Microorganism discriminating apparatus and method

Abstract

To provide an apparatus and a method for discriminating microorganisms capable of simply discriminating the microorganisms living in a plurality of kinds of culture media in a short time by using an electrode sensor.SOLUTION: The microbe identification device 1 includes a plurality of types of media 13a to 13d in which microbes can be cultured, a plurality of electrodes sensors 11a to 11d in which anode electrodes and cathode electrodes are integrally formed via insulators, a measuring unit 20 that measures, for each medium, a galvanic current flowing between the electrodes in a state in which at least the electrodes are in contact with each medium, and an analyzing unit 30 that stores current values measured by the measuring unit 20 and identifies microbes based on a change in the current values for each medium.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a microorganism discrimination device and method that can quantitatively discriminate the types of living microorganisms such as bacteria using an electrode sensor. [Background technology]

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

[0003] Various methods have been proposed for tracking microbial growth, including optical methods that use light transmitted through a sample containing microorganisms, electrochemical methods that measure thermal and electrical changes associated with the metabolic activity of microorganisms, and methods that measure changes in dissolved oxygen concentration due to microbial respiration.However, due to limitations on the amount of microorganisms that can be measured, the most common method is the culture method, in which microorganisms are cultured on agar in a petri dish and the amount of microorganisms is examined.However, these culture methods have issues such as the need to consume large amounts of materials such as petri dishes and agar, and the long culture time (1-2 days).

[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 1. 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] Patent Document 2 proposes using such an electrode sensor to measure the amount of microorganisms themselves by measuring the current flowing between the anode and cathode electrodes, 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] Japanese Patent Application Laid-Open No. 2016-10403 Summary of the Invention [Problem to be solved by the invention]

[0007] The inventors have focused on the metabolites excreted by living microorganisms and have made it possible to detect microorganisms in a culture medium using an electrode sensor, but there is room for further study regarding quantitatively determining the type and amount of microorganisms.

[0008] That is, when identifying the species of living microorganisms cultured in a medium, conventional methods have been to visually inspect the cultured colonies or to use genetic or biochemical methods, but this has the drawback of being time-consuming and expensive. Furthermore, when quantitatively identifying microorganisms, the number of microbial colonies produced by culture is generally used as an index, but the culture requires time, which has the drawback of being time-consuming and expensive.

[0009] SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a microorganism discrimination device and method that can easily and quickly discriminate between microorganisms living in multiple types of culture media using an electrode sensor. [Means for solving the problem]

[0010] The microorganism discrimination device according to the present invention comprises a plurality of types of culture media capable of cultivating microorganisms, a plurality of electrode sensors each having an anode electrode body and a cathode electrode body integrally formed via an insulator, a measurement unit that measures, for each culture medium, the galvanic current flowing between at least both electrode bodies of the electrode sensor while the electrode body is in contact with the culture medium, and an analysis unit that stores the current value measured by the measurement unit and discriminates microorganisms based on changes in the current value for each culture medium.

[0011] The microorganism discrimination method of the present invention is a method for discriminating microorganisms in a culture medium using an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, in which at least both electrode bodies of the electrode sensor are brought into contact with each of a plurality of types of culture medium in which microorganisms can be cultured, the galvanic current between the two electrode bodies is measured for each culture medium while the microorganisms are cultured in the culture medium, and the microorganisms are discriminated based on the change in the measured current value for each culture medium. [Effects of the Invention]

[0012] According to the present invention, the electrode sensor is placed in contact with each of a plurality of types of culture medium, the current value generated in the electrode sensor is measured, and the microorganisms are identified based on the change in the current value for each culture medium, thereby enabling the type and quantity of microorganisms in the culture medium to be accurately determined in a short period of time. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a schematic diagram illustrating the configuration of a microorganism discrimination device according to the present invention. [Figure 2] 2A and 2B are a plan view and a partially enlarged cross-sectional view of an electrode sensor. [Figure 3] 10 is a graph showing an example of a response test. [Figure 4A] 10 is a graph showing the transition of the current value of the culture medium alone and the transition of the current value of the liquid in which Escherichia coli is suspended for each culture medium. [Figure 4B] 10 is a graph showing the transition of the current value of the culture medium alone and the transition of the current value of the liquid in which Escherichia coli is suspended for each culture medium. [Figure 5]10 is a table showing whether or not there was a change in current value for each microorganism test result. [Figure 6] 10 is a graph showing the change in current value for each suspension using LB culture medium. [Figure 7] 1 is a graph showing the change in current value when Escherichia coli is suspended at a concentration of 10 6 cfu / milliliter in a diluted solution obtained by diluting an LB culture solution. [Figure 8] 1 is a graph showing the adsorption of hydrogen sulfide in saline, LB culture medium, and diluent. [Figure 9] 1 is a graph showing the change in current value when Escherichia coli is suspended in a 20% diluted solution of LB culture medium at a concentration of 10 6 cfu / milliliter. [Figure 10] 10 is a graph showing changes in current value in the case of a suspension of E. coli and river water. [Figure 11] 1 is a graph showing a calibration curve representing the relationship between 100 nA arrival time and E. coli concentration. [Figure 12] 10 is a graph showing changes in current value when a response test is performed on a suspension of Escherichia coli in a sterile environment and outside the sterile environment. DETAILED DESCRIPTION OF THE INVENTION

