Live bacteria quantifying device, quantifying method, and kit for quantifying live bacteria

Abstract

Provided is a live bacteria quantifying method comprising: a deposition step in which bacteria is placed in a measuring vessel containing a redox substance that is permeable to the cell membranes of the live bacteria to be measured, that is oxidized or reduced within the cells, and that is deposited within the cells, and the bacteria and redox substance are mixed for a prescribed period of time; a current peak measurement step in which the measurement solution is electrochemically measured after the deposition step to determine the current peak of the residual redox substance; and a quantitative evaluation step in which the current peak difference is determined between the current peak of the residual redox substance measured in the current peak measurement step and the current peak of the initial redox substance measured in advance, and the live bacteria are quantitatively evaluated from the current peak difference.

Description

Apparatus, method and kit for quantifying live bacteria

[0001] The present invention relates to an apparatus, a method and a kit for quantifying viable bacteria.

[0002] Widely used methods for quantifying small viable bacteria (microorganisms) measuring only a few micrometers in size include the culture method, in which colonies formed by the growth of single cells on a plate are counted, and microscopy using fluorescent labels. The culture method requires a culture period of at least one day. Both the culture method and microscopy require skilled techniques and expensive equipment. Another method involves spectroscopic and electrochemical measurements that focus on the dissolved oxygen consumed by bacterial aerobic respiration. While these respiration-focused measurements are simple, absolute evaluation is difficult because oxygen solubility varies significantly depending on the environment. Another method involves incorporating the cell membrane-permeable formazan dye MTT (a type of tetrazolium salt, 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into the cells of viable bacteria, culturing them to change color (from yellow to purple), and then measuring the spectral absorption wavelength. This method usually requires incubation for 5 hours or more.

[0003] Patent Document 1 discloses a method for detecting bacteria by reacting a mediator that reacts in response to the action of an enzyme present in the cell membrane of the bacteria to be detected in a sample with the enzyme, and then detecting or quantifying the reacted mediator by an electrochemical method. Examples of mediators include benzoquinone, 2,6-dimethylbenzoquinone, methoxybenzoquinone, and vitamin K3 (menadione).

[0004] Non-Patent Document 1 discloses a method in which cell membrane-permeable MTT is taken up into bacterial cells, cultured, and then the cell membrane is disrupted on an electrode to electrochemically measure intracellular MTT formazan (which is sometimes called "MTT formazan" after reduction and discoloration). This method requires centrifuging the bacteria in the measurement solution, transferring only the bacteria onto a large-area electrode, disrupting the cell membrane, and drying on the electrode, resulting in a complex and lengthy procedure.

[0005] Patent No. 4448223

[0006] Author: Kengo Ishiki, Dung Q. Nguyen, Aya Morishita, Hiroshi Shiigi, and Tsutomu Nagaoka, “Electrochemical Detection of Viable Bacterial Cells Using a Tetrazolium Salt'', Analytical Chemistry 2018 Vol. 90 pages 10903-10909

[0007] The present invention provides an apparatus, a method and a kit for quantifying viable bacteria that enable electrochemical evaluation of bacterial activity using a redox substance (for example, an artificial dye) more simply than conventional techniques.

[0008] The first viable cell quantification device measures a preset current peak difference (ΔI Ｐ ) and current peak difference (ΔI Ｐ a data storage unit that stores first or second verification data including the number of viable bacteria corresponding to the number of viable bacteria; and a measurement solution containing redox substances that are cell membrane permeable to the viable bacteria to be measured and that are oxidized or reduced within the cells and deposited within the cells of the viable bacteria, by electrochemical measurement (sweeping a voltage within a predetermined range (from a positive potential to a negative potential or from a negative potential to a positive potential)), and detecting at least one current peak (I Ｐ１ a current peak measurement unit for measuring the current peak (I) of the remaining redox substances obtained by measuring the measurement solution after deposition on the live bacteria with the current peak measurement unit; Ｐ１ ) and the at least one current peak (I Ｐ１ The current peak (I) of the initial redox substance before deposition on the live bacteria corresponds to a preset value (for example, acquired via an input means or a communication means, stored in a data storage unit, and measured in advance by a current peak measurement unit in a state where the redox substance is not in contact with the live bacteria). Ｐ０ ) and the current peak difference (ΔI Ｐ ) and calculate the current peak difference (ΔI Ｐ ) (current peak difference (ΔI Ｐand a quantitative evaluation unit that collates the first or second collation data from the data storage unit (as a search key) and quantitatively evaluates the viable bacteria. Examples of a "measurement solution containing residual redox substances" include (1) a measurement solution obtained by placing viable bacteria in a measurement container containing redox substances that are cell membrane permeable to the viable bacteria to be measured and that are oxidized or reduced within the cells to deposit (or become insolubilized) within the cells, mixing the viable bacteria for a predetermined period of time (e.g., 20, 30, or 40 minutes), and allowing the bacteria to deposit; (2) a supernatant obtained by centrifuging the measurement solution after the deposition; and (3) a measurement solution in a suspended state containing viable bacteria and residual redox substances (a mixed suspension or a suspension in the state of being mixed).

[0009] The second viable cell quantification device measures the current peak (I Ｐ１ ) and the current peak of the remaining redox species (I Ｐ１ a data storage unit that stores first or second verification data including the number of viable bacteria corresponding to the number of viable bacteria; and a measurement solution containing residual redox substances that have not been taken up by the viable bacteria, which is electrochemically measured (by sweeping a voltage within a predetermined range (from a positive potential to a negative potential or from a negative potential to a positive potential)) to measure a current peak (I Ｐ１ a current peak measurement unit for determining the current peak (I Ｐ１ and a quantitative evaluation unit that compares the first or second comparison data from the data storage unit using the first or second comparison data as a key and quantitatively evaluates the viable bacteria. Since the current peak of the redox substances initially contained in the measurement solution is known in advance, the current peak of the remaining redox substances (I Ｐ１ ) makes it possible to quantitatively evaluate the number of viable bacteria.

