Microorganism quantity determination device and method

Abstract

To provide a microorganism quantity determination device and method that can determine a microorganism quantity present in a medium easily in a short time using an electrode sensor.SOLUTION: A microorganism quantity determination device 1 includes: an electrode sensor 10 in which an anode electrode body and a cathode electrode body are integrally formed via an insulator; a culture medium 11 in which microorganisms can be cultured; a measurement unit 12 that measures a galvanic current flowing between at least both electrode bodies of the electrode sensor 10 while they are in contact with the culture medium 11; and an analysis unit 13 that stores a current value measured by the measurement unit 12 and determines a microorganism quantity based on time data corresponding to a change in the current value.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a microbial amount determining device and method that can quantitatively determine the amount of living microorganisms such as bacteria using an electrode sensor. [Background technology]

[0002] In the fields of food processing, medicine, and nursing care, there is a need to prevent food poisoning and infectious diseases caused by bacteria and other microorganisms, and it is therefore necessary to constantly monitor microorganisms in the actual working environment. For this reason, the development of technology to detect microorganisms is underway.

[0003] When tracking the growth of microorganisms, optical methods using light transmitted through a sample containing microorganisms and electrochemical methods that measure thermal and electrical changes associated with the metabolic activity of microorganisms have been proposed, but because there are limitations to the amount of microorganisms that can be measured, a culture method is generally used in which microorganisms are cultured on agar in a petri dish and the amount of microorganisms is examined. However, such a culture method has issues such as the need to consume large amounts of materials such as petri dishes and agar, and the long culture time (1 to 2 days).

[0004] The present inventors have proposed a microorganism detection device that detects a current change caused by the 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 shown in Patent Document 1. By using such an electrode sensor, it has become possible to detect microorganisms easily and accurately by detecting minute current changes caused by metabolites produced by microorganisms in the culture medium.

[0005] Patent Document 2 proposes using such an electrode sensor to measure the amount of microorganisms themselves by measuring the current flowing between an anode electrode and a cathode electrode, but it has not been confirmed that the amount of microorganisms can be directly measured from changes in current. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6804063 [Patent Document 2] JP 2016-10403 A Summary of the Invention [Problem to be solved by the invention]

[0007] The inventors have focused on the metabolites excreted by living microorganisms and made it possible to detect microorganisms in a culture medium using an electrode sensor, but there is room for further study on the amount of living microorganisms cultured in a culture medium. That is, the amount of living microorganisms cultured in a culture medium is generally measured by the number of microbial colonies produced by culture, but the relevance of such an index to the amount of living microorganisms cultured in a culture medium needs to be further studied.

[0008] Therefore, an object of the present invention is to provide a microbial amount determining device and method that can determine the amount of surviving microorganisms in a culture medium simply and quickly using an electrode sensor. [Means for solving the problem]

[0009] The microbial quantity determination 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, a culture medium capable of 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 microbial quantity based on time data corresponding to the change in the current value. Furthermore, the analysis unit determines the microbial quantity based on time data from the start of contact with the culture medium to the time when the current value reaches a predetermined value during the period in which the current value increases and reaches its maximum value. Furthermore, the analysis unit determines the microbial quantity based on time data from the start of contact with the culture medium to the time when the current value changes the most during the period in which the current value increases and reaches its maximum value. Furthermore, the electrode sensor has a metal thin film formed on the anode electrode body with a thickness of 10 μm or more.

[0010] The method for determining the amount of microorganisms according to the present invention uses an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, brings at least both electrode bodies into contact with a culture medium in which microorganisms can be cultured, measures the galvanic current between the two electrode bodies, and determines the amount of microorganisms based on time data corresponding to the change in the measured current value. Effect of the Invention

[0011] According to the present invention, the amount of viable microorganisms can be determined easily and quickly by measuring the current value generated in the electrode sensor while the electrode sensor is in contact with the culture medium and determining the amount of microorganisms based on the time data corresponding to the change in the current value. [Brief description of the drawings]

[0012] [Figure 1] 1 is a schematic diagram of a microbial quantity determination device according to the present invention; [Diagram 2] 3A and 3B are a plan view and a partially enlarged cross-sectional view of an electrode sensor. [Diagram 3] 11 is a graph showing an example of measurement results regarding a galvanic current flowing between both electrode bodies. [Figure 4] 1 is a graph showing the measurement results in Example 1. [Figure 5A] 1 is a graph showing the correlation between the number of viable bacteria and the time required to reach a predetermined current value in Example 1. [Figure 5B] 1 is a graph showing the correlation between the number of viable bacteria and the time required to reach a predetermined current value in Example 1. [Figure 6] 1 is a graph showing the correlation between the number of viable bacteria and the time required to reach the maximum current value in Example 1. [Figure 7] 1 is a graph showing the correlation between the number of viable bacteria and the maximum current value in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] 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, but 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.