[0014] 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.

[0015] 1 is a schematic diagram of a microorganism discrimination device according to an embodiment of the present invention. The microorganism discrimination device 1 is equipped with a plurality of measurement units 10, each of which has an electrode sensor 11 formed by integrating an anode electrode body and a cathode electrode body via an insulator, and which is placed in a ventilated and closed container 12; in this example, the device is equipped with four measurement units 10a to 10d. The number of measurement units can be adjusted appropriately depending on the type of microorganism to be measured.

[0016] Culture media 13a-13d capable of cultivating microorganisms are placed in the measurement units 10a-10d, respectively, and the measurement unit 20 measures the galvanic current flowing between the electrodes of the electrode sensors 11a-11d while bringing them into contact with the culture media 13a-13d, respectively, for each culture medium. The analysis unit 30 stores the current values ​​measured by the measurement unit 20 and identifies the microorganisms based on changes in the current value for each culture medium. The display unit 40 displays the analysis results of the analysis unit 30.

[0017] 2A and 2B are a plan view and a partially enlarged cross-sectional view, respectively, of the electrode sensor 11. In FIG. 2B, a cross section in the left-right direction is shown in the area enclosed by a circle.

[0018] The electrode sensor 11 has a structure similar to that of a known ACM sensor, with an insulator 111 layered on the upper surface of a rectangular anode electrode body 110, and a cathode electrode body 112 layered on the upper surface of the insulator 111. A comb-shaped electrode portion in which a plurality of narrow notches are arranged in parallel in a comb shape is formed in the central portion of the insulator 111 and the cathode electrode body 112, and the surface of the anode electrode body 110 is exposed in the notches of the comb-shaped electrode portion. When the culture medium 13 is brought into contact with both electrode bodies of the electrode sensor 11, a galvanic current flows between the anode electrode body 110 and the cathode electrode body 112 exposed in the notches.

[0019] A galvanic current is generated between the two electrode bodies because an oxidation reaction occurs on the electrode surface of the anode electrode body 110 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 112, 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 112 has a more noble potential than the electrode surface of the anode electrode body 110 under environmental conditions in which an electrochemical reaction occurs.

[0020] In the electrode sensor 11, the two electrode bodies are integrally formed in the interdigital electrode portion so as to be opposed to each other with an insulator interposed therebetween, and a galvanic current is generated by causing an electrochemical reaction at the contact points of 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 between the two electrode bodies opposed to each other with the culture medium interposed therebetween, and it is known that the detection sensitivity increases as the distance between the boundary areas becomes narrower.

[0021] 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.

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

[0023] The insulator 111 is made of a non-conductive insulating material that electrically insulates the anode electrode body 110 and the cathode electrode body 112. 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.

[0024] A metal thin film is preferably formed on the electrode surfaces of the anode electrode body 110 and the cathode electrode body 112. 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, it is preferable that the surfaces of the anode electrode body and the cathode electrode body have a low resistance value, specifically, a resistance value of 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 for the anode electrode body 110, the metal used for the cathode electrode body 112 may be a metal with a composition that results in a more noble potential than silver. Furthermore, when silver is used for the anode electrode body 110, 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 more likely to occur over long periods of use.

[0026] The anode electrode body 110 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 by sintering a conductive paste. Wet film formation processes include electroplating, electroless plating, and composite plating, while dry film formation processes include vapor deposition and sputtering.