[0010] In the first and second viable cell quantification devices, at least one current peak is determined for the initial and remaining redox substances. Furthermore, the current peak for the initial redox substances may be set in accordance with a predetermined mixing time (deposition time). For example, if the mixing (deposition) time is 10 minutes, the current peak for the initial redox substances at 10 minutes may be selected, and if the mixing (deposition) time is 30 minutes, the current peak for the initial redox substances at 30 minutes may be selected. Furthermore, the quantitatively evaluated viable cell count may be on the order of 10. Furthermore, the quantified viable cell count may include multiple types of bacteria. In this case, multiple types of bacteria are quantified simultaneously.

[0011] The first and second viable cell quantification devices are configured to calculate the evaluation results (e.g., viable cell count, current peak difference (ΔI Ｐ ), the current peak of the remaining redox species (I Ｐ１ )) in a storage medium, and an output unit that outputs the evaluation results obtained by the quantitative evaluation unit or the evaluation results stored in the storage medium to output means. Examples of "output" include displaying on a display means, outputting to a printer and printing, transmitting to an external device, and storing in a storage medium. The external device or storage medium may be connected locally or to the Internet. The first and second viable cell quantification devices may be small and portable.

[0012] The first and second viable bacteria quantification devices may have an electrode tip to which a preset amount of the redox substance is attached. The electrode tip may be a disposable type. The current peak measurement unit of the first and second viable bacteria quantification devices immerses the electrode tip in the measurement solution, and then electrochemically measures (sweeps a voltage within a predetermined range (from a positive potential to a negative potential or from a negative potential to a positive potential)) the measurement solution containing the remaining redox substance not taken up by the viable bacteria, and measures the current peak (I Ｐ１ ) may be obtained.

[0013] In the first and second viable bacteria quantification devices, the current peak measurement unit electrochemically measures (at least sweeps a voltage within a predetermined range (from a positive potential to a negative potential)) a measurement solution containing remaining redox substances that have not been taken up by viable bacteria, and measures the current peak (I Ｐ１ In other words, the current peak may be determined from the reduction current. In the first and second viable bacteria quantification devices, the current peak measurement unit electrochemically measures (at least sweeps a voltage within a predetermined range (from a negative potential to a positive potential)) the measurement solution containing the remaining redox substances that have not been taken up by the viable bacteria, and determines the current peak (I Ｐ１ ) may be determined. That is, a current peak may be determined from the oxidation current. The current peaks determined may be one or more.

[0014] The first viable cell quantification method includes a deposition step in which viable bacteria to be measured are placed in a test solution containing redox substances that are cell membrane permeable and that are oxidized or reduced intracellularly to deposit (or become insolubilized) within the cells of the viable bacteria, and the test solution is mixed for a predetermined time (e.g., 20, 30, or 40 minutes), thereby incorporating and depositing the redox substances within the cells of the viable bacteria; and after the deposition step, the test solution (in a suspended state containing viable bacteria and remaining redox substances) is electrochemically measured (by sweeping a predetermined range of voltage (from a positive potential to a negative potential or from a negative potential to a positive potential)) to measure at least one current peak (I Ｐ１ a current peak measuring step for determining at least one current peak (I Ｐ１ ) and the at least one current peak (I Ｐ１ ) corresponding to a preset initial redox species current peak (I Ｐ０ ) and the current peak difference (ΔI Ｐ ) and calculate the current peak difference (ΔI Ｐ A first quantitative evaluation step of quantitatively evaluating the viable bacteria from the current peak (I Ｐ１ and a second quantitative evaluation unit that quantitatively evaluates the viable bacteria from the current peak difference (ΔI Ｐ ) or current peak (I Ｐ１The number of viable bacteria corresponding to the current peak difference (ΔI Ｐ ) or current peak (I Ｐ１ The data (data for comparison) of the number of live bacteria corresponding to the current peak difference (ΔI Ｐ ) or current peak (I Ｐ１ ) may be used as a key to compare the first or second comparison data and quantify the viable bacterial count. The current peak measurement step may include a voltage application step of applying a predetermined range of voltages (from a positive potential to a negative potential or from a negative potential to a positive potential) according to electrochemical measurement to the working electrode and the reference electrode of the electrode tip or the single electrode, and a current response measurement step of determining the current peak (peak height) of the current response measured by applying the predetermined range of voltages (and the potential (peak potential) at the current peak). The current peak of the initial redox substance may be set corresponding to a predetermined mixing time (deposition time). For example, if the mixing (deposition) time is 10 minutes, the current peak of the initial redox substance at 10 minutes may be selected, and if the mixing (deposition) time is 30 minutes, the current peak of the initial redox substance at 30 minutes may be selected. The quantitatively evaluated viable bacterial count may be on the order of 10 digits. The quantified viable bacterial count may include multiple species of bacteria. In this case, multiple species of bacteria are quantified simultaneously. The current peak measurement step (or current response measurement step) involves electrochemically measuring (at least sweeping a voltage within a predetermined range (from a positive potential to a negative potential)) a measurement solution containing residual redox substances that have not been taken up by live bacteria, and measuring the current peak (I Ｐ１ In other words, the current peak may be determined from the reduction current. In the current peak measurement step (or current response measurement step), the current peak measurement unit electrochemically measures (at least sweeps a voltage in a predetermined range (from a negative potential to a positive potential)) the measurement solution containing the remaining redox substances that have not been taken up by the viable bacteria, and determines the current peak (I Ｐ１) may be determined. That is, a current peak may be determined from the oxidation current. The current peak to be determined may be one or more types. The first viable cell quantification method is a method for quantifying viable cells, which is based on the evaluation results (e.g., viable cell count, current peak difference (ΔI Ｐ ), the current peak of the remaining redox species (I Ｐ１ ) in a storage medium, and an output step of outputting the evaluation results obtained in the quantitative evaluation step or the evaluation results stored in the storage medium to an output means. Examples of "output" include displaying on a display means, outputting to a printer and printing, transmitting to an external device, and storing in a storage medium. The external device or storage medium may be connected locally or via the Internet.