[0014] 1 is a schematic diagram of a microbial quantity determining device according to an embodiment of the present invention. The microbial quantity determining device 1 includes an electrode sensor 10 in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, a culture medium 11 in which microorganisms can be cultured, a measuring unit 12 that measures the galvanic current flowing between the electrodes of the electrode sensor 10 while both electrodes are in contact with the culture medium 11, and an analyzing unit 13 that determines the microbial quantity based on time data corresponding to changes in the measured current value.

[0015] In this example, the electrode sensor 10 and culture medium 11 are placed in a storage container 2. The storage container 2 is constructed by attaching a rectangular cover plate 14 and a bottom plate 15 to the upper and lower openings of a cylinder 16, and the electrode sensor 10 is placed on the upper surface of the bottom plate 15. An input port is formed in the center of the cover plate 14, and a plug 17 is inserted into the input port to seal it.

[0016] The culture medium 11 is introduced into the container 2 by dropping it through the opening after removing the plug 17. The introduced culture medium 11 is placed on the upper surface of the electrode sensor 10 and comes into contact with the anode electrode body and the cathode electrode body of the electrode sensor 10. After the culture medium 11 is introduced, the opening of the cover plate 14 is closed with the plug 17 to prevent contamination from the outside.

[0017] 2A and 2B are a plan view and a partially enlarged cross-sectional view of the electrode sensor 10. FIG. 2B shows a cross section taken along the left-right direction in a region enclosed by a circle.

[0018] The electrode sensor 10 has a structure similar to that of a known ACM sensor, in which an insulator 10b is laminated in layers on the upper surface of a rectangular anode electrode body 10a, and a cathode electrode body 10c is laminated in layers on the upper surface of the insulator 10b. A comb-shaped electrode portion in which a plurality of narrow notches are arranged in parallel in a comb shape is formed in the center of the insulator 10b and the cathode electrode body 10c, and the surface of the anode electrode body 10a is exposed in the notches of the comb-shaped electrode portion. Then, by bringing the culture medium 11 into contact with both electrode bodies of the electrode sensor 10, a galvanic current flows between the anode electrode body 10a and the cathode electrode body 10c exposed in the notches.

[0019] A galvanic current is generated between the two electrodes by the electrode surface of the anode electrode body 10a exposed in the notch being a site where an oxidation reaction occurs to generate electrons, and the electrode surface of the comb-shaped electrode portion of the cathode electrode body 10c being a site where a reduction reaction occurs to lose electrons. Therefore, the electrode surface of the cathode electrode body 10c is set to a combination of dissimilar metals for both electrodes so that it has a higher potential than the electrode surface of the anode electrode body 10a under environmental conditions in which an electrochemical reaction occurs.

[0020] In the electrode sensor 10, the two electrode bodies are integrally formed in the comb-shaped 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 part in contact with the two electrode bodies with a culture medium interposed between them. The output of the generated galvanic current is proportional to the length of the boundary of the contact area with the culture medium at the boundary part of 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 parts becomes narrower.

[0021] For this reason, in the above-mentioned example, the electrode sensor is arranged such that the anode electrode body and the cathode electrode body are stacked with an insulator interposed therebetween, but is not limited to this configuration. For example, the electrode sensor may be arranged such that both electrode bodies are arranged in parallel on a substrate, the boundary portion where the electrodes are arranged facing each other is formed in a comb-like shape, and the formed narrow electrode portions are arranged in parallel alternately.

[0022] It is also possible to form such a comb-tooth electrode structure so as to surround a stick-shaped substrate, or to form a comb-tooth electrode structure on the inside of a cylindrical substrate to create a compact electrode sensor, and the compact electrode sensor may be inserted into a culture medium in a sealed container to determine the presence of microorganisms.