[0027] The cathode electrode body 112 has a conductive material containing a metal material at least on the electrode surface, and when a plate-shaped body is used as the anode electrode body 110 serving as the substrate, the cathode electrode body 112 is preferably formed by a printing method using a conductive paste, as shown in Fig. 2. Forming the cathode electrode body by such a printing method allows the layer to be formed thick, at 5 to 100 µm, which reduces the electrical resistance and enables the current generated between the two electrode bodies to be measured accurately and stably.

[0028] 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 letterpress 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 are also possible and are not particularly limited.

[0029] In the electrode sensor 11, a galvanic current flows due to electrochemical reactions that occur between the anode electrode body 110 and the cathode electrode body 112 due to trace components contained in the culture medium 13 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 electrodes.

[0030] (Culture medium) H2S → HS - +H + (cathode electrode body) O2+2H2O+4e - →4OH - (anode electrode body) 2Ag+H2S → Ag2S+2H + +2e - 2Ag+HS - →Ag2S+H + +2e - 2Ag+S 2- →Ag2S+2e -

[0031] In the above reaction, a change in the concentration of hydrogen sulfide causes a change in the current. Furthermore, since an increase or decrease in the galvanic current value occurs only when microorganisms are alive in the culture medium, it is possible to determine whether the microorganisms in the culture medium are alive or dead by observing the change in the galvanic current.

[0032] Furthermore, when measuring metabolites produced by microorganisms, the environmental conditions remain the same and no reagents are used, so it is possible to measure in their natural state without damaging the microorganisms, and the state of the microorganisms can be understood in real time.

[0033] It is preferable to use a plurality of types of culture medium 11 that can culture microorganisms, and to use a combination of media that can culture specific microorganisms in order to distinguish the type of microorganism.

[0034] As for the relationship between a culture medium and a specific microorganism, as described below, a response test using an electrode sensor is performed for a combination of a culture medium and a microorganism, and reference data can be obtained in advance based on the obtained response characteristics.

[0035] Examples of reference data include data on the presence or absence of culture, data on changes in current value during culture, and data that serves as an index for changes in current value.

[0036] As a combination of culture media, it is advisable to combine media with different reference data, such as media capable of culturing a specific microorganism and media capable of culturing microorganisms other than the specific microorganism. By combining media with different characteristics, it becomes possible to identify microorganisms even in samples containing any microorganism.

[0037] Furthermore, in the case of a medium capable of cultivating only specific microorganisms, if one type of medium is used, the microorganisms can be identified by their response characteristics based on changes in current value. In the case of a medium capable of cultivating multiple types of microorganisms, if the culture process for each microorganism is different, it is possible to identify each microorganism based on the difference in response characteristics. For example, if multiple types of microorganisms have different timings for starting metabolism, the difference in timing will be reflected in changes in current value, and the microorganisms can be identified based on the response characteristics of each microorganism.

[0038] Furthermore, hydrogen sulfide, which is generated as a metabolite of the above-mentioned microorganisms, is adsorbed to proteins and peptides contained in the culture medium, and the reduction in hydrogen sulfide is thought to affect the measurement results. Therefore, it is preferable to use a culture medium for discrimination, which has been diluted as necessary to reduce the concentration of proteins and peptides based on the results of the response test.

[0039] Regarding the culture medium, multiple types of culture medium can be selected according to the microorganism to be identified based on reference data for the microorganism, and a culture kit for identifying a specific microorganism can be created by, for example, using a ventilated, closed container, dividing the interior into multiple spaces, and sealing each culture medium in each compartment.The culture kit can efficiently perform the identification process by directly introducing culture medium prepared in advance for microbial identification into the measurement unit.

[0040] As the medium, known media such as liquid media, solid media, gel media, and membrane media can be used.

[0041] Bacterial media include broth, broth gelatin, nitrate, milk, cornmeal, MR-VP, tomato juice, TSI, SIM, glucose-broth, and YM.

[0042] 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.

[0043] 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.

[0044] In addition to the media mentioned above, other media used for research purposes include complete bacterial medium, 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.

[0045] The microorganisms to be identified may be any type of living microorganism that produces metabolites, including bacteria, actinomycetes, fungi, etc. For example, it is also possible to identify bacteria that cause food poisoning or infectious diseases.