[0015] (Electrode Chip) The electrode tip has a working electrode, a counter electrode, and a reference electrode, or two electrodes: a working electrode and a reference electrode. The electrode tip is electrically connected to a power source. For example, the current peak is determined from the current waveform when a predetermined range of voltage (from a positive potential to a negative potential or from a negative potential to a positive potential) is applied (swept) to the electrode tip in response to a command from the current peak measurement unit. A predetermined amount of redox substance may be attached to the electrode tip. An electrode tip with an attached redox substance may be placed in a test solution containing live bacteria and a solvent, the redox substance dissolved, and the mixture mixed for a predetermined period of time to perform electrochemical measurement. After dissolving the redox substance attached to the electrode tip in the test solution, the live bacteria may be added to the test solution and mixed for a predetermined period of time. An electrode tip without an attached redox substance may be placed in a test solution containing live bacteria, a redox substance, and a solvent to perform electrochemical measurement. The electrode tip may be an electrode made of metal, carbon, conductive glass, or the like, or an electrode formed by metal plating or printing with conductive ink. The amount of the attached redox substance may be of multiple types.

[0016] The current peak measurement unit may include three electrodes, namely, a working electrode, a counter electrode, and a reference electrode, or a single electrode, namely, a working electrode and a reference electrode. The current peak measurement unit may include: a voltage application control unit that applies a voltage within a predetermined range (from a positive potential to a negative potential or from a negative potential to a positive potential) according to electrochemical measurement to the electrode tip or the working electrode and the reference electrode of the single electrode; and a current response measurement unit that determines the current peak (peak height) of the current response measured by applying a voltage within the predetermined range (and the potential (peak potential) at the time of the current peak).

[0017] The data storage unit may temporarily store the first and second matching data, may acquire the first and second matching data from an external device, or may be configured to allow the first and second matching data to be updated.

[0018] The first viable cell quantification kit includes a redox substance that is permeable to the cell membrane of the viable bacteria to be measured and that is oxidized or reduced intracellularly to deposit (or become insolubilized) within the cells. The first viable cell quantification kit includes a redox substance and a solvent. The redox substance and the solvent may be individually packaged in packages or containers. The solvent container may contain the redox substance and be used as a measurement container. The second viable cell quantification kit includes a measurement solution containing a solvent and a redox substance that is permeable to the cell membrane of the viable bacteria to be measured and that is oxidized or reduced intracellularly to deposit (or become insolubilized) within the cells. The measurement solution may be packaged in a container or package. The container or package may be used as a measurement container. The first and second viable cell quantification kits are used for measurements using the above-mentioned quantification device and / or quantification method. The first and second viable cell quantification kits may include multiple packages containing different amounts of the redox substance. The first and second viable cell quantification kits may include multiple packages containing different types of the redox substance. The first and second viable cell quantification kits may include a plurality of measurement containers with different concentrations of redox substances.The first and second viable cell quantification kits may include a plurality of measurement containers with different types of redox substances.

[0019] (Live Bacteria) Live bacteria are living cells, and examples thereof include Escherichia coli (e. coli), Salmonella enterica (S. enterica), Pseudomonas aeruginosa (P. aeruginosa), and Staphylococcus aureus (S. aureus).

[0020] (Redox Substances) Redox substances are substances that are cell membrane permeable and are oxidized or reduced within cells to deposit within the cells. Examples of redox substances include artificial dyes. Examples of artificial dyes include tetrazolium salts. Tetrazolium salts include, for example, MTT (3-(4,5-di-MethylThiazol-2-yl)-2,5-diphenylTetrazolium bromide), CTC (5-cyano-2,3-ditolyl INT (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium) chloride), XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), NBT (nitro blue tetrazolium), WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium), 2,3,5-Triphenyltetrazolium Chloride, Tetranitro Blue Tetrazolium, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymetho xyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), m-Tolyltetrazolium Red, m-Tolyltetrazolium Red, 2,3-Diphenyl-5-ethyltetrazolium Chloride, 2,3-Diphenyl-5-carboxytetrazolium Chloride, 2,3,5-Triphenyltetrazolium Iodide, o-Tolyltetrazolium Red, Tetrazolium Violet, p-Tolyltetrazolium Red, 2,3,5-Triphenyltetrazolium Bromide, and the like.

[0021] (Oxidation-reduction substance) The solvent of the measurement solution is, for example, tap water, pure water, purified water, etc. These solvents may be sterilized (for example, at 121° C. for 20 minutes).

[0022] Another disclosed computer program performs each step of the viable cell quantification method when executed by at least one processor. Another disclosed storage medium storing the computer program performs each step of the viable cell quantification method when executed by at least one processor. Another disclosed information processing device includes at least one processor and a memory (which may be the storage medium) storing a computer program executed by the processor, and performs each step of the viable cell quantification method when executed by the at least one processor. The information processing device is not particularly limited and examples include smartphones, tablets, smartwatches, wearable computers, personal computers, local servers, cloud servers, etc., and a single or a combination of multiple information processing devices may be configured to be connectable via wired and / or wireless communication means.

[0023] The viable cell quantification device may be configured to include, for example, a dedicated circuit, firmware, a processor and a memory storing a processing program, a display, a bus, an input / output interface, a communication device, and the like.

[0024] [Effects] The present invention has the following effects: (1) Electrochemical evaluation of bacterial activity can be easily performed. (2) By electrochemically quantifying the remaining redox substances in the measurement solution, a long-term culture process is unnecessary, and the number of viable bacteria can be measured quickly. (3) The present invention can be applied to hygiene and quality control in the medical, pharmaceutical, and food fields, environmental assessment (biofilms), and the fields of pre-disease and specific disease prevention (intestinal flora).

