Cell activity measuring device, cell activity measuring method, and cell activity measuring kit

Abstract

Provided is a cell activity measuring method that can, unlike conventional absorbance measurements or electrochemical measurements, evaluate the cell activity and the number of active cells by optically measuring scattered light from an insoluble reduced substance that becomes insoluble after being reduced in cells. This cell activity measuring method comprises: a deposition step for inputting measurement target cells in a measurement solution containing a soluble substance that has cell membrane permeability with respect to the measurement target cells and that can be reduced in active cells to form an insoluble reduced substance that is deposited in the active cells, and causing uptake of the soluble substance, formation of the insoluble reduced substance, and deposition of the same in the active cells; and, after the deposition step, an optical determination step for determining the cell activity level of respective cells on the basis of a specific color obtained by scattered light from the insoluble reduced substance in the cells in a deposited state.

Description

Cellular activity measuring device, measuring method and measuring kit

[0001] The present invention relates to a device and method for measuring cell activity and a kit for the same, and more particularly to a device and method capable of evaluating cell activity by optical testing using, for example, a non-soluble reducing substance that becomes non-soluble after reduction.

[0002] Cellular activity is an essential evaluation metric for hygiene and quality control in the medical, pharmaceutical, and food industries. For example, due to the small size of bacterial cells (a few micrometers), culture methods that observe colonies formed by the growth of single cells on a plate or flow cytometry are widely used for evaluation. However, these methods have drawbacks, such as long culture times (more than one day), the need for skilled techniques, and expensive equipment. To address these issues, spectroscopic and electrochemical measurements focusing on the dissolved oxygen consumed by bacterial aerobic respiration have been used. However, these methods are not sensitive enough to evaluate the activity of single cells. In animal cells, in addition to fluorescent imaging of signal transduction and electrochemical measurements of cellular respiration, methods have been established to visualize intracellular temperature distribution using thermosensitive fluorescent labels. Individual methods for measuring cellular activity focusing on fluorescence, dissolved oxygen, and reaction heat have been developed. While high-resolution measurements are possible due to the large size of cells, quantitative evaluation of activity has been difficult due to the short and unstable fluorescence lifetime and the dependence of dissolved oxygen on the environment, such as temperature and dissolved substances.

[0003] 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 live bacteria, culturing them to cause a color change (yellow to purple), and then measuring the spectral absorption wavelength. This method typically requires culturing for 5 hours or more. For example, Patent Document 1 discloses that mitochondrial functionality can be determined by using MTT, which is reduced by the metabolism of mitochondrial enzymes to produce a blue compound (MTT formazan), and determining the concentration of MTT formazan by colorimetric analysis (absorbance measurement).

[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] Non-Patent Document 2 describes a method in which MTT, a redox substance, is taken up by live bacteria and living cells or amoebas equivalent to live bacteria, MTT formazan is produced and deposited within the live bacteria, and the MTT formazan accumulated within the cells is measured electrochemically. In this method, the current peak measurement observes the MTT formazan production in all cells present in the culture system, and measurement of individual cells is not possible.

[0006] Non-Patent Document 3 describes that live bacteria reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to produce insoluble particles that exhibit strong resonantly scattered light, and that there is a linear relationship between the number of live bacteria and the intensity of the resonantly scattered light. In this document, a bacterial dispersion (suspension) is used, and the intensity of elastically scattered light, which has the same wavelength as the incident light, is significantly affected by scattering by the cells themselves and by changes in scattering due to dispersion and aggregation. Furthermore, it is not possible to measure individual cells.

[0007] Special Publication No. 2017-52238

[0008] 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, pp. 10903-10909 Authors: "Hikaru Ikeda, Satohiro Itagaki, Shigeki Nishii, Yojiro Yamamoto, Yasuhiro Sadanaga, Hiroshi Shiigi", "Electrochemical measurement of microbial activity", Review of Polarography 2022, Vol. 68, No. 1, pp. 15-25 Author “Yu Jun Shi, Jun Chen, Ming Xu” A New Method for Antimicrobial Susceptibility Testing of Vitro-cultured Bacteria by Means of Resonance Light Scattering Technique, J. Microbiol. Biotechnol 2008, Vol. 18, No. 1, pp. 118-123

[0009] Unlike conventional absorbance measurements or electrochemical measurements, the present invention provides a cell activity measurement device, measurement method, and cell activity kit that can evaluate the cell activity and number of active cells on a cell-by-cell basis by optically measuring scattered light from insoluble reduced substances that become insoluble (non-soluble) after reduction within cells.

[0010] The method for measuring cell activity disclosed herein may include a deposition step in which the cells to be measured are placed in a measurement solution containing a soluble substance that is cell membrane permeable to the cells to be measured and that can be reduced within the active cells to form an insoluble reducing substance that deposits (or crystallizes (aggregates) and becomes insoluble) within the active cells, thereby allowing the soluble substance to be taken up within the active cells to form and deposit the insoluble reducing substance, and an optical determination step in which, after the deposition step, the cell activity of each cell is determined based on the specific color of scattered light from the insoluble reducing substance within the cells in a deposited state.

[0011] The deposition step is performed for a predetermined time period set according to various conditions, and may include, for example, a time period during which the measurement solution containing a soluble substance capable of forming a non-soluble reducing substance is mixed with, contacted with, or left to stand with the cells to be measured. The optical determination step may include, after the deposition step, an optical observation step in which the deposited cells (including active cells and inactive cells) are observed by generating scattered light using an optical observation means. In the optical observation step, a sample may be collected from the measurement solution containing the deposited cells, for example, a suspension or a precipitate after centrifugation, and this sample may be observed using an optical observation means. Examples of the optical observation means include a dark-field microscope and a bright-field microscope.