[0046] Specifically, Staphylococcus genus (e.g., Staphylococcus aureus, including 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.), Shigella genus (e.g., Vibrio cholerae, Vibrio parahaemolyticus, etc.), Examples include bacteria of the genus Shigella (e.g., Shigella sonnei, Shigella dysenteriae, Shigella flexneri, Shigella boydii, etc.), genus Clostridium (e.g., Clostridium botulinum, etc.), genus Listeria (e.g., Listeria monocytogenes, etc.), genus Mycobacterium (e.g., Mycobacterium tuberculosis, etc.), genus Cryptosporidium (e.g., Cryptosporidium parvum, etc.), genus Coxiella (e.g., Coxiella burnetii, etc.), genus Francisella (e.g., Francisella tularensis, etc.), genus Bacillus (e.g., Bacillus cereus, Bacillus anthracis, etc.), genus Yersinia (e.g., Yersinia pestis, etc.), etc.

[0047] In particular, preferred target microorganisms for the purpose of food hygiene inspection include methicillin-resistant Staphylococcus aureus (MRSA), Campylobacter (e.g., Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Campylobacter sputum, etc.), Salmonella (e.g., Salmonella typhi, Salmonella paratyphi A, Salmonella enteritidis, Salmonella typhimurium, Salmonella cholera suis, Salmonella dublin, etc.), enterohemorrhagic Escherichia coli (e.g., O111, O157, etc.), Vibrio (e.g., Vibrio cholerae, Vibrio parahaemolyticus, etc.), Legionella (e.g., Legionella pneumophila, etc.), Bacillus cereus (e.g., Bacillus cereus), etc.

[0048] The measurement unit 20 uses a resistanceless ammeter connected between the anode electrode assembly 110 and the cathode electrode assembly 112 of each measurement unit to constantly measure the galvanic current generated between the anode electrode assembly 110 and the cathode electrode assembly 112. The measurement current value can then be output to the analysis unit 30. The measurable current range is preferably set to 0.1 nA to 1000 nA.

[0049] The measuring section 20 has a function of measuring on multiple channels, and is configured to simultaneously measure current changes in multiple measurement units and output the results to the analyzing section 30.

[0050] The analysis unit 30 stores reference data for multiple types of culture media, and performs a storage process for storing the current value output from the measurement unit 20 at predetermined time intervals, and a discrimination process for discriminating microorganisms based on the stored reference data and changes in the measured current value. For example, software for performing the storage process and discrimination process can be installed in an information processing device such as a personal computer, thereby realizing such processes by the analysis unit 30.

[0051] The storage process is preferably performed at short time intervals to measure minute current changes, 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.

[0052] In the analysis process, the change in the measured current value of each culture medium is analyzed, and the type of microorganism can be determined based on a combination of the reference data of each culture medium and the change in the current value.

[0053] For example, the probability of the presence of a specific microorganism increases based on the presence or absence of a change in the current value of a culture medium in which the specific microorganism can be cultured, and accurate identification becomes possible by combining this with the presence or absence of a change in the current value of another culture medium. Furthermore, the presence of a specific microorganism can also be identified by analyzing the correlation between the measured change in current value and reference data based on the current value measured when the specific microorganism is cultured in the same culture medium.

[0054] FIG. 3 is a graph showing an example of a response test. In this example, E. coli solution (LB culture medium, 10 5 The graph shows the temporal changes in the sensor output current Igal (nA) and OD600 in the measurement unit during a response test using 1000 cfu / ml. The OD600 of the E. coli solution increased rapidly after a 60-minute induction period. The rate of increase then decreased around 240 minutes, but the increase gradually increased. Based on the changes in OD600, the growth rate of E. coli was in the logarithmic growth phase from 60 to 240 minutes, and then in the stationary phase from 400 minutes onward. The sensor output current Igal was below 10 nA until 140 minutes after the start of the test, began to increase at approximately 160 minutes, reached a maximum at 390 minutes, and then decreased. The sensor output current Igal began to increase approximately 100 minutes after the OD600 began to increase. It can be seen that the sensor output current Igal began to increase during the logarithmic growth phase of E. coli and reached a maximum during the deceleration phase before transitioning to the stationary phase.

[0055] Thus, 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, and therefore the time at which the current value reaches a predetermined value correlates with the amount of microorganisms. Furthermore, since the time at which the rate at which the current value increases is at its maximum also correlates 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, and therefore the process of determining microorganisms can be performed efficiently. [Example]

[0056] The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

[0057] <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. 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. The thickness of the silver metal thin film formed on the surface of the substrate was measured with a micrometer (manufactured by Mitutoyo Corporation) and was found to be 12 μm.

[0058] 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 printing a resin paste (manufactured by Henkel Ablestick Japan Co., Ltd.) made of epoxy resin material in a comb-like pattern and then heating and curing it at 150°C for 1 hour in a nitrogen atmosphere. The resin layer was 20 μm thick.