[0025] 1 is a functional block diagram for explaining an example of the function of a viable cell quantification device; FIG. 2 is a diagram showing an example of matching data; FIG. 3 is a diagram showing an example of matching data; FIG. 4 is a flow chart of a viable cell quantification method; FIG. 5 is a diagram showing a current response curve, an initial MTT current peak, and a residual MTT current peak of Example 1; FIG. 6 is a diagram showing a current peak difference according to the viable cell concentration (E. coli) of Example 1; FIG. 7 is a diagram showing peak intensity of a comparative example; FIG. 8 is a diagram showing peak intensity according to the viable cell concentration (E. coli) of a comparative example; FIG. 9 is a diagram showing a current response curve for E. coli, showing four current peaks; FIG. 10 is a diagram showing a change in the difference between the four current peaks according to the stirring time for E. coli; FIG. 11 is a diagram showing a current response curve for another example (E. coli), showing four current peaks; FIG. 12 is a diagram showing a change in the difference between the four current peaks according to the stirring time for another example (E. coli); FIG. 13 is a diagram showing a current response curve for another example (E. coli), showing four current peaks; FIG. 14 is a diagram showing a change in the difference between the four current peaks according to the stirring time for another example (E. coli); FIG. 1 is a diagram showing changes in four current peak differences corresponding to stirring time in another example (Salmonella); FIG. 2 is a diagram showing a current response curve and four current peaks in another example (Staphylococcus aureus); FIG. 3 is a diagram showing changes in four current peak differences corresponding to stirring time in another example (Staphylococcus aureus); FIG. 4 is a diagram showing a current response curve and four current peaks in another example (Pseudomonas aeruginosa); FIG. 5 is a diagram showing changes in four current peak differences corresponding to stirring time in another example (Pseudomonas aeruginosa); FIG. 6 is a diagram showing a current response curve and four current peaks in another example (four types of bacteria); FIG. 7 is a diagram showing changes in four current peak differences corresponding to stirring time in another example (four types of bacteria); FIG. 8 is a diagram showing a current response curve and one current peak in another example (NBT); FIG. 9 is a diagram showing changes in one current peak difference corresponding to stirring time in another example (NBT). ΔI for each concentration of E. coli Ｐ ΔI per cell for each bacterium. Ｐ FIG.

[0026] (Embodiment 1) A viable bacteria quantification device 1 will be described with reference to Figure 1. The viable bacteria quantification device 1 includes a data accumulation unit 11, a current peak measurement unit 12, a quantitative evaluation unit 13, a memory 14, a data storage unit 15, an electrode tip connection unit 102, a display unit 103, an input operation unit 104, a control unit 105, and a communication unit 109.

[0027] The data storage unit 11 includes a storage medium and stores a preset current peak difference (ΔI Ｐ ), current peak difference (ΔI Ｐ ) and accumulates matching data including viable cell counts corresponding to the identified bacteria. FIG. 2A shows an example of the first matching data. The first matching data includes identification information, bacteria, redox substances, initial current peak, residual current peak, current peak difference, and viable cell count. The viable cell count in the first matching data indicates the cell count of the bacteria. FIG. 2B shows an example of the second matching data. The second matching data includes identification information, redox substances, initial current peak, residual current peak, current peak difference, and viable cell count. The viable cell count in the second matching data indicates the cell count of all types of bacteria.

[0028] The current peak measurement unit 12 electrochemically measures (sweeps from a predetermined positive potential to a negative potential or from a negative potential to a positive potential) a measurement solution containing remaining redox substances that have not been taken up by live bacteria, and measures at least one current peak (I Ｐ１ ) is determined. The current peak measurement unit 12 has a voltage application control unit 121 that applies a voltage within a predetermined range according to electrochemical measurement to the working electrode 22 and reference electrode 23 of the electrode tip 20, and a current response measurement unit 122 that determines the current peak (peak height) of the current response measured by applying a voltage within the predetermined range (and the potential at the current peak (peak potential)). Examples of electrochemical measurements include measurement methods using electrochemical techniques such as differential pulse voltammetry, normal pulse voltammetry, linear sweep voltammetry, stripping voltammetry, cyclic voltammetry, potentiometry, and amperometry.

[0029] The voltage application control unit 121 applies a predetermined constant voltage between the working electrode 22 and the counter electrode 24 of the electrode tip 20. The current response measurement unit 122 measures a current value corresponding to the constant voltage applied to the working electrode 22 and the counter electrode 24. Once a current value is obtained at the working electrode 22, the voltage application control unit 121 applies a voltage within a predetermined range, repeating equilibrium and sweep, between the working electrode 22 and the reference electrode 23 according to electrochemical measurement. The current response measurement unit 121 measures the current value at the working electrode 22. The current response measurement unit 121 detects the measured current peak. The potential at the detected current peak is called the peak potential. The current response measurement unit 121 performs baseline fitting on the current response measurement data to determine a baseline, and detects the peak height and BG value based on the baseline. The current response measurement unit 122 may also calculate the peak height of the measured current peak and the area of ​​the current peak curve.

[0030] The quantitative evaluation unit 13 is configured to measure the current peak (I Ｐ０ ) and the current peak (I Ｐ１ ) and the current peak difference (ΔI Ｐ ) and calculate the current peak difference (ΔI Ｐ ) to compare the data with the first or second comparison data in the data storage unit 11 and quantitatively evaluate the viable bacteria.

[0031] The data storage unit 15 stores the evaluation results obtained by the quantitative evaluation unit 13 in the memory 14. The evaluation results include, for example, the number of viable bacteria, the current peak difference (ΔI Ｐ ), the current peak of the remaining redox species (I Ｐ１ The display unit 103 displays the evaluation results obtained by the quantitative evaluation unit 13 or the evaluation results stored in the memory 14 on a monitor (not shown). The input operation unit 104 has an input interface, and an operator inputs necessary operation data and instructions.

[0032] The control unit 105 has the function of integrating and executing operation processing, input / output data processing, and control of each functional unit of the viable cell quantification device 1. The communication unit 109 has wireless or wired communication means and transmits the evaluation results obtained by the quantitative evaluation unit 13 or the evaluation results stored in the memory 14 to an external device. The communication unit 109 also receives matching data from the external device. The control unit 105 sends the matching data to the data storage unit 11 via the internal bus of the device and stores it.

[0033] The electrode tip connection section 102 has a structure to which the electrode tip 20 is electrically attached. The electrode tip 20 has three electrodes: a working electrode 22, a reference electrode 23, and a counter electrode 24. A suspension of the measurement solution is dropped onto the specimen drop areas E of the three electrodes, and a voltage within a predetermined range is applied to measure the current response. The electrode tip 20 may be disposable, or may be washed and reused.