[0012] The predetermined time for the deposition step can be any time, for example, 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or more. The deposition step and the optical evaluation step can be performed at any time interval (e.g., 10-minute intervals) so that changes in the deposition state can be observed over time. The predetermined time may include, for example, the time required for mixing, contact time, or standing time. For example, it may be the total time for mixing and standing time. A "measurement solution containing cells in a deposited state" can be, for example, (1) a measurement solution obtained by placing the cells to be measured in a measurement solution containing a soluble substance that is cell membrane permeable to the cells to be measured and can be reduced in the cells to form an insoluble reduced substance that deposits (or becomes insoluble) within the cells, and allowing the cells to settle for a predetermined time; (2) a precipitate obtained by centrifuging the measurement solution after the deposition; or (3) a suspension after the predetermined time has elapsed. The "cells" may include multiple types of bacteria, multiple types of cells, or animal cells. The cells to be measured may include both activated and inactivated cells, and activated cells may be inactivated during the deposition and optical determination steps.

[0013] The deposition step may include a step of preparing a measurement solution and a measurement target. The deposition step may include a step of adding the measurement target to the measurement solution, and mixing, contacting, and leaving the mixture stationary for a predetermined period of time. The deposition step may include a culture step of shaking culture or static culture at a predetermined temperature (e.g., 290 K to 320 K) during the mixing step (including mixing, contacting, leaving the mixture stationary after mixing, and leaving the mixture stationary after contact). The deposition step may include a centrifugation step of performing a predetermined centrifugation treatment.

[0014] The optical determination step may include an active cell counting step of counting the number of active cells (or inactive cells) indicated by the specific color in a predetermined area (e.g., a unit area of ​​a predetermined frame within a field of view) by image analysis or visual inspection. The optical determination step may also include a viability determination step of determining the viability of individual cells based on the presence or absence or luminance (brightness) of the specific color by image analysis or visual inspection. "Scattered light of a specific color" indicates that a non-soluble reducing substance (e.g., MTT formazan) has crystallized (deposited) within the cell. The specific color varies depending on the type of non-soluble reducing substance, type of bacteria, type of cell, and type of measurement solution, and may be, for example, red, orange, yellow, purple, blue, or green.

[0015] The optical evaluation step may include an imaging step of acquiring an observation image (scattered light image) using an imaging device. The optical evaluation step may also include an active cell number calculation step of analyzing the observation image (scattered light image) acquired in the imaging step and calculating at least the number of active cells based on the number of non-soluble reducing substances crystallized (deposited) within the cells. The optical evaluation step may also include a cell activity evaluation step of analyzing the observation image (scattered light image) acquired in the imaging step and determining the cell activity of individual cells based on the size (crystallization area, spread) of the non-soluble reducing substances crystallized (deposited) within individual cells (including active cells and inactive cells) or the size of crystallization (deposition) over a predetermined time period (any time or change over time) during the deposition step. For example, the cell activity is determined to be high in proportion to the crystallization area relative to the deposition time from the deposition step to optical evaluation. Typically, the crystallization area increases in proportion to the deposition time. Even if the deposition time is short, if the crystallization area is large, the cell activity is judged to be high. The "deposition time" may be any time, for example, from more than 0 minutes to 120 minutes.

[0016] The optical evaluation step may include a light scattering spectrum measurement step in which a measurement sample (e.g., a suspension) is irradiated with light and the spectrum is measured using scattered light. The light scattering spectrum measurement step may use, for example, Raman scattering spectroscopy, Brillouin scattering spectroscopy, or may involve measuring an extinction spectrum. The extinction spectrum is the sum of the absorption spectrum and the scattering spectrum, and scattering characteristics can be evaluated by analyzing the extinction spectrum. The light scattering spectrum measurement step may also include a spectral cell activity evaluation step in which the cell activity of individual cells is determined based on the intensity of a specific color wavelength that indicates the deposited crystallized product. For example, light scattering spectra are measured over the deposition time from the deposition step to the optical evaluation, and the cell activity is determined to be high in proportion to the intensity of the specific color wavelength (e.g., peak height, peak area, peak area ratio at two wavelengths).

[0017] The first cell activity measuring device of the present disclosure may include an active cell number calculating unit that performs image analysis of the scattered light image obtained by placing cells to be measured in a test solution containing a soluble substance that is cell membrane permeable and can be reduced within the active cells to form a non-soluble reduced substance that deposits within the active cells, irradiating the cells with light so that the soluble substance is taken up within the active cells to form the non-soluble reduced substance and deposit it, and then calculating at least the number of active cells based on the number of non-soluble reduced substances crystallized within each cell. The cell activity measuring device may also include a cell activity determining unit that performs image analysis of the scattered light image obtained by the imaging device and determines the cell activity of each cell based on the size of the non-soluble reduced substance crystallized (deposited) within the cells or the size of crystallization over a predetermined time period (an arbitrary time period or a change over time). A sample, for example, a suspension or a precipitate after centrifugation, may be collected from the test solution containing the deposited cells, and the sample may be observed using an optical observation device. Examples of the optical observation device include a dark-field microscope and a bright-field microscope. The image analysis in the viable cell number calculation unit may include, for example, a process of counting the number of crystallizations shown in a specific color within the cells after binarization processing. The image analysis in the cell activity determination unit may include, for example, a process of determining the area of ​​crystallizations shown in a specific color within the cells from the number of pixels after binarization processing.

[0018] The second cell activity measuring device of the present disclosure may include an optical observation means for irradiating light onto the cells in a state in which the cells to be measured are in a measurement solution containing a soluble substance that is cell membrane permeable to the cells and can be reduced within the active cells to form a non-soluble reduced substance that deposits within the active cells, thereby causing the soluble substance to be taken up within the active cells, forming and depositing the non-soluble reduced substance, and generating scattered light; an imaging device for capturing the scattered light generated by the optical observation means and generating a scattered light image; an active cell number calculation unit for performing image analysis of the scattered light image obtained by the imaging device and calculating at least the number of active cells based on the number of non-soluble reduced substances that have crystallized within each cell; and a cell activity determination unit for performing image analysis of the scattered light image obtained by the imaging device and determining the cell activity of each cell based on the size of the non-soluble reduced substance that has crystallized (deposited) within the cells or the size of the crystallization (deposition) over a predetermined time (an arbitrary time or change over time).

[0019] The active cell number calculation unit of the first and second cell activity measuring devices may calculate the active cell rate (viable cell rate). The active cell rate can be calculated using the following formula (1): Active cell rate (%) = number of cells that exhibited crystallization (formazan production) / total number of cells (1) Total number of cells = number of active cells + number of inactive cells + number of cells determined to be other cells Number of cells that exhibited crystallization = number of active cells The number of cells that exhibited crystallization may be counted for each elapsed time of mixing for a predetermined period of time (including static state), and the active cell rate may be calculated, and the cell activity may be determined from the fluctuations thereof.