[0059] Next, a conductive layer that would become the 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 in a comb shape 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.

[0060] The resulting electrode sensor has a cathode electrode body made of a conductive layer 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 sandwiched between them.In a circular area with a radius of 35 mm in the center of the cathode electrode body, 1 mm wide notches are arranged in a comb-like pattern at 1 mm intervals, and no resin layer is formed in the notched areas, resulting in an electrode structure in which the silver metal thin film is exposed.

[0061] <About the discrimination device> The discrimination device used was assembled as a measurement unit with the configuration shown in Figure 1. The electrode sensor was placed on the top surface of a resin bottom plate, with the cathode electrode assembly facing up, and a glass cylinder 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 tightly attached, thereby assembling the storage container. A breathable silicone stopper was inserted into the opening in the center of the cover plate to removably seal it. 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 during measurement.

[0062] One end of a conductive wire was connected to the exposed portion of the anode electrode body and the cathode electrode body of the electrode sensor of each measurement unit 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 discriminating the stored measurement results were installed in the information processing device in advance.

[0063] <About the culture medium> The medium used had the following composition: (LB culture solution) The LB culture medium was prepared by suspending 10.0 g of tryptone, 5.0 g of yeast extract, and 5.0 g of NaCl in 1 L of purified water and dissolving the suspension under stirring. The suspension was then sterilized with high-pressure steam at 121°C for 15 minutes using an autoclave.

[0064] (YPD culture solution) YPD culture medium was prepared by suspending 10.0 g of yeast extract, 20.0 g of peptone, and 20.0 g of dextrose in 1 L of purified water, boiling the mixture for 1 minute while stirring, and then sterilizing the mixture with high-pressure steam at 121°C for 15 minutes in an autoclave.

[0065] (Baird-Parker culture medium) Baird-Parker broth was prepared by suspending 10.0 g of casein pancreatic digest, 1.0 g of yeast extract, 5.0 g of meat extract, 10.0 g of sodium pyruvate, 12.0 g of L-glycine, and 5.0 g of lithium chloride in 1 L of purified water and dissolving the suspension with stirring. The suspension was then sterilized by high-pressure steam at 121°C for 15 minutes using an autoclave. Next, 1.0 ml of 1.0% potassium tellurite solution was added and mixed, followed by the addition of 2.0 ml of 50% egg yolk solution.

[0066] (Desoxycholate broth) Desoxycholate culture medium was prepared by suspending 10.0 g of peptone, 10.0 g of teatella, 1.0 g of sodium desoxycholate, 5.0 g of sodium chloride, 2.0 g of dipotassium hydrogen phosphate, 2.0 g of ammonium ferric citrate, and 0.033 g of neutral red in 1 liter of purified water and dissolving the suspension by heating with stirring.

[0067] <Preparation of microorganisms> The following microorganisms were used: (E. coli) This was prepared using commercially available E. coli (E. coli DH5α). 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 each culture medium to remove any metabolites that had been generated, and then resuspended in each culture medium. The bacterial count in 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 Each culture medium was diluted and adjusted to a predetermined number of bacteria, based on the standard of equivalent to cfu / milliliter.

[0068] (yeast) The yeast (Fukui Prefecture-developed yeast FK-501) was used and prepared in the same manner as for E. coli. (Staphylococcus aureus) The obtained Staphylococcus aureus was used for preparation in the same manner as for Escherichia coli.

[0069] <About response testing> The measurement unit was placed in a thermostatic chamber at 37°C, and the bacterial solution could be poured in through an opening at the top of the container. During the test, the opening was sealed with a culture stopper or similar to allow ventilation. After dropping 10 ml of bacterial solution prepared to a specified concentration onto the top surface of the electrode sensor, the galvanic current flowing between the anode and cathode electrodes was continuously measured at 1-minute intervals using an ammeter.

[0070] <Measuring hydrogen sulfide concentration> In the response test, the concentration of hydrogen sulfide in the culture medium was measured by quantitative analysis using an ion chromatograph (manufactured by Thermo Fisher Scientific Co., Ltd.; ICS-3000 electrochemical detector used) and a calibration curve method.

[0071] [Example 1] A suspension of Escherichia coli, yeast, or Staphylococcus aureus was prepared in each culture medium, and a response test was performed for each microorganism. The concentration of the microorganism used in the test was 10 5 cfu / ml or 10 6The culture solution or suspension was then placed in a measuring unit and tested. The test results are shown in Figures 4A and 4B.