[0034] The voltage peak measurement unit 12, the voltage application control unit 121, the current response measurement unit 122, the quantitative evaluation unit 13, the data storage unit 15, the control unit 105, the display unit 103, and the input operation unit 104 may be composed of a dedicated circuit, firmware, a computer program, and hardware (processor, memory, etc.).

[0035] (Viable Bacteria Quantification Method) The viable bacteria quantification method includes the following processing steps. In the deposition step (S1), the viable bacteria to be measured are placed in a measurement container containing a redox substance that is cell membrane permeable and that is oxidized or reduced intracellularly to deposit (or become insolubilized) within the cells, and the viable bacteria are mixed for a predetermined period of time (e.g., 20, 30, or 40 minutes). The redox substance in the measurement solution permeates the cell membrane of the viable bacteria and diffuses into the cells. The redox substance, for example, MTT, is reduced to MTT formazan by electron carriers within the viable bacteria, such as NADH (nicotinamide adenine dinucleotide), NADPH (nicotinamide adenine dinucleotide phosphate), coenzymes, menaquinone, and ubiquinone. Because MTT formazan is insoluble, it deposits within the cells and does not redissolve in the measurement solution. In the current peak measurement step (S2), after the deposition step, the measurement solution (in a suspended state containing live bacteria and residual redox substances) is subjected to electrochemical measurement (sweeping a voltage within a predetermined range (from a positive potential to a negative potential or from a negative potential to a positive potential)) to measure at least one current peak (I Ｐ１ ) is determined. The current peak measurement step may include a voltage application step in which a predetermined range of voltages (from a positive potential to a negative potential or from a negative potential to a positive potential) according to cyclic voltammetry measurement is applied to the working electrode and reference electrode of an electrode tip or a single electrode, and a current response measurement step in which the current peak (peak height) of the current response measured by applying a voltage within the predetermined range (and the potential at the current peak (peak potential)) is determined. Electrochemical detection means used in the current peak measurement step include, for example, devices that measure current, voltage, electrical resistance, and impedance, and methods using electrochemical techniques such as differential pulse voltammetry, normal pulse voltammetry, linear sweep voltammetry, stripping voltammetry, cyclic voltammetry, potentiometry, and amperometry, which are controlled by software installed in the devices. The quantitative evaluation step (S3) involves determining the current peak (I Ｐ０ ) and the current peak (I Ｐ１ ) and the current peak difference (ΔIＰ ) and calculate the current peak difference (ΔI Ｐ The amount of redox substances in the measurement solution, such as MTT, decreases in proportion to the amount of bacteria taken in, and as the number of bacteria increases, the amount of redox substances remaining in the measurement solution decreases, from which the number of viable bacteria can be estimated.

[0036] The viable cell quantification method may further include the following steps: A data storage step (S4) stores the evaluation results (e.g., viable cell count, current peak difference (ΔI Ｐ ), the current peak of the remaining redox species (I Ｐ１ )) into memory. In the output step (S5), the evaluation results obtained in the quantitative evaluation step or the evaluation results stored in the memory are output to output means. Each of the above steps may be realized using the viable bacteria quantification device 1 of embodiment 1, or may be realized by a general electrochemical measurement device or information processing device.

[0037] [Viable Bacterial Quantification Kit] A measurement solution containing a solvent and a redox substance that is cell membrane permeable to the viable bacteria to be measured and that is oxidized or reduced intracellularly to become deposited (or insolubilized) within the cells is packaged in a measurement container. After the viable bacteria are placed in the measurement container and mixed, the resulting suspension can be measured. When the kit contains only a redox substance, the viable bacteria and water (solvent) can be placed in the container, the redox substance can be added and mixed, and the resulting suspension can be measured. The package is preferably a container that can seal the contents and prevent deterioration of their physical properties, such as a plastic bottle container, a film packaging container, or a glass container.

[0038] (Example) The example was carried out under the following conditions. Oxidation-reducing substances: MTT (operation procedures 1 to 3), NBT (operation procedure 4) Initial concentration of MTT and NBT in the measurement solution: 0.10 mM Solvent for the measurement solution: Sterilized pure water Live bacteria: Escherichia coli K12 (operation procedures 1 to 3), Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa (operation procedure 3) Live bacteria concentration in the measurement solution: 1 x 10 ３ , 1×10 ４ , 1×10 ５ , 1 x 10 ６ , 1×10７ [CFU mL －１ ] Deposition time: 37℃ 1 to 10, 20, 30, 40, 50, 60 minutes (contact mixing time) Electrochemical measurement: Cyclic voltammetry measurement device Electrodes: Glassy carbon electrode as working electrode, platinum coil electrode as counter electrode, silver / silver chloride electrode as reference electrode Sweep voltage: +0.2V to -0.4V Voltage application: Sweep from +0.6V to -0.6V, sweep from -0.6V to +0.6V

[0039] <Operation Procedure 1: Example 1> (1) Place Escherichia coli K12 into a container containing an MTT-containing measurement solution. (2) Mix for 40 minutes. MTT permeates the bacterial cell membrane, is reduced within the cell, and becomes MTT formazan, which is then deposited. (3) Remove the suspension from the measurement container and drop it onto the electrode tip. (4) Perform cyclic voltammetry measurement. Apply a voltage from +0.2 V to -0.4 V, and measure the current response. (5) From the current response, determine the remaining MTT current peak (I Ｐ１ ) [μA]. A current peak can be confirmed around -0.12 V (see Figure 4A). (6) The initial MTT current peak (I Ｐ０ ) and the remaining MTT current peak (I Ｐ１ ) and the current peak difference (ΔI Ｐ ) is calculated. Ｐ =I Ｐ０ -I Ｐ１ (7) Current peak difference (ΔI Ｐ The viable bacteria are quantitatively evaluated from the current peak difference (ΔI Ｐ The amount of MTT that is taken up by and deposited in viable bacteria is calculated from the calculated amount of MTT. The number of viable bacteria is calculated from the calculated amount of MTT. The amount of MTT that can be taken up by viable bacteria is set in advance (determined experimentally).