[0020] A third disclosed cell activity measuring device may include an optical observation means for irradiating light onto the cells in a state in which the cells are in a state in which the cells are taken up into a measurement solution containing a soluble substance that is cell membrane permeable and can be reduced within the active cells to form a non-soluble reduced substance that deposits within the active cells, thereby causing the soluble substance to be taken up into the active cells, forming and depositing the non-soluble reduced substance, thereby producing scattered light; a light scattering spectrum measuring device for measuring a spectrum using the scattered light; and a spectral cell activity determining unit for determining the cell activity of each cell based on the intensity or change in intensity of a specific color wavelength obtained from the light scattering spectrum measuring device. The "intensity of cell activity" is proportional to the size of the crystallization area and is proportional to the scattered light intensity and the intensity of the spectrally analyzed peak wavelength (peak height, peak area, peak area ratio at two wavelengths).

[0021] The spectral cell activity determination step and the cell activity determination unit may determine cell activity from a peak area ratio obtained by dividing the peak area of ​​a first wavelength (e.g., 670 nm) by the peak area of ​​a second wavelength (e.g., 600 nm) in the scattered light spectrum of a single cell. The average value of the peak area ratios for each single cell may be used as the average peak area ratio to determine the average peak area ratio cell activity. Multiple thresholds may be set for the peak area ratio (average peak area ratio) to classify cell activity. By setting multiple thresholds, cell activity can be classified into levels such as low, medium, and high, allowing, for example, the evaluation of injured bacteria.

[0022] The first, second, and third cell activity measuring devices may include a storage medium for storing the results obtained by the activated cell number calculation unit (e.g., activated cell number, activated cell rate), the results obtained by the cell activity determination unit (e.g., cell activity), and the results obtained by the spectral cell activity determination unit (e.g., cell activity, peak intensity of a wavelength indicating a specific color). The cell activity measuring device may also include an output unit for outputting the results obtained by the activated cell calculation unit (e.g., activated cell number, activated cell rate), the results obtained by the cell activity determination unit (e.g., cell activity), the results obtained by the spectral cell activity determination unit (e.g., cell activity, peak intensity of a wavelength indicating a specific color), or data 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.

[0023] The first cellular activity measurement kit is used in the above-mentioned cellular activity measurement method or cellular activity measurement device, and includes a soluble substance that is cell membrane permeable to the cells to be measured and can be reduced within the active cells to form an insoluble reducing substance that deposits (or becomes insolubilized) within the cells. The kit may also include an immobilization carrier to which the soluble substance is attached. The immobilization carrier may include, for example, a hydrophilic polymer, a hydrophobic polymer, or various surface treatment layers. The immobilization carrier may be formed on the surface of a plate or the inner surface of a container. The solvent for dissolving the soluble substance may be individually packaged in a package or container. The container for the solvent may be used as a measurement container by putting the soluble substance in it. The cellular activity measurement kit may also include a solvent for the soluble substance.

[0024] The first live bacteria measurement kit is used in the above-mentioned cell activity measurement method or cell activity measurement device, and may include a plurality of packages, measurement containers, or plates containing the soluble substance. The content, type, and concentration of the soluble substance relative to the solvent contained in the plurality of packages or measurement containers may be different from each other.

[0025] (Cells to be measured) The cells are living cells, and examples thereof include Escherichia coli (e. coli), Salmonella enterica (S. enterica), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), Serratia marcescens, Bacillus cereus, Staphylococcus epidermidis, Pseudomonas fluorescens, other bacterial cells, and animal cells.

[0026] (Soluble Substances) Soluble substances are cell membrane permeable and active, and are reduced within the cells, where they are deposited and insolubilized to form insoluble reduced substances. Examples of soluble 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.

[0027] (Solvent of Measurement Solution) 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).

[0028] Another disclosed computer program, when executed by at least one processor, performs each step (excluding the deposition step) of the above-described method for measuring cellular activity. Another disclosed storage medium storing the computer program, when executed by at least one processor, performs each step (excluding the deposition step) of the above-described method for measuring cellular activity. Another disclosed information processing device includes at least one processor and a memory (which may be the above-described storage medium) storing a computer program executed by the processor, and when executed by the at least one processor, performs each step (excluding the deposition step) of the above-described method for measuring cellular activity. The information processing device is not particularly limited and may, for example, be a smartphone, tablet, smartwatch, wearable computer, personal computer, local server, cloud server, etc., and a single or a combination of information processing devices may be configured to be connectable via wired and / or wireless communication means.

[0029] The activated cell number calculation unit, cell activity determination unit, spectral cell activity determination unit, and output unit of the cell activity measurement device may be configured with, for example, a dedicated circuit, firmware, a memory storing a processor and a processing program, a display, a bus, an input / output interface, a communication device, etc. The imaging device may have a camera function with an imaging element, a memory for storing captured images as digital data, and a communication means for transmitting the digital data to the outside.

[0030] [Effects] (1) By observing individual cells using optical means, it is possible to easily determine whether a cell is active or inactive at the cell level. Furthermore, the intensity of cell activity can be easily determined from the size of the crystallized area per cell and the scattered light intensity over time (spectral intensity per cell). (2) As formazan deposition and crystallization (aggregation) progress within cells, the activity of a single cell can be determined from the size and spectral intensity of formazan at the cell level. (3) Activity based on cell metabolism can be evaluated. For example, cell activity, fermentation control of yeast, and the activity of eukaryotic and other animal cells can be evaluated at the single-cell level. (4) The intensity of specific wavelengths in the spectrum obtained under a dark-field microscope can be measured with high sensitivity, allowing for quantitative evaluation. This is also useful for evaluating injured bacteria, whose activity varies slightly between individual cells, as well as the effects of antibacterial agents, disinfectants, and antibiotics, and for evaluating drug-resistant bacteria. (5) By setting a threshold for the intensity of specific wavelengths in the spectrum, it is possible to perform batch counting based on cell activity using image analysis. (6) In the conventional MTT assay, the average evaluation of all bacterial cells contained in the dispersion was performed, and it was not possible to evaluate individual cells. However, in the present invention, the activity of individual cells can be measured quickly, and the degree of cell activity can be accurately determined.

