maximum value, it is possible to determine the viable cell count based on the time data until the predetermined current value is reached.

[0129] Therefore, by using a calibration curve that shows such a correlation depending on the combination of the type of microorganism and the culture medium as a reference for determination, it becomes possible to determine the amount of microorganisms efficiently in a short time.

[0130] Fig. 12 is a graph showing the relationship between the viable cell count and the time data up to the point where the current value changes the most. Fig. 12 clearly shows that there is a positive correlation between the viable cell count and the time data up to the point where the current value changes the most. This shows that it is possible to determine the viable cell count based on the time data up to the point where the current value changes the most.

[0131] 13 is a graph showing the relationship between the number of viable bacteria and the maximum current value. 7 At cfu / ml or less, the change in maximum current value relative to the change in viable cell count is small, and the correlation with the viable cell count is weaker than in the case of the time data described above.

[0132] When the strength of this correlation is examined using a correlation coefficient, the common logarithm of the viable cell count and the time data both show a strong positive correlation of 0.95 or more, while the common logarithm of the viable cell count and the maximum current value show a positive correlation of approximately 0.7. Therefore, in the determination process, time data that has a correlation with the viable cell count is obtained, and the viable cell count is determined based on the obtained time data, thereby making it possible to efficiently obtain more accurate determination results.

[0133] 1...electrode sensor, 2...specimen, 3...detection unit, 4...container, 10...anode electrode body, 11...insulator, 12...cathode electrode body, 100...microorganism quantity determination device, 50...container, 51...electrode sensor, 52...culture medium, 53...measurement unit, 54...analysis unit, 55...cover plate, 56...bottom plate, 57...cylinder, 58...stopper

Claims

1. A sulfur component detection method for detecting sulfur components in a specimen using an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed with an insulator interposed therebetween, in which, while the specimen is in contact with both electrode bodies, a galvanic current flowing between the two electrode bodies due to an electrochemical reaction that occurs between the sulfur components in the specimen and the anode electrode body is measured, and the sulfur components in the specimen are detected based on the measured current value.

2. The method for detecting sulfur components according to claim 1, wherein the specimen contains sulfur compounds derived from biological tissue.

3. A method for detecting sulfur components according to claim 1 or 2, wherein the sample contains sulfur compounds derived from metabolic products of living organisms.

4. A method for detecting sulfur components as described in claim 1 or 2, wherein the anode electrode body uses a silver plate or at least the exposed surface that comes into contact with the sample is coated with silver, and a sulfur compound is produced on the exposed surface as a result of an electrochemical reaction that occurs between the silver and the sulfur component.

5. The method for detecting a sulfur component according to claim 4, wherein at least the exposed surface of the anode electrode body is covered with a layer of silver having a thickness of 10 μm or more.

6. A sulfur component detection device comprising an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed with an insulator interposed therebetween, and a detection unit which measures a galvanic current flowing between the two electrode bodies due to an electrochemical reaction occurring between the sulfur components in the sample and the anode electrode body while at least both electrode bodies of the electrode sensor are in contact with the sample, thereby detecting a change in current value due to the sulfur components in the sample.

7. A method for regenerating an electrode sensor used in the sulfur component detection method described in claim 4, which removes sulfur compounds produced on the exposed surface by a reduction reaction caused by passing an electric current through the anode electrode body in an electrolyte.

8. A method for regenerating an electrode sensor used in the method for detecting sulfur components as described in claim 4, comprising contacting at least the exposed surface of the anode electrode body with a sulfuric acid solution containing thiourea to remove sulfur compounds formed on the exposed surface.

9. A microbial quantity determination device that detects sulfur components in the metabolism of microorganisms to determine the amount of microorganisms, comprising an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, a culture medium for culturing microorganisms, a measurement unit that measures the galvanic current flowing between at least both electrode bodies of the electrode sensor while in contact with the culture medium, and an analysis unit that stores the current value measured by the measurement unit and determines the amount of microorganisms based on time data corresponding to changes in the current value.

10. The microbial quantity determination device according to claim 9, wherein the analysis unit determines the microbial quantity based on time data from the start of contact with the culture medium to the time when a predetermined value is reached during the period in which the current value increases and reaches its maximum value.

11. The microbial quantity determination device according to claim 9, wherein the analysis unit determines the microbial quantity based on time data from the start of contact with the culture medium to the point at which the current value changes the most during the period in which the current value increases and reaches its maximum value.

12. A method for determining the amount of microorganisms by detecting sulfur components in the metabolism of microorganisms, comprising contacting at least both an anode electrode body and a cathode electrode body with a culture medium for culturing microorganisms using an electrode sensor formed integrally with an insulator, measuring the galvanic current between the two electrode bodies, and determining the amount of microorganisms based on time data corresponding to the change in the measured current value.

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