[0040] 10 ３ From 10 ７ [CFU mL －１ ], the current peak difference (ΔI Ｐ ) was reduced (see FIG. 4B). ４ It can only be quantified at this concentration.

[0041] <Operation Procedure 2: Example 2> (1) E. coli K12 (the number of viable bacteria in the entire measurement solution in FIG. 6A : 7.3×10) was added to a container containing an MTT-containing measurement solution. ５ CFUmL －１ , in FIG. 7A, the number of viable bacteria in the entire measurement solution: 2.0 × 10 ７ CFUmL －１ ) is added. (2) Mix for 1 to 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, and 60 minutes. MTT permeates the bacterial cell membrane, is reduced within the cell, and becomes MTT formazan, which is then deposited. (3) After mixing for 1 to 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, and 60 minutes, the suspension is removed from the measurement container and dropped onto the electrode tip. (4) Cyclic voltammetry measurements are performed. The voltage is swept from +0.6 V to -0.6 V, and then from -0.6 V to +0.6 V, and the current response is measured. (5) From the current response, the remaining MTT current peak (I Ｐ１ ) [μA]. In the reduction sweep, current peaks can be confirmed near -0.1 V and -0.4 V, and in the oxidation sweep, current peaks can be confirmed near 0 V and +0.4 V (see Figures 6A and 7A). (6) Initial MTT current peak (I Ｐ０ ) and the remaining MTT current peak (I Ｐ１ ) and the current peak difference (ΔI Ｐ ) is calculated at each current peak. Ｐ =I Ｐ０ -I Ｐ１ In FIG. 6B, the current peak differences (ΔI Ｐ 7B shows the current peak differences (ΔI Ｐ (7) Current peak difference (ΔI Ｐ The viable bacteria are quantitatively evaluated from the current peak difference (ΔI Ｐ The amount of MTT that is taken up by and deposited in viable bacteria is calculated from the calculated amount of MTT. The number of viable bacteria is calculated from the calculated amount of MTT. The amount of MTT that can be taken up by viable bacteria is set in advance (determined experimentally).

[0042] According to the results of Operation Procedure 2, if the mixing time is 1 minute or more, the current peak difference (ΔI Ｐ It was confirmed that the number of viable bacteria can be quantified from 10 minutes or more, and that quantification can be performed with greater accuracy if the time is 10 minutes or more. It was also confirmed that the accuracy of quantification increases in proportion to the concentration of viable bacteria.

[0043] <Operation Procedure 3: Example 3> (1) Escherichia coli K12 (the number of viable bacteria in the entire measurement solution in FIG. 8A : 2.7×10) was added to a container 1 containing an MTT-containing measurement solution. ９ CFUmL －１ ), Salmonella in container 2 (the number of live bacteria in the entire measurement solution in FIG. 9A: 1.9×10 ９ CFUmL －１ ), Staphylococcus aureus in container 3 (the number of live bacteria in the entire measurement solution in FIG. 10A: 1.0×10 ９ CFUmL －１ ), and Pseudomonas aeruginosa in container 4 (the number of live bacteria in the entire measurement solution in FIG. 11A: 1.3 × 10 ９ CFUmL －１ ) were placed in the container 5 containing the MTT-containing measurement solution, and the resulting mixture was allowed to settle on each bacterium. In addition, Escherichia coli K12, Salmonella, Staphylococcus aureus, and Pseudomonas aeruginosa (the total viable cell count in the entire measurement solution in FIG. 12A: 10 ９ CFUmL －１ ) and deposit it. (2) Mix for 1 to 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, and 60 minutes. MTT permeates the bacterial cell membrane, is reduced inside the cell, and becomes MTT formazan, which deposits. (3) After mixing for 1 to 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, and 60 minutes, remove the suspension from measurement containers 1 to 5, and drop it onto the electrode tip. (4) Perform cyclic voltammetry measurement. Sweep from +0.6 V to -0.6 V, and then sweep from -0.6 V to +0.6 V, and measure the current response. (5) From the current response, determine the remaining MTT current peak (I Ｐ１ ) [μA]. In the reduction sweep, current peaks can be confirmed near -0.1 V and -0.4 V, and in the oxidation sweep, current peaks can be confirmed near 0 V and +0.4 V (see Figures 8A to 12A). (6) Initial MTT current peak (I Ｐ０) and the remaining MTT current peak (I Ｐ１ ) and the current peak difference (ΔI Ｐ ) is calculated at each current peak. Ｐ =I Ｐ０ -I Ｐ１ 8B to 12B, the current peak differences (ΔI Ｐ (7) Current peak difference (ΔI Ｐ The viable bacteria are quantitatively evaluated from the current peak difference (ΔI Ｐ The amount of MTT that is taken up by and deposited in viable bacteria is calculated from the calculated amount of MTT. The number of viable bacteria is calculated from the calculated amount of MTT. The amount of MTT that can be taken up by viable bacteria is set in advance (determined experimentally).

[0044] According to the results of Operation Procedure 3, if the mixing time is 1 minute or more, the current peak difference (ΔI Ｐ It was also confirmed that multiple types of live bacteria can be quantified simultaneously.

[0045] <Operation Procedure 4: Example 4> (1) E. coli K12 (viable cell count in the entire measurement solution: 10 ８ ~10 ９ Degree CFUmL －１ ) and allow it to deposit on the bacteria. (2) Mix for 10, 20, 30, 40, 50, and 60 minutes. NBT permeates the bacterial cell membrane, is reduced inside the cells, and becomes NBT formazan, which deposits. (3) After mixing for 10, 20, 30, 40, 50, and 60 minutes, remove the suspension from the measurement container and drop it onto the electrode tip. (4) Perform cyclic voltammetry measurements. Sweep from +0.2 V to -0.6 V, and then sweep from -0.6 V to +0.6 V, and measure the current response. (5) From the current response, determine the remaining NBT current peak (I Ｐ１ ) [μA]. In the reduction sweep, one current peak can be confirmed near -0.2 V (see Figure 13A). (6) Initial NBT current peak (I Ｐ０ ) and the remaining NBT current peak (IＰ１ ) and the current peak difference (ΔI Ｐ ) is calculated at one current peak. Ｐ =I Ｐ０ -I Ｐ１ In FIG. 13B, the current peak difference (ΔI Ｐ (7) Current peak difference (ΔI Ｐ The viable bacteria are quantitatively evaluated from the current peak difference (ΔI Ｐ The amount of NBT that has been taken up by and deposited in the viable bacteria is calculated from the calculated amount of NBT. The number of viable bacteria is calculated from the calculated amount of NBT. The amount of NBT that can be taken up by the viable bacteria is preset (determined experimentally).