[0031] 1 is a flow chart showing an example of steps in a method for measuring cell activity. FIG. 1 is a flow chart showing an example of steps in a method for measuring cell activity. FIG. 2 is a functional block diagram for explaining an example of the function of a device for measuring cell activity. FIG. 3 is a diagram showing a scattered light image (0 min) of Example 1. FIG. 4 is a diagram showing a scattered light image (10 min) of Example 1. FIG. 5 is a diagram showing a scattered light image (40 min) of Example 1. FIG. 6 is a diagram showing the results of image analysis of the scattered light image (40 min) of Example 1. FIG. 7 is a diagram showing the peak intensity of the scattered light spectrum of Example 1. FIG. 8 is a diagram showing a scattered light image (60 min) after autoclaving treatment of Example 2. FIG. 9 is a diagram showing a scattered light image (60 min) after UV irradiation treatment (15 min) of Example 2. FIG. 10 is a diagram showing a scattered light image (60 min) of Example 2. FIG. 11 is a diagram showing a scattered light image (60 min) after alcohol treatment of Example 2. FIG. 12 is a diagram showing scattered light images (10, 30, 60, 180 min) using MTT 2.4 mM of Example 3. 1 is a diagram showing peak intensity fluctuations (blank, 30, 60, 180 minutes) of the scattered light spectrum of Example 3. FIG. 2 is a diagram showing scattered light images (10, 30, 60 minutes) of Example 3 using 0.1 mM MTT. FIG. 3 is a diagram showing scattered light images (10, 30, 60 minutes) of Example 3 using 0.5 mM MTT. FIG. 4 is a diagram showing a scattered light image and scattered light spectrum of Example 4. FIG. 5 is a diagram showing a scattered light image and scattered light spectrum of Example 4. FIG. 6 is a diagram showing a scattered light image and scattered light spectrum of Example 4. FIG. 7 is a diagram showing a scattered light image and scattered light spectrum of Example 4. FIG. 8 is a diagram showing a scattered light image and scattered light spectrum of Example 4. FIG. 9 is a diagram showing a scattered light image and scattered light spectrum of Example 4. FIG. 10 is a diagram showing a viable cell count evaluation of Example 5 and the culture method. FIG. 11 is a diagram showing a viable cell count evaluation of Example 6 and the culture method. FIG. 12 is a diagram showing an example of an immobilization carrier plate. FIG. 13 is a diagram showing an example of an immobilization carrier plate. FIG. 14 is a diagram showing an example of an immobilization carrier plate. FIG. 1 shows an example of evaluation of injured bacteria.

[0032] (Embodiment 1) Figure 1A illustrates an example of the steps of a method for measuring cell activity. (S1) Deposition Step: A measurement solution is prepared (preparation step S1-1). The measurement solution contains a soluble substance that is cell membrane permeable to the cells to be measured and can be reduced within the cells to form an insoluble reduced substance that deposits and becomes insoluble within the active cells. The solvent for the measurement solution is purified water. Measurement targets include bacterial cells or animal cells.

[0033] Next, the object to be measured is added to the measurement solution (mixing step S1-2). Then, the mixture is cultured at a predetermined temperature (e.g., 290K to 320K) (culture step S1-3). During the culture step, soluble substances permeate the cell membranes of the cells and diffuse into the cells. Insoluble reducing substances, such as MTT, are reduced to MTT formazan by intracellular electron carriers such as NADH (nicotinamide adenine dinucleotide), NADPH (nicotinamide adenine dinucleotide phosphate), coenzymes, menaquinone, and ubiquinone. Because MTT formazan is insoluble, it deposits within individual cells and does not dissolve again in the measurement solution.

[0034] The cultured measurement solution is then centrifuged in a centrifuge (centrifugation step S1-4), and the precipitate is then collected (precipitate collection step S1-5).

[0035] Instead of the step of adding the object to be measured to the measurement solution (mixing step), the following step may be included: a step of dropping a liquid containing the cells to be measured onto the surface of an immobilization carrier plate carrying a soluble substance (e.g., MTT). The soluble substance permeates the cell membrane of the cells and diffuses into the cells. For example, MTT, since MTT formazan is insoluble, is deposited within individual cells and does not dissolve again in the measurement solution.

[0036] (S2) Optical Determination Step After the deposition step, the precipitate after centrifugation is observed for scattered light using a dark-field microscope or a bright-field microscope (optical observation step). The number of active cells indicated by scattered light of a specific color is counted visually in a predetermined area (for example, a predetermined frame within the field of view) (active cell number counting step S2-1). This allows the number of active cells to be measured. The active cell rate is calculated using the following formula (1): Active cell rate (%) = number of cells that exhibit crystallization (formazan production) / total number of cells (1) Total number of cells = number of active cells + number of inactive cells + number of cells determined as other cells Number of cells that exhibit crystallization = number of active cells The number of cells that exhibit crystallization may be counted for each predetermined time (any time or change over time) after mixing and leaving the mixture to stand, and the active cell rate is calculated, and the cell activity may be determined from the fluctuations.

[0037] FIG. 1B illustrates an example of another method for measuring cell activity. (S1) The deposition step is the same as that shown in FIG. 1A. (S21) Optical Observation Step After the deposition step, scattered light images of the precipitate obtained after centrifugation are captured with an imaging device using a dark-field microscope or a bright-field microscope (imaging step S21-1). The scattered light images are analyzed or visually inspected to calculate the number of active cells and the number of inactive cells based on the number of insoluble reducing substances crystallized (deposited) within each cell (viable cell count calculation step S21-2). Viability of each cell is determined based on the presence or absence of a specific color or its brightness (brightness) using image analysis or visual inspection (viability determination step). Inactive cells may be determined as dead cells, and others as live cells. The scattered light images are analyzed or visually inspected to determine the cell activity of each cell based on the size of the insoluble reducing substances crystallized (deposited) within each cell (crystallization area) or the size of crystallization (deposition) over a specified time period (cell activity determination step S21-3).