[0072] Figures 4A and 4B are graphs showing the change in current value for each culture medium alone and for a suspension of E. coli, with the vertical axis representing current and the horizontal axis representing time. Figure 4A(a) shows the case of LB culture medium, and Figure 4A(b) shows the case of YPD culture medium. Figure 4B(a) shows the case of Baird-Parker culture medium, and Figure 4B(b) shows the case of desoxycholate culture medium.

[0073] Furthermore, response tests were performed on yeast and Staphylococcus aureus using the prepared suspensions in the same manner as for E. coli. The test results are shown in a table in Figure 5.

[0074] Figure 5 shows the test results for each microorganism, showing whether or not there was a change in the current value. A circle in the table indicates that there was a difference in the change in the current value compared to when only the culture medium was used, and an cross in the table indicates that there was no difference in the change in the current value compared to when only the culture medium was used.

[0075] With E. coli, an increase in current was observed in the LB culture medium, but no such increase in current was observed in the YPD culture medium or the Baird-Parker culture medium. However, an increase in current was observed in the desoxycholate culture medium, similar to that observed in the LB culture medium.

[0076] For yeast, an increase in current value was observed only in the case of YPD culture medium, but not in the case of other culture mediums. For Staphylococcus aureus, an increase in current value was observed in the case of LB culture medium and Baird-Parker culture medium, but not in the case of other culture mediums.

[0077] The increase in current value is thought to be due to the electrode sensor detecting the small amount of hydrogen sulfide released into the suspension as the microorganisms grow. Therefore, a medium in which the current value increases in the response test is a medium in which the microorganism can be cultivated, and a medium in which no increase in current value is observed is a medium in which the microorganism cannot be cultivated. As shown in Figure 5, reference data can be obtained to indicate whether or not the combination of microorganism and medium can be cultivated.

[0078] Based on this reference data, when microorganisms are cultured in the discrimination device shown in Figure 1 using four types of culture media, namely LB culture medium, YPD culture medium, Baird-Parker culture medium, and desoxycholate culture medium, each culture medium has a different microbial response characteristic, and therefore it becomes possible to discriminate the type of microorganism from the combination of changes in the current value of each culture medium.

[0079] [Example 2] LB culture medium and YPD culture medium were used to culture 10 E. coli and 10 yeast. 6 A mixed suspension was prepared at a concentration of cfu / ml. The mixed suspension was subjected to a response test.

[0080] In the case of a suspension containing a mixture of E. coli and yeast, a change in current value was confirmed in both the LB culture medium and the YPD culture medium. The test results for the LB culture medium are shown in Figure 6.

[0081] Figure 6 shows the change in current value for a suspension of only E. coli, a suspension of only yeast, and a suspension of a mixture of E. coli and yeast, using LB culture medium, with the vertical axis representing current value and the horizontal axis representing time.

[0082] As shown in Figure 5, in the case of LB culture medium, a change in current value was observed in E. coli but not in yeast. Conversely, in the case of YPD culture medium, a change in current value was observed in yeast but not in E. coli. However, as shown in Figure 6, when the two cultures were mixed, a change in current value was observed in both cultures.

[0083] Therefore, it is clear that by combining media capable of culturing different microorganisms, it is possible to distinguish the type of any microorganism.

[0084] [Example 3] Using diluted LB broth, E. coli was cultured at 10 6 A suspension containing the E. coli was prepared at a concentration of 1000 cfu / ml and a response test was performed. The LB culture medium was diluted with 0.5% saline, and the E. coli suspension was prepared using dilutions of LB culture medium at concentrations of 50%, 60%, 70%, 80%, 90%, and 100%. The test results are shown in Figure 7.

[0085] In Figure 7, even when the amount of microorganisms is constant, the maximum current value decreases as the concentration of the culture medium decreases, but in the period until the maximum value is reached, the current value starts to rise at the same time and increases at almost the same rate. This shows that it is possible to determine the amount of microorganisms by using an index corresponding to the change in current value during the period from the start of measurement until the current value increases and reaches its maximum value.

[0086] [Example 4] A quantitative analysis was conducted on the hydrogen sulfide adsorption characteristics of the culture medium. LB culture medium, a diluted solution of LB culture medium diluted with 0.5% saline to a concentration of 20%, and 0.5% saline were prepared as samples. Commercially available sodium sulfide was added to each sample to a concentration of 15.6 μmol / L. The added sodium sulfide hydrolyzes to generate hydrogen sulfide.