[0023] The insulator 10b is made of a non-conductive insulating material that electrically insulates the anode electrode body 10a and the cathode electrode body 10c. There are no particular limitations on the insulating material, and examples of the insulating material 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 later.

[0024] A metal thin film is preferably formed on the electrode surfaces of the anode electrode body 10a and the cathode electrode body 10c. 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. In addition, it is preferable that the surfaces of the anode electrode body and the cathode electrode body have a low resistance value, specifically, it is preferable to set the resistance value to 0.1 Ω or less. The surface resistance value can be measured by connecting measurement terminals to both ends of the center line of the electrode body.

[0025] When silver is used as the metal of the anode electrode body 10a, the metal used in the cathode electrode body 10c may be a metal having a composition that has a more noble potential than silver. When silver is used in the anode electrode body 10a, 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 the silver metal thin film is thinner than 10 μm, problems such as peeling of the thin film are likely to occur when used for a long period of time.

[0026] The anode electrode body 10a 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, dry film formation, or sintering a conductive paste. Examples of wet film formation include electroplating, electroless plating, and composite plating, and examples of dry film formation include vapor deposition and sputtering.

[0027] The cathode electrode body 10c has a conductive material including a metal material at least on the electrode surface, and when a plate-shaped body is used as the anode electrode body 10a serving as the substrate, the cathode electrode body 10c is preferably formed by a printing method using a conductive paste, as shown in Fig. 2. By forming the cathode electrode body by such a printing method, the layer thickness can be formed to be as thick as 5 to 100 µm, the electrical resistance value decreases, and the current generated between the two electrode bodies can be measured accurately and stably.

[0028] In addition, 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 letterpress printing, lithographic printing, intaglio printing, stencil printing, electrostatic printing, inkjet printing, and laser printing. The electrode sensor shown in FIG. 2 can be created by screen printing, which is a stencil printing method, but other printing methods may also be used, and are not particularly limited.

[0029] In the electrode sensor 10, a galvanic current flows due to electrochemical reactions that occur between the anode electrode body 10a and the cathode electrode body 10c due to trace components contained in the culture medium 11 that contacts the two electrodes and metabolites produced by living microorganisms. For example, in the case of hydrogen sulfide, a typical metabolite of microorganisms, the following reaction is thought to occur, causing a change in the current flowing between the two electrodes. Medium; H2S → HS - +H + Cathode electrode body; O2+2H2O+4e - →4OH - Anode electrode: 2Ag+HS - →Ag2S+H + +2e -

[0030] In the above reaction, a change in the concentration of 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. In addition, since an increase or decrease in the galvanic current value occurs only when microorganisms are alive in the culture medium, it is also possible to determine whether the microorganisms in the culture medium are alive or dead based on the change in the galvanic current. Furthermore, when measuring the metabolites produced by the microorganisms, the environmental conditions are left unchanged and no reagents are used, so that it is possible to measure in a natural state without damaging the microorganisms, and the state of the microorganisms can be grasped in real time.

[0031] The medium 11 is not particularly limited as long as it is capable of culturing a wide range of microorganisms, and any known medium such as a liquid medium, a solid medium, a gel medium, or a membrane medium can be used.

[0032] Bacterial media include broth, broth gelatin, nitrate, milk, cornmeal, MR-VP, tomato juice, TSI, SIM, glucose-broth, and YM.

[0033] Fungal media include oatmeal medium, wort agar, wort, soil extract, PGY medium, Fowell acetate medium (medium for yeast spore formation), V-8 juice medium, yeast extract, fermentation test medium, yeast assimilation medium, acid production medium, high osmotic pressure medium, starch production medium, and arbutin decomposition medium.

[0034] Examples of media for algae include AF6 medium, C medium, URO medium, VT medium, medium for marine algae, ESM medium, f / 2 medium, IMR medium, MNK medium, and Daigo artificial seawater medium.

[0035] In addition to the above media, other media used for research purposes include complete medium for bacteria, Lennox medium (L medium), LB medium, minimal medium for E. coli (Davis medium), minimal salts medium for E. 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.