[0038] 1C illustrates an example of another method for measuring cell activity. (S1) The deposition step is the same as the preparation step, mixing step, and culturing step. The suspension is collected without centrifugation (suspension collection step S1-6).

[0039] (S22) Optical Observation Step After the deposition step, the suspension is irradiated with light and the spectrum is measured using scattered light (light scattering spectrum measurement step S22-1). Based on the spectral peak (the intensity of the wavelength of a specific color), the cell activity of each cell is determined (spectral cell activity determination step S22-2).

[0040] The cell activity measuring device 1 will be described with reference to Figure 2. The cell activity measuring device 1 comprises an acquisition unit 20, an active cell number calculation unit 21, a cell activity determination unit 22, and a spectrum cell activity determination unit 23. The acquisition unit 20 captures scattered light images obtained by a dark-field microscope 7 or a bright-field microscope with an imaging device 8, and acquires this scattered light image data. The acquisition unit 20 also acquires light scattering spectrum data obtained by a light scattering spectrum measuring device 9.

[0041] The active cell number calculation unit 21 performs image analysis on the scattered light image and calculates the number of active cells based on the number of crystallized (deposited) insoluble reducing substances within the cells. This image analysis may, for example, involve counting the number of crystallized cells indicated by a specific color within the cells after binarization. The active cell number calculation unit 21 may calculate the active cell rate using the following formula (1): Active cell rate (%) = Number of cells showing crystallization (formazan formation) / Total number of cells (1) Total number of cells = Number of active cells + Number of inactive cells + Number of other cells Number of cells showing crystallization = Number of active cells The number of cells showing crystallization may be counted for each predetermined time (any time or change over time) after mixing and leaving the cells to stand, and the active cell rate may be calculated. The cell activity of each individual cell may then be determined based on the fluctuations in the active cell rate.

[0042] The cell activity determination unit 22 performs image analysis of the scattered light image and determines the cell activity of each cell based on the size of the insoluble reducing substance crystallized (deposited) within the cell (crystallization area) or the size of the crystallization (deposition) relative to the predetermined mixing time. The image processing here may be, for example, a process of determining the area of ​​crystallization shown in a specific color within the cell from the number of pixels after binarization.

[0043] The spectral cell activity determination unit 23 determines the cell activity of each cell based on the spectral peak (the intensity of the wavelength of a specific color) obtained from the light scattering spectrum measurement device 9 .

[0044] The cell activity measuring device 1 may be composed of a cloud server, an on-premise server, an information processing device, etc. The acquisition unit 20 may be composed of a wireless communication means, a wired communication means, or a recording medium reading device. The display unit 10 has a function of displaying various data on a monitor. The input operation unit 11 has an input interface through which an operator inputs necessary operation data and instructions. The control unit 12 has a function of integrating and executing operation processing, input / output data processing, and control of each functional unit. The memory 13 stores various data. The output unit 14 may have a communication means for transmitting various data to the outside and a printer control function for printing. The memory 13 may store programs for executing various controls, and the processor may read out the programs and execute the processing.

[0045] The memory 13 stores various data including the results obtained by the active cell number calculation unit 21 (e.g., active cell number, active cell rate), the results obtained by the cell activity determination unit 22 (e.g., cell activity), and the results obtained by the spectral cell activity determination unit 23 (e.g., cell activity, peak intensity of a wavelength indicating a specific color). The output unit 14 outputs various data including the results obtained by the active cell number calculation unit 21 (e.g., active cell number, active cell rate), the results obtained by the cell activity determination unit 22 (e.g., cell activity), and the results obtained by the spectral cell activity determination unit 23 (e.g., cell activity, peak intensity of a wavelength indicating a specific color).

[0046] The acquisition unit 20, the activated cell number calculation unit 21, the cell activity determination unit 22, the spectral cell activity determination unit 23, the control unit 12, the display unit 10, and the input operation unit 11 may be composed of dedicated circuits, firmware, computer programs, and hardware (processors, memories, etc.).

[0047] [Cell Activity Measurement Kit] A measurement solution containing a soluble substance and a solvent that can form an insoluble reducing substance that is cell membrane permeable to the cells to be measured and that can be reduced within the active cells and deposited (or insolubilized) within the cells is packaged in a measurement container. After the measurement target is placed in the measurement container and mixed, the resulting suspension or precipitate can be measured. When the kit contains only a soluble substance, the measurement target and water (solvent) can be placed in the container, the soluble substance can be added and mixed, and the resulting suspension or precipitate can be measured. The package is preferably a container that can seal the contents and prevent their physical properties from deteriorating, such as a plastic bottle container, a film packaging container, or a glass container.

[0048] An example of an immobilization carrier plate is shown in Figure 10A. An immobilization carrier is formed on the surface of a glass plate. Examples of immobilization carriers include hydrophilic polymers, hydrophobic polymers, and various surface modification layers. Examples of hydrophilic polymers include agar, agarose gel, polyvinyl alcohol, and polyvinylpyrrolidone. Examples of hydrophobic polymers include polypyrrole, polyaniline, polythiophene, PEDOT, and polyvinyl chloride. Examples of surface modification include hydrophilization treatment using plasma and silane coupling agents (Figure 10A(b)).

[0049] (Example 1: Escherichia coli K12) Example 1 was carried out under the following conditions. Soluble substance: MTT Initial concentration of MTT in measurement solution: 0.10 mM Glucose 0.1% (w / v) 5 mL Solvent of measurement solution: Sterilized pure water Cells to be measured: Escherichia coli K12 Cell concentration in measurement solution: 1 x 10 １２ [CFU / mL] 5 μL Deposition time: 37°C 10, 40, 60 minutes (constant temperature incubator) Centrifugation time: 10,000 x G, 10 minutes Dark field microscope: Nikon Light scattering spectrum analyzer: Ocean Optics

[0050] <Operation Procedure 1> (1) Place E. coli K12 in a container containing an MTT-containing measurement solution. (2) Incubate for 10, 40, and 60 minutes. MTT permeates the cell membrane of E. coli K12, is reduced within the cells, and precipitates as MTT formazan. (3) Centrifuge the measurement containers after incubation for 10, 40, and 60 minutes, and collect the precipitates. (4) Visually observe each precipitate using a dark-field microscope. Capture scattered light images using an imaging device.