[0087] Then, 0.5 ml samples were taken immediately after the addition, and 0.5 and 20 minutes later, and quantitative analysis was performed using ion chromatography to measure the hydrogen sulfide concentration. The measurement results are shown in Figure 8. In Figure 8, the concentrations of hydrogen sulfide in 0.5% saline, LB culture medium, and the 20% diluted solution are shown in bar graphs.

[0088] It was confirmed that the concentration of hydrogen sulfide in the sample decreased slightly after addition of 0.5% saline. In the case of LB culture medium, the concentration of hydrogen sulfide decreased significantly compared to 0.5% saline, decreasing to about half immediately after addition and reaching nearly zero after 20 minutes. In the case of the 20% diluted solution, the decrease in hydrogen sulfide concentration was more gradual, decreasing to about three-quarters immediately after addition and to about half after 20 minutes.

[0089] The decrease in hydrogen sulfide concentration in 0.5% saline solution is thought to be due to adsorption to glassware and oxidation by dissolved oxygen, while the decrease in hydrogen sulfide concentration in LB culture medium is thought to be due to adsorption to proteins and peptides in the LB culture medium as well.

[0090] Therefore, when identifying microorganisms based on hydrogen sulfide in the culture medium, the concentration of the culture medium affects the response characteristics, so it is preferable to use a culture medium with an optimal concentration based on the magnitude of the current value output from the electrode sensor.

[0091] [Example 5] A response test similar to that in Example 3 was carried out on a 20% diluted solution of LB culture medium. The test results are shown in Figure 9. For comparison, Figure 9 graphs the changes in current value for the 50% diluted solution of Example 3 and the LB culture medium, along with the change for the 20% diluted solution.

[0092] In the case of the 20% diluted solution, the timing of the increase in the current value was almost the same, but because the effect of hydrogen sulfide adsorption was small, the rate of increase in the current value was faster than in the 50% diluted solution and LB culture medium, and it was confirmed that the time it took for the current value to reach 100 nA was approximately 20 minutes earlier.

[0093] Therefore, when the culture medium concentration is set low to reduce the effect of hydrogen sulfide adsorption, the maximum current value decreases, but the amount of microorganisms can be determined based on the time data until the current value reaches a predetermined value during the period from the start of measurement until the current value increases and reaches its maximum value, thereby enabling accurate determination processing.

[0094] [Example 6] The microorganisms contained in unknown samples were identified based on the response characteristics of the culture medium using different concentrations of microorganisms. 2 cfu / ml, 10 3 cfu / ml and 10 4 A suspension was prepared by mixing the bacteria at a concentration of 1000 cfu / milliliter. The same LB medium was also used to prepare a sample solution by mixing it at a 1:1 ratio with a sample of water taken directly from a river (located in Kawai Washizuka-cho, Fukui City, in the Kitagawa River, a Class A river in the Kuzuryu River system, Fukui Prefecture). Response tests were performed on the prepared suspension and sample solution. The test results are shown in Figure 10.

[0095] In FIG. 10, a change accompanied by an increase in the current value was confirmed in both the suspension and the sample solution, indicating the possibility that E. coli was present in the sample solution.

[0096] Figure 11 shows a calibration curve that shows the relationship between the time when the current value reached 100 nA and the concentration of E. coli during the period from the start of measurement until the current value increased and reached its maximum value. In Figure 11, the vertical axis represents the time when the current value reached 100 nA, and the horizontal axis represents the concentration on a logarithmic scale.

[0097] The concentration was calculated from the sample liquid arrival time using the calibration curve, and was found to be 2.0 x 10 3 The sample solutions were mixed at a 1:1 ratio, so the concentration in the river water was doubled to 4.0 x 10 3 Calculated as cfu / milliliter.

[0098] When the river water was examined for E. coli using a petri dish culture method, 3.3 × 10 3 cfu / milliliter, which was confirmed to be in close agreement with the test results.

[0099] Therefore, the type and amount of microorganisms contained in an unknown sample can be determined using a calibration curve, which is the response characteristic of LB culture medium to E. coli. By using the data from such a calibration curve as reference data, microorganisms can be identified based on the measurement results and the reference data.