[0046] (Comparative Example) As a comparative example, evaluation was performed by spectroscopic measurement (conventional method). Artificial dye: MTT Initial concentration of MTT in measurement solution: 0.10 mM Solvent of measurement solution: Sterilized pure water Live bacteria: Escherichia coli K12 Live bacteria concentration in measurement solution: 1 x 10 ３ , 1×10 ４ , 1×10 ５ , 1×10 ６ , 1×10 ７ [CFU mL －１ ] Culture time: 37°C 40 minutes, 5 hours Spectrophotometric measurement: Absorbance meter

[0047] <Operation Procedure 1: Comparative Example 1> (1) Place E. coli K12 in a container containing an MTT-containing measurement solution. (2) Cultivate. (3) Measure the absorption spectrum of the measurement solution using a spectrophotometer. A peak intensity appears in the 580 nm wavelength region (see Figure 5A). (4) From the peak intensity at 580 nm, determine the concentration of MTT formazan deposited on live bacteria. ΔA in Figure 5A indicates the absorbance of MTT formazan.

[0048] In a 5-hour culture, 10 ４ From 10 ７ [CFU mL －１ ], the peak intensity was confirmed at E. coli concentrations up to 10 ３At a concentration of 1000, the peak intensity was low and quantification was not possible (see FIGS. 5A and 5B). At 40 minutes of stirring, the peak intensity was below the detection limit and no live bacteria could be confirmed, making it impossible to quantify the amount of live bacteria.

[0049] (Examples demonstrating correlation with official methods) Official methods set standards and criteria for specific foods. These are generally set out in the Notification Act, and individual test methods are set out in the Notification Act in response to cases where food hygiene problems have arisen. Only methods specified in the Notification Act and Notification Act are understood to be official methods. Laboratories that perform official methods must not only follow the test method protocol (such as the type of culture medium and test procedures), but also demonstrate that they are capable of conducting appropriate tests, including the specifications of the equipment used and the skills of the personnel. Internal quality control (internal accuracy control) is a mandatory requirement for laboratories, and ISO 17025 certification is required. Petrifilm testing, one of the standard testing methods described in the "Food Hygiene Inspection Guidelines: Microorganisms" supervised by the Ministry of Health, Labor and Welfare, is widely used in the field as a simple (rapid) method due to its good correlation with official methods. 3M Petrifilm ＴＭ The viable cell count measurement plate (AC plate) is a test method certified by the international third-party organizations AOAC and AFNOR, the former as AOAC-OMA (Official Methods of Analysis) (certification numbers 986.33, 989.10, 990.12) using FDA BAM as the reference method, and the latter as AFNOR (certification number 3M 01 / 01-09 / 89) using NF ISO 4833-1 (Oct 2013) as the reference method has been confirmed as valid. In addition, it is a standard testing method listed in the Food Sanitation Inspection Guidelines in Japan, and since there is a good correlation with the official method, a comparison was made between the simple method using AC plates and the present invention.

[0050] (Example) The example was carried out under the following conditions. Oxidation-reducing substance: MTT Initial concentration of MTT in the measurement solution: 0.10 mM Solvent of the measurement solution: Sterilized phosphate buffer Viable bacteria: Escherichia coli K12 Viable bacteria concentration in the measurement solution: 10 ７ [CFU mL －１] Deposition time: 37℃ 60 minutes (contact mixing time) Electrochemical measurement: Cyclic voltammetry measurement device Electrodes: Glassy carbon electrode as working electrode, platinum coil electrode as counter electrode, silver / silver chloride electrode as reference electrode Sweep voltage: Sweep from +0.2V to -0.3V, and from -0.3V to +0.7V Official method: 3M Petrifilm ＴＭ 1.0 mL of the MTT-containing measurement solution was dropped onto a plate for measuring viable cell count (AC plate), which was then cultured for 24 hours at 37° C. The number of colored colonies formed on the sheet was counted.

[0051] <Operation Procedure 5: Example 5> (1) Place 4.2 x 10 Escherichia coli K12 into each container containing the MTT-containing measurement solution. ２ , 4.2 × 10 ３ , 4.2 × 10 ４ , 4.2 × 10 ５ , 4.2 × 10 ６ , 4.2 × 10 ７ [CFU mL －１ ] and mixed for 60 minutes. MTT permeates the bacterial cell membrane, is reduced within the cell, and becomes MTT formazan, which is then deposited. (2) Cyclic voltammetry measurements are performed. A voltage is applied from +0.2 V to -0.3 V, and the current response is measured. (3) From the current response, the remaining MTT current peak (I Ｐ１ ) [μA]. A current peak can be confirmed around -0.12 V. (4) The initial MTT current peak (I Ｐ０ ) and the remaining MTT current peak (I Ｐ１ ) and the current peak difference (ΔI Ｐ ) is calculated (see Table 1). Ｐ =I Ｐ０ -I Ｐ１ (5) ΔI for each concentration of E. coli Ｐ Plot the above to create a calibration curve (see Figure 14).