[0051] Figure 3A shows a scattered light image of the E. coli K12 sample before addition to the test solution. The contours of the E. coli K12 cell membrane can be identified and distinguished. Figure 3B shows a scattered light image of a precipitate cultured for 10 minutes. In addition to the cell membrane, crystallized areas of MTT formazan can be identified and distinguished by a specific color (red). By counting the specific color, the number of active E. coli K12 cells can be determined. Furthermore, the area and brightness of the specific color differ depending on the cell. This allows the intensity of activity in a single cell to be determined. Figure 3C shows a scattered light image of a precipitate cultured for 40 minutes. The scattered light image of Figure 3C after 40 minutes of culture shows a greater number of crystallized MTT formazan deposited within the cells than the scattered light image of Figure 3B after 10 minutes of mixing. It can also be seen that the intensity and area of ​​the specific color per cell differ. Figure 3D shows a scattered light image of a precipitate cultured for 60 minutes. The scattered light image of Figure 3D after 60 minutes of incubation shows a greater number of crystallized MTT formazan deposited within the cells than the scattered light image of Figure 3C after 40 minutes of incubation. It can also be seen that the intensity and area of ​​the specific color per cell are different. (5) Count the number of cells that exhibit the specific crystallized color (red). Calculate the percentage of active cells.

[0052] Figure 3E(a) shows the results of image analysis of the scattered light image (40 minutes). Note that Figure 3E uses a different image from the image in Figure 3C. Figure 3E(b) shows the results of extracting MTT formazan (red) from image (a) after image processing (binarization) using the active cell number calculation unit. The number of MTT formazan (red) cells was 97. Figure 3E(c) shows the results of extracting cells from image (a) after image processing (binarization) using the active cell number calculation unit. The total number of cells was 173. Meanwhile, the total number of cells visually observed in image (a) was 249, and the number of MTT formazan (red) cells was 91. It was confirmed that the visual results and the image processing results were equivalent.

[0053] <Operation Procedure 2> (1) Place E. coli K12 into a container containing an MTT-containing measurement solution. (2) Incubate for 40 minutes. MTT permeates the cell membrane of E. coli K12, is reduced within the cells, and precipitates as MTT formazan. (3) After 40 minutes of incubation, collect the suspension from the measurement container. (4) Measure each suspension using a light scattering spectrometer. (5) Determine the peak and peak area at a wavelength (670 nm) of a specific color (red) in each spectrum. The intensity of cell activity is determined in proportion to the peak area.

[0054] Figure 4 shows the results of scattering spectrum analysis. The graph shows the ratio of the 500 nm and 670 nm peak areas in a single cell divided by the 600 nm peak area. The ratio of the 500 nm peak area divided by the 600 nm peak area was constant (approximately 0.2) for each cell, but the ratio of the 670 nm peak area divided by the 600 nm peak area increased from 0 to 0.8 with increasing size and number of MTT formazan molecules in the cells. These results indicate a correlation between the size of the peak area at a specific wavelength (670 nm) and the crystallization area (specific color), allowing the intensity of cellular activity to be determined from spectral analysis.

[0055] Example 2 shows an example in which no inactivation treatments were performed, and a reference example in which high-pressure steam sterilization treatment using an autoclave, ultraviolet irradiation treatment, and alcohol treatment were performed. Soluble substance: MTT Initial concentration of MTT in measurement solution: 0.10 mM Glucose 0.1% (w / v) 5 mL Solvent of measurement solution: Sterilized pure water Cells to be measured: Escherichia coli K12 Cell concentration in measurement solution: 3.2 x 10 １１ [CFU / mL] 1 μL Deposition time: 37°C, 60 minutes (constant temperature incubator) Centrifugation time: 10,000 x G, 3 minutes Dark field microscope: Nikon Light scattering spectrum analyzer: Ocean Optics Autoclave: Tomy Seiko Co., Ltd.; conditions: 0.2 MPa, 121°C, 20 minutes Ultraviolet irradiation device: Panasonic Healthcare Co., Ltd.; irradiation time: 15 minutes

[0056] <Operation Procedure 1> (1-0) Dispersion of E. coli K12 was added to 3.2 × 10 １１ (1-1) The E. coli K12 dispersion was autoclaved, and the number of CFU / mL was adjusted to about 10 １１ (1-2) The E. coli K12 dispersion was irradiated with UV light for 15 minutes, and the concentration of the bacteria was determined by visual observation to be approximately 10 １１ (1-3) The E. coli K12 sample was dispersed in 70% ethanol water, left for 30 minutes, centrifuged (×10,000 G, 3 minutes), the ethanol was removed, and the sample was dispersed in sterilized water. １１ (2) Three microtubes containing 1 mL of nutrient bouillon (NB) medium prepared to 0.10 mM MTT and 0.1% (w / v) glucose were placed in a 37°C incubator, and the E. coli dispersions (1-1), (1-2), and (1-3) and (1-0) were added to a concentration of 3.2 × 10 cells / mL. １１1 μL of each E. coli dispersion solution (CFU / mL) was added. (3) After 1 hour, the mixture was centrifuged to obtain a precipitate (5°C, 10,000 G, 3 minutes). (4) Sterile water was added, the mixture was dispersed, and then centrifuged (5°C, 10,000 G, 3 minutes). (5) The supernatant was discarded, the mixture was dispersed in sterile water, and then centrifuged (5°C, 10,000 G, 3 minutes). (6) The above steps (4-5) were repeated. (7) The supernatant was discarded, and the resulting precipitate was dispersed in 1 mL of sterile water. (8) 10 μL of each E. coli dispersion solution was dropped onto a glass slide, and the mixture was left to stand in a refrigerator at 4°C for 17 hours and dried. (9) Each glass slide was observed under a transmission dark-field microscope (exposure time 60 ms).