[0036] The amount of microorganisms can be easily determined by taking a specimen from an environment where it is necessary to quantitatively determine the amount of microorganisms and adding it to the culture medium 11. The specimen can be used as is, or the microorganisms may be concentrated as necessary, which allows for a simple operation. In addition, it is preferable to select a combination of the culture medium and the metal material used for the electrode body so that the current flowing between the two electrode bodies is as stable and low as possible when the two electrode bodies are in contact with a culture medium containing no microorganisms.

[0037] The microorganisms to be determined may be any type of living microorganism that produces metabolites, including bacteria, actinomycetes, fungi, etc. For example, the causative bacteria of food poisoning or infectious diseases can also be determined.

[0038] Specifically, Staphylococcus spp. (e.g., Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA)), Campylobacter spp. (e.g., Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter sputum, etc.), Salmonella spp. (e.g., Salmonella typhi, Salmonella paratyphi A, Salmonella enteritidis, Salmonella typhimurium, Salmonella cholera suis, Salmonella dublin, etc.), Escherichia spp. (e.g., enterohemorrhagic Escherichia coli (O111, O157, etc.)), Vibrio spp. (e.g., Vibrio cholerae, Vibrio parahaemolyticus, etc.), Legionella spp. (e.g., Legionella pneumophila, etc.), Shigella spp. Examples of bacteria that may be included in the list include bacteria of the genus Shigella (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.), and bacteria of the genus Yersinia (e.g., Yersinia pestis, etc.).

[0039] In particular, preferred target microorganisms for the purpose of food hygiene inspection include methicillin-resistant Staphylococcus aureus (MRSA), Campylobacter spp. (e.g., Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter sputum, etc.), Salmonella spp. (e.g., Salmonella typhi, Salmonella paratyphi A, Salmonella enteritidis, Salmonella typhimurium, Salmonella cholera suis, Salmonella dublin, etc.), enterohemorrhagic Escherichia coli (O111, O157, etc.), Vibrio spp. (e.g., Vibrio cholerae, Vibrio parahaemolyticus, etc.), Legionella spp. (e.g., Legionella pneumophila, etc.), Bacillus cereus (e.g., Bacillus cereus), etc.

[0040] The measurement unit 12 uses a non-resistance ammeter connected between the anode electrode body 10a and the cathode electrode body 10c to constantly measure the galvanic current generated between the anode electrode body 10a and the cathode electrode body 10c. Then, the measurement unit 12 can output the measured current value to the analysis unit 13. The measurable current range is preferably set to 0.1 nA to 1000 nA.

[0041] The measuring unit 12 can be made compact and portable by supplying power from a battery such as a battery pack or from an AC adapter from a commercial power source. The measuring unit 12 can also be provided with a function for measuring on multiple channels, and current changes of multiple specimens can be measured in parallel.

[0042] The analysis unit 13 performs a storage process for storing the current value output from the measurement unit 12 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, such processes of the analysis unit 13 can be realized by installing software for performing the storage process and the determination process in an information processing device such as a personal computer.

[0043] In order to measure minute changes in current, it is preferable to perform the storage process at short time intervals, specifically, the time interval should be set to 0.1 to 60 seconds. Then, the current value is stored together with the time data that has elapsed since the start of measurement.

[0044] In the analysis process, the stored changes in current value are analyzed, and the current change that correlates with the amount of viable microorganisms is identified, and the time data at that point in time is obtained, thereby making it possible to determine the amount of microorganisms.

[0045] FIG. 3 is a graph showing an example of the measurement result of the galvanic current flowing between the two electrodes. If the measurement start point is the time when the specimen is put into the medium and the medium contact is started, the current value is hardly increased immediately after the start, but the current value increases with time as the amount of metabolites from the living microorganisms put into the medium increases. Then, the current value reaches a peak and then gradually decreases. As described above, such a change in the current value changes due to the metabolites produced by the microorganisms, and therefore it is considered to have a correlation with the amount of living microorganisms. Therefore, it is possible to identify the current change that is correlated with the amount of microorganisms in the current value change, and to determine the amount of living microorganisms from the time data at the specified time point.

[0046] In the case of the example of the measurement results shown in Fig. 3, the amount of microorganisms can be determined based on time data T1 from the start of contact with the medium to the time when the current value reaches a predetermined value I1 during the period when the current value increases and reaches its maximum value. The amount of microorganisms may also be determined based on time data T2 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.