[0100] In this example, response tests for E. coli suspensions and sample solutions with different concentrations can be simultaneously measured and analyzed using the discrimination device. Alternatively, a response test for the suspension can be performed in advance to obtain reference data, and analysis can be performed based on the measurement results of the sample solution and the reference data.

[0101] [Example 7] LB medium was used to incubate 10 E. coli cells. 5 A suspension containing the mixture was prepared at a concentration of cfu / milliliter. The prepared suspension was placed in each of two measurement units, one of which was placed inside the sterile room and the other outside the sterile room, and a response test was performed. The test results are shown in Figure 12.

[0102] In Figure 12, when the response test was conducted in a sterile environment, the current value increased as E. coli grew from approximately 200 minutes after the start of measurement, reached a maximum current value at approximately 400 minutes, and then decreased. On the other hand, when the response test was conducted outside of a sterile environment, the current value increased until it reached a maximum value, just as in the sterile environment, and then decreased, but an increase in the current value was confirmed again from approximately 600 minutes. This is thought to be because, outside of a sterile environment, it is possible that airborne bacteria were mixed in during the response test, and the current value increased as the airborne bacteria grew.

[0103] Therefore, when multiple types of microorganisms are cultured in one type of culture medium and the timing of their growth differs, the timing of hydrogen sulfide metabolism associated with the growth of each microorganism is clearly reflected in changes in the current value, making it possible to distinguish between multiple types of microorganisms even in one type of culture medium. [Explanation of symbols]

[0104] 1... Microorganism discrimination device, 10a to 10d... Measurement unit, 11a to 11d... Electrode sensor, 12a to 12d... Storage container, 13a to 13d... Culture medium, 20... Measurement section, 30... Analysis section, 40... Display section, 110... Anode electrode body, 111... Insulator, 112... Cathode electrode body

Claims

1. A microorganism discrimination device comprising: a plurality of types of culture media capable of cultivating microorganisms; a plurality of electrode sensors each having an anode electrode body and a cathode electrode body integrally formed with an insulator interposed therebetween; a measurement unit that measures, for each culture medium, the galvanic current flowing between at least both electrode bodies of the electrode sensor while the electrode bodies are in contact with the culture medium; and an analysis unit that stores the current values ​​measured by the measurement unit and discriminates microorganisms based on changes in the current value for each culture medium.

2. 2. The microorganism discrimination device according to claim 1, wherein the analysis unit discriminates microorganisms based on reference data based on current values ​​measured by culturing specific microorganisms in advance in at least one type of culture medium and on changes in current values ​​for each of the culture media.

3. 3. The microorganism discrimination device according to claim 1, wherein the analysis unit discriminates microorganisms based on time data corresponding to changes in the current value during a period from the start of measurement until the current value increases and reaches a maximum value.

4. 3. A culture kit for use in the microorganism discrimination device according to claim 1, comprising a combination of a plurality of types of culture media, including the media capable of culturing different microorganisms.

5. A microorganism discrimination device comprising: a culture medium capable of cultivating multiple types of microorganisms; an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed with an insulator interposed therebetween; a measurement unit that measures the galvanic current flowing between at least both electrode bodies of the electrode sensor while the culture medium is in contact with the both electrode bodies; and an analysis unit that stores the current value measured by the measurement unit and discriminates multiple types of microorganisms based on changes in the current value.

6. A method for identifying microorganisms in a culture medium using an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, in which at least both electrode bodies of the electrode sensor are brought into contact with a plurality of types of culture medium capable of cultivating microorganisms, and the galvanic current between the two electrode bodies is measured for each culture medium while culturing the microorganisms in the culture medium, and the microorganisms are identified based on the change in the measured current value for each culture medium.

7. 7. The method for identifying microorganisms according to claim 6, wherein the microorganisms are identified based on reference data based on current values ​​measured by culturing specific microorganisms in advance in at least one of the culture media and on changes in current values ​​for each of the culture media.

8. 7. The method for distinguishing microorganisms according to claim 6, wherein the microorganisms are distinguished based on time data corresponding to the change in the current value during the period from the start of measurement until the current value increases and reaches its maximum value.

9. A method for distinguishing microorganisms in a culture medium using an electrode sensor in which an anode electrode body and a cathode electrode body are integrally formed via an insulator, the method comprising contacting at least both electrode bodies of the electrode sensor with a culture medium capable of cultivating multiple types of microorganisms, measuring the galvanic current between the two electrode bodies while culturing the multiple types of microorganisms in the culture medium, and distinguishing the multiple types of microorganisms based on changes in the measured current value.

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