[0057] FIG. 5A shows a scattered light image of autoclaved E. coli K12. E. coli K12 cells can be seen, but the specific color (red) of MTT formazan crystallized areas cannot be confirmed. FIG. 5B shows a scattered light image of ultraviolet-irradiated E. coli K12. E. coli K12 cells can be seen, but the specific color (red) of MTT formazan crystallized areas can be confirmed to a small extent. FIG. 5C shows a scattered light image of E. coli K12 that has not been subjected to either of the above treatments. The specific color (red) of MTT formazan crystallized areas within the E. coli K12 cells can be confirmed. FIG. 5D shows a scattered light image of alcohol-treated E. coli K12. E. coli K12 cells can be seen, but the specific color (red) of MTT formazan crystallized areas cannot be confirmed.

[0058] The total number of cells in the field of view and the number of cells showing red scattered light, which indicates the formation of MTT formazan, were counted from images of untreated E. coli K12, E. coli K12 treated with ultraviolet light for 15 minutes, autoclaved E. coli K12, and alcohol-treated E. coli K12 observed under a transmitted light dark-field microscope (n=3).

[0059] Without treatment, cells divided in response to the conditions, resulting in the highest total cell count and the highest number of cells that produced formazan. In contrast, with UV irradiation treatment, both the total cell count and the number of cells that produced formazan were low. With autoclaving and alcohol treatment, all cells died, and no formazan production, which indicates cell activity, was observed.

[0060] Example 3 In Example 3, 4T1 cells were used. Soluble substance: MTT Cells to be measured: 4T1 cells Dark-field microscope: manufactured by Nikon Corporation Light scattering spectrum analyzer: manufactured by Ocean Optics

[0061] <Procedure 1> (1) Seed 4T1 cells (1 x 10) into a 48-well plate. ５ cells / well, ×14 wells) (2) After 20 hours, the medium and MTT reagent were replaced. PBS (phosphate-buffered saline) containing 10 mg / mL MTT was diluted 10-fold with DMEM (Dulbecco's Modified Eagle Medium) and replaced with 0.25 mL (MTT 2.4 mM, ×6 wells). Blank: DMEM was left unreplaced (×2 wells). (3) 10, 30, 60, and 180 minutes after the addition of MTT, the medium in each well was aspirated using a heat-sterilized Pasteur (the timing of adding the reagent was staggered, and the operations after washing were performed simultaneously). (4) The cells were washed twice with 0.5 mL / well of PBS. (5) Add 0.2 mL of trypsin per well, shake well, and incubate at 37°C for 3 minutes. (6) Observe under a microscope to confirm that the cells have detached. (7) Tap the plate and add 0.2 mL of DMEM. (8) Transfer the entire volume to a microtube and centrifuge (1500 rpm, 2 minutes). (9) Aspirate the liquid using a heat-sterilized Pasteur tube and tap. (10) Add 100 μL of sterilized water (1 well per condition, 7 wells in total) or 100 μL of colorless medium (1 well per condition, 7 wells in total) and disperse by pipetting. (11) Drop onto a MAS-coated slide. (12) Observe under a dark-field microscope.

[0062] Figure 6A shows scattered light images of precipitates cultured for 10, 30, 60, and 180 minutes. It was confirmed that the crystallized areas of MTT formazan became stronger as the incubation time increased, as a specific color (red) became stronger. Figure 6B shows the scattered light spectrum. (a) It was confirmed that the intensity of the 700 nm peak increased in proportion to the incubation time. (b) The peak area ratio (peak area at a specific wavelength relative to the area in the entire wavelength range) for wavelengths of 500 nm, 600 nm, and 700 nm is shown. At 700 nm, the blank was 0, but it rapidly increased in proportion to the incubation time. In contrast, it was confirmed that at 600 nm, it rapidly decreased in proportion to the incubation time.

[0063] <Operation Procedure 2> Differences from Operation Procedure 1 above are shown in (2) and (3), with the exception that the rest is the same as Operation Procedure 1. (2) After 20 hours, replace the medium with the MTT reagent. (i): Replace 0.25 mL of PBS containing 2 mg / mL MTT diluted 10 times with DMEM (MTT 0.5 mM). (ii): Replace 0.25 mL of PBS containing 0.4 mg / mL MTT diluted 10 times with DMEM (MTT 0.1 mM). (3) 10, 30, and 60 minutes after the addition of MTT in (i) and (ii), aspirate the medium from each well with a heat-sterilized Pasteur (the timing of adding the reagent was staggered, and the operations after washing were performed simultaneously in parallel).

[0064] Figure 7A shows scattered light images of precipitates cultured for 10, 30, and 60 minutes in (i). It was confirmed that the crystallized areas of MTT formazan showed a specific color (red) that became stronger with increasing incubation time. Figure 7B shows scattered light images of precipitates cultured for 10, 30, and 60 minutes in (ii). It was confirmed that the crystallized areas of MTT formazan showed a specific color (red) that became stronger with increasing incubation time. Figures 7B(a), (b), and (c) are images taken with a dark-field microscope, and Figures 7B(d) and (e) are images taken with a bright-field microscope. Even in Figures 7A and 7B, where the MTT concentration is lower than in Figure 6A, it was confirmed that the crystallized areas of MTT formazan showed a specific color (red).

[0065] (Example 4) Measurements were carried out for Salmonella, Serratia, Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Pseudomonas fluorescens under the following conditions: Soluble substance: MTT Initial concentration of MTT in measurement solution: 0.10 mM Glucose 0.1% (w / v) 1 mL Solvent of measurement solution: Sterilized pure water Cells to be measured: Salmonella, Serratia, Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Pseudomonas fluorescens Cell concentration in measurement solution: 1 x 10 １２ [CFU / mL] 5 μL Deposition time: 37°C, 60 minutes (constant temperature incubator) Centrifugation time: 10,000×G, 3 minutes Dark field microscope: Nikon Light scattering spectrum analyzer: Ocean Optics

[0066] <Operation Procedure 4> (1) 1 μL of each bacterial dispersion was added to 1 mL of NB medium (0.1% (w / v)) containing glucose with an MTT concentration of 0.10 mM. (2) Each was cultured for 60 minutes in a 37°C incubator. MTT permeates the cell membrane, is reduced within the cell, and precipitates as MTT formazan. (3) After culture, the cells were centrifuged (10,000×G, 3 minutes) and the supernatant was replaced with sterile water, a process repeated three times. (4) The sample was dropped onto a glass slide, dried, and then observed under a dark-field microscope (exposure time 100 ms). A scattered light image was captured using an imaging device. (5) The scattered light spectrum of the bacterial cells was measured using a spectrometer.