[0047] During the period from when the current value increases to when it reaches its maximum value, the rate at which the current value increases increases as the amount of surviving microorganisms increases, so the time to reach a predetermined value is correlated with the amount of microorganisms. In addition, the time at which the rate at which the current value increases is at its maximum is also correlated with the amount of microorganisms, so the amount of microorganisms can be determined based on time data having such a correlation. In this determination process, the amount of microorganisms can be determined early within the period from when the current value increases to when it reaches its maximum value, so the determination process can be performed efficiently.

[0048] It is also possible to perform a comprehensive judgment by combining multiple pieces of time data having such correlations. Also, by analyzing the correlation between the amount of microorganisms and time data in advance according to the combination of the medium and the microorganism, the judgment process can be performed more accurately and efficiently based on the analysis results.

[0049] For the measurement results, an approximation curve may be obtained 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 obtained from the approximation curve. In addition, a noise removal process such as a moving average method or a low-pass filter may be performed. EXAMPLES

[0050] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

[0051] [Example 1] <About electrode sensors> 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, 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 surface of the substrate was 12 μm when measured with a micrometer (manufactured by Mitutoyo Corporation).

[0052] Next, a resin layer was formed as an insulator 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 in a comb shape, and then heating and curing it at 150°C for 1 hour in a nitrogen atmosphere. The thickness of the resin layer was 20 μm.

[0053] Next, a conductive layer that would become a cathode electrode body was formed so as to be laminated 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 as a comb-shaped conductive material so as to overlap the resin layer, and then heated and cured at 130°C for 1 hour in a nitrogen atmosphere to form the conductive layer. The thickness of the conductive layer was 20 μm.

[0054] 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 thin metal film of silver is formed, with an insulator made of a resin layer interposed between them, and in a circular area of ​​35 mm radius in the central part of the cathode electrode body, 1 mm wide notches are arranged in a comb shape at intervals of 1 mm, where no resin layer is formed, and the thin metal film of silver is exposed.

[0055] <About the judgment device> The determination device was assembled and used as shown in FIG. 1. In a sterile chamber, an electrode sensor was placed on the upper surface of a resin bottom plate with the cathode electrode body facing up, and a glass cylinder (inner diameter 42 mm, height 32 mm) was placed on the cathode electrode body via a silicon 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 fixed with fasteners consisting of bolts and nuts so that each part was in close contact with each other, and the storage container was assembled. A silicon plug was inserted into the input port opened in the center of the cover plate to removably seal it. Each part of the assembled storage container was autoclaved at 121°C for 15 minutes and placed in a thermostatic bath at 37°C.

[0056] One end of a conductive wire was connected to the anode electrode body and the cathode electrode body of the electrode sensor at the portion exposed outside 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. The zero-resistance ammeter was connected to an information processing device consisting of a personal computer and set 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.

[0057] <About the medium> The medium used had the following composition: Tryptone; 10.0 g (BD Biomedicals) Yeast Extract: 5.0g (BD Biosciences) ·NaCl; 5.0g (Wako) The medium was mixed with 1,000 ml of distilled water and sterilized with high-pressure steam at 121° C. for 15 minutes using an autoclave.

[0058] <Preparation of microorganisms> The medium was prepared using commercially available Escherichia coli (E. coli DH5α) as the microorganism to be added. 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 any 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 using a spectrophotometer, and the bacterial count at OD = 0.1 was 10 7 The LB culture medium was diluted to the desired number of bacteria, based on the standard of cfu / milliliter. The number of live bacteria was 1, 10, and 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 Ten samples of cfu / ml were prepared.

[0059] <Measurement method> After dropping 10 milliliters 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 37°C and measured continuously for 1000 minutes. The measurement results for each sample are shown in Figure 4.

[0060] <Determination method> In Figure 4, the vertical axis represents current value (nA) and the horizontal axis represents time (min). The time when the current value starts to rise and the time when 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 the current value.

[0061] 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 value 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 value was analyzed. For the time when the current value changes the most, the difference between the sampled current values ​​during the target period was calculated, and the time data showing the maximum value was used.