[0052]

[0053] <Operation Procedure 6: Example 6> (1) 100 g of minced chicken meat was left to rot at 30°C for 50 hours, and then dispersed in 100 mL of sterilized water. 50 μL of the dispersion was added to a container containing an MTT-containing measurement solution and mixed for 60 minutes. MTT permeates the bacterial cell membrane, is reduced within the cell, and becomes MTT formazan, which is then deposited. (2) Cyclic voltammetry measurements were performed. A voltage was applied from +0.2 V to -0.3 V, and the current response was measured. The remaining MTT current peak (I Ｐ１ ) [μA]. A current peak can be confirmed around -0.12 V. (3) The initial MTT current peak (I Ｐ０ ) and the remaining MTT current peak (I Ｐ１ ) and the current peak difference (ΔI Ｐ ) was calculated, and ΔI was calculated on the calibration curve created in <Operation Procedure 5>. Ｐ Plot the values ​​and calculate the number of viable bacteria (see Table 2). Ｐ =I Ｐ０ -I Ｐ１ (4) The same test solution is applied to 3M Petrifilm. ＴＭ The test sample was dropped onto a plate for measuring viable bacteria (AC plate), and the number of viable bacteria was determined by colony counting (see Table 1). (5) The number of bacteria obtained by this method and the standard test method was 1.08±0.02×10 ６ CFUmL －１ and 1.17 ± 0.03 × 10 ６ CFUmL －１ The error was within 10%. (6) The measurement solution contained general viable bacteria, lactic acid bacteria, psychrophilic bacteria, and fungi.

[0054]

[0055] <Operation Procedure 7: Example 7> (1) E. coli K12, E. coli O26, Salmonella, Staphylococcus aureus, and Staphylococcus epidermidis are individually placed in containers containing MTT-containing measurement solutions and mixed for 60 minutes. MTT permeates the bacterial cell membrane, is reduced within the cells, and becomes MTT formazan, which is then deposited. (3) Cyclic voltammetry measurement is performed. A voltage is applied from +0.2 V to -0.3 V, and the current response is measured. (4) The remaining MTT current peak (I Ｐ１) [μA]. A current peak can be confirmed around -0.12 V. (5) The initial MTT current peak (I Ｐ０ ) and the remaining MTT current peak (I Ｐ１ ) and the current peak difference (ΔI Ｐ ) is calculated. Ｐ =I Ｐ０ -I Ｐ１ (6) Current peak difference (ΔI Ｐ (7) The same test solution is then applied to 3M Petrifilm. ＴＭ The solution was dropped onto a plate for measuring viable cell count (AC plate), and the number of viable cells was determined by colony counting (see Table 3). (8) ΔI of each of Escherichia coli K12, Escherichia coli O26, Salmonella, Staphylococcus aureus, and Staphylococcus epidermidis Ｐ Dividing by the number of live bacteria obtained by colony counting gives ΔI per cell. Ｐ The ratios of E. coli O26, Salmonella, Staphylococcus aureus, and Staphylococcus epidermidis were calculated using E. coli K12 as the standard (1.00) (see Table 3). The ratios of E. coli O26, Salmonella, Staphylococcus aureus, and Staphylococcus epidermidis were 0.94 to 1.21, which was similar to E. coli K12 (see Figure 15).

[0056]

[0057] REFERENCE SIGNS LIST 1 viable bacteria quantification device 12 current peak measurement unit 121 voltage application control unit 122 current response measurement unit 13 quantitative evaluation unit 14 memory 15 data storage unit 103 display unit 104 input operation unit 105 control unit 109 communication unit 20 electrode tip 22 working electrode 23 reference electrode 24 counter electrode

Claims

1. a deposition step of placing the live bacteria to be measured in a measurement solution containing an oxidation-reduction substance that is cell membrane permeable to the live bacteria and that is oxidized or reduced within the cells and deposited within the cells of the live bacteria, and mixing the live bacteria for a predetermined period of time to incorporate the oxidation-reduction substance into the cells of the live bacteria and deposit it therein; a current peak measuring step of electrochemically measuring the measurement solution after the depositing step to obtain a current peak due to a residual redox substance remaining in the measurement solution; At least one current peak (I Ｐ１ ), and the at least one current peak (I Ｐ１ ) corresponding to a preset initial redox species current peak (I Ｐ０ ) and the current peak difference (ΔI Ｐ ) and the current peak difference (ΔI Ｐ A quantitative evaluation step of quantitatively evaluating the live bacteria from the Viable bacteria quantification method.

2. a data storage unit that stores preset matching data; The live bacteria to be measured are placed in a measurement solution containing a redox substance that is cell membrane permeable to the live bacteria and that is oxidized or reduced within the cells and deposited within the cells of the live bacteria, and the live bacteria are mixed for a predetermined period of time. After the redox substance is taken up and deposited within the cells of the live bacteria, the measurement solution containing the remaining redox substance that has not been taken up by the live bacteria is electrochemically measured, and at least one current peak (I Ｐ１ A current peak measurement unit for determining At least one current peak (I Ｐ１ ), and the at least one current peak (I Ｐ１ ) corresponding to a preset initial redox species current peak (I Ｐ０ ) and the current peak difference (ΔI Ｐ ) and the current peak difference (ΔI Ｐ A quantitative evaluation unit that compares the comparison data of the data storage unit and quantitatively evaluates the live bacteria. Viable bacteria quantification device.

3. 2. The method for quantifying viable bacteria according to claim 1, wherein in the current peak measurement step, the measurement solution is electrochemically measured using an electrode tip to which a preset amount of the redox substance is attached, and a current peak of the residual redox substance remaining in the measurement solution is obtained.

4. A kit for quantifying live bacteria used in the method for quantifying live bacteria according to claim 1, or a kit for quantifying live bacteria used in the apparatus for quantifying live bacteria according to claim 2, The substance has a redox property that is cell membrane permeable to the live bacteria to be measured and is oxidized or reduced within the cells to be deposited within the cells. The viable bacteria are placed in a measurement solution containing the redox substances and mixed for a predetermined time, and the redox substances are taken up and deposited in the cells of the viable bacteria. Then, the measurement solution containing the remaining redox substances that have not been taken up by the viable bacteria is electrochemically measured, and at least one current peak (I Ｐ１ ) is used for current peak measurement to obtain Kit for quantifying live bacteria.

5. The current peak measurement unit uses an electrode tip to which a preset amount of the redox substance is attached to perform electrochemical measurement of a measurement solution containing remaining redox substances that have not been taken up by live bacteria, and measures at least one current peak (I Ｐ１ 3. The viable bacteria quantification apparatus according to claim 2, wherein the viable bacteria quantification apparatus determines the amount of viable bacteria present in the viable bacteria.