[0062] 5A and 5B are graphs showing the time from the start of contact with the medium until the current value reaches a predetermined value. The predetermined current value is set to 10, 20, 30, 50, 100, 200, 300 nA and the maximum current value, respectively, with the vertical axis showing the time (minutes) until each predetermined value is reached and the horizontal axis showing the number of viable bacteria (cfu / milliliter). In FIG. 6A and FIG. 6B, it is clearly shown that there is a positive correlation between the number of viable bacteria and the time data until the predetermined current value is reached. Furthermore, the time data until the maximum current value is reached also shows a positive correlation, indicating that during the period until the current value increases and reaches the maximum value, it is possible to determine the number of viable bacteria based on the time data until the predetermined current value is reached.

[0063] Therefore, by using a calibration curve showing such a correlation according to the combination of the type of microorganism and the medium as a standard, it is possible to efficiently determine the amount of microorganisms in a short period of time.

[0064] Fig. 6 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. 6 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.

[0065] FIG. 7 is a graph showing the relationship between the number of viable bacteria and the maximum current value. 7Below cfu / ml, the change in maximum current value relative to the change in the 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.

[0066] When the strength of this correlation is examined using a correlation coefficient, the common logarithm of the viable bacteria count and the time data both show a strong positive correlation of 0.95 or more, while the correlation with the maximum current value shows a positive correlation of about 0.7. Therefore, in the determination process, time data that has a correlation with the viable bacteria count is obtained, and the viable bacteria count is determined based on the obtained time data, thereby making it possible to efficiently obtain more accurate determination results.

[0067] <Example 2> Four types of electrode sensors, in which the thickness of the silver metal thin film formed on the surface of the substrate as the anode electrode body was 5, 8, 10, and 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 and no metal thin film.

[0068] Using the same medium and microorganisms as in Example 1, 8 A sample of cfu / milliliter was prepared, and the current value was measured using the same determination device as in Example 1. From the obtained measurement results, looking at the relationship between the silver film thickness and the maximum current value, the maximum current value was 400 nA when the film thickness was 5 μm and 8 μm, while it was 610 nA when the film thickness was 10 μm, 650 nA when the film thickness was 12 μm in Example 1, 650 nA when the film thickness was 15 μm, and 650 nA when a silver plate was used.

[0069] 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.

[0070] Therefore, by using a calibration curve showing such a correlation according to the combination of the type of microorganism and the medium as a standard, it is possible to efficiently determine the amount of microorganisms in a short period of time.

[0071] <Comparative Example 1> The determination device was equipped with an electrode sensor manufactured in the same manner as in Example 1. The medium and microorganisms were the same as those in Example 1. 7 Samples were prepared with a concentration of cfu / milliliter, and 70 v / v % ethanol was added to the samples before use.

[0072] When the current value was measured in the same manner as in Example 1, no increase in the current value was observed during the measurement period, as shown in Fig. 4. Since all E. coli was killed by adding ethanol to the sample, there was no increase in the current value, and it was confirmed that the increase in the current value was due to surviving microorganisms. [Explanation of symbols]

[0073] Reference Signs List 1... microbial quantity determination device, 2... storage container, 10... electrode sensor, 10a... anode electrode body, 10b... insulator, 10c... cathode electrode body, 11... culture medium, 12... measurement section, 13... analysis section, 14... cover plate, 15... bottom plate, 16... cylinder, 17... stopper

Claims

1. A microbial quantity determination device comprising: an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator; a culture medium in which microorganisms can be cultured; 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.

2. The microbial amount determining device according to claim 1 , wherein the analysis unit determines the microbial amount based on time data from the start of contact with the culture medium to the time when the current value increases and reaches a maximum value.

3. The microbial amount determining device according to claim 1 , wherein the analysis unit determines the microbial amount based on time data from the start of contact with the medium to the point at which the current value changes the most during the period from when the current value increases to when it reaches its maximum value.

4. 2. The microbial quantity determining device according to claim 1, wherein the electrode sensor has a metal thin film formed on the anode electrode body having a thickness of 10 [mu]m or more.

5. A method for determining the amount of microorganisms, which uses an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, contacts at least both electrode bodies with a culture medium in which microorganisms can be cultured, measures the galvanic current between the two electrode bodies, and determines the amount of microorganisms based on time data corresponding to the change in the measured current value.

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