[0067] (Salmonella) Figure 8A(a) shows a scattered light image of Salmonella before adding MTT. The outline of the cell membrane of Salmonella can be identified. Figure 8A(b) shows the scattered light spectrum. Figure 8A(c) shows a scattered light image of Salmonella after observing the precipitate cultured for 60 minutes. The crystallized areas of MTT formazan were confirmed as a specific color (red). Figure 8A(d) shows the scattered light spectrum. A peak at a wavelength of 680 nm was confirmed, confirming the difference in spectrum from Figure 8(b).

[0068] (Serratia marcescens) Figure 8B(a) shows a scattered light image of Serratia marcescens before it was added to MTT. The outline of the cell membrane of Serratia marcescens can be identified. Figure 8B(b) shows the scattered light spectrum. Figure 8B(c) shows a scattered light image of Serratia marcescens, observing the precipitate after culturing for 60 minutes. The areas where MTT formazan had crystallized were confirmed as a specific color (reddish purple). Figure 8B(d) shows the scattered light spectrum. A peak was confirmed at wavelengths of 690-700 nm, confirming the difference in spectrum from Figure 8B(b).

[0069] (Bacillus cereus) Figure 8C(a) shows a scattered light image of Bacillus cereus before it was added to MTT. The outline of the cell membrane of Bacillus cereus can be identified. Figure 8C(b) shows the scattered light spectrum. Figure 8C(c) shows a scattered light image of Bacillus cereus after culturing for 60 minutes and observing the precipitate. The areas where MTT formazan had crystallized were confirmed as a specific color (red). Figure 8C(d) shows the scattered light spectrum. A peak at wavelengths of 670-680 nm was confirmed, confirming the difference in spectrum from Figure 8C(b).

[0070] (Staphylococcus aureus) Figure 8D(a) shows a scattered light image of Staphylococcus aureus before adding MTT. The outline of the cell membrane of Staphylococcus aureus can be identified and distinguished. Figure 8D(b) shows the scattered light spectrum. Figure 8D(c) shows a scattered light image of Staphylococcus aureus after observing the precipitate that was cultured for 60 minutes. The area where MTT formazan crystallized was confirmed as a specific color (red). Figure 8D(d) shows the scattered light spectrum. A peak at wavelengths of 670-680 nm was confirmed, confirming the difference in spectrum from Figure 8D(b).

[0071] (Staphylococcus epidermidis) Figure 8E(a) shows a scattered light image of Staphylococcus epidermidis before adding MTT. The outline of the cell membrane of Staphylococcus epidermidis can be identified. Figure 8E(b) shows the scattered light spectrum. Figure 8E(c) shows a scattered light image of Staphylococcus epidermidis after observing the precipitate that was cultured for 60 minutes. The areas where MTT formazan had crystallized were confirmed as a specific color (red). Figure 8E(d) shows the scattered light spectrum. A peak was confirmed at wavelengths of 670-680 nm, confirming the difference in spectrum from Figure 8E(b).

[0072] (Pseudomonas aeruginosa) Figure 8F(a) shows a scattered light image of Pseudomonas aeruginosa before addition to MTT. The outline of the Pseudomonas aeruginosa cell membrane can be identified and distinguished. Figure 8F(b) shows the scattered light spectrum. Figure 8F(c) shows a scattered light image of Pseudomonas aeruginosa after observing the precipitate cultured for 60 minutes. The crystallized areas of MTT formazan were confirmed as a specific color (red). Figure 8F(d) shows the scattered light spectrum. A peak at wavelengths of 670-680 nm was confirmed, confirming the difference in spectrum from Figure 8F(b).

[0073] (Pseudomonas fluorescens) Figure 8G(a) shows a scattered light image of Pseudomonas fluorescens before addition to MTT. The outline of the Pseudomonas fluorescens cell membrane can be identified and distinguished. Figure 8G(b) shows the scattered light spectrum. Figure 8G(c) shows a scattered light image of Pseudomonas fluorescens after observing the precipitate cultured for 60 minutes. The areas where MTT formazan had crystallized were confirmed as a specific color (red). Figure 8G(d) shows the scattered light spectrum. A peak at wavelengths of 660-670 nm was confirmed, confirming the difference in spectrum from Figure 8G(b).

[0074] (Example 5: Evaluation of viable cell count) The viable cell count contained in spoiled minced chicken meat was calculated and compared with the culture method. Example 5 was carried out under the following conditions. Soluble substance: MTT Deposition time: 37°C, 15 minutes (constant temperature incubator) Centrifugation time: 10,000 x G, 10 minutes Dark field microscope: manufactured by Nikon Corporation Light scattering spectrum analyzer: manufactured by Ocean Optics

[0075] <Operation Procedure 1> (1) 25 g of minced chicken meat was left in a 37°C incubator for 18 hours. (2) The mixture was dispersed in 225 mL of sterilized water and filtered. (3) The viable bacterial count in the filtered sample was evaluated using Petrifilm (AC). (4) MTT was added to NB medium containing 0.1% (w / v) glucose, and the concentration was adjusted to 0.5 mM. (5) 100 μL of the sample was added to the medium prepared in (3) above, and the mixture was allowed to stand at 37°C for 15 minutes. (6) Centrifugal washing was repeated twice, and the mixture was dispersed in 1.0 mL of sterilized water. (7) 1.0 μL of the dispersion liquid from (5) above was dropped onto a slide glass and dried in a refrigerator at 4°C for 5 hours. (8) The mixture was observed under a dark-field microscope (x100, exposure time 100 ms).

[0076] The number of cells forming MTT formazan was counted in multiple fields of view by visual inspection and image processing. The total number of bacteria was estimated taking into account the multiple fields of view. An example of scattered light image data is shown in Figure 9A. The number of cells was counted in multiple fields of view 1 to 8, and the average was 6.8. The area of ​​the field of view was calculated using the scale bar, and the cell number (viable bacteria number) was converted taking into account the ratio to the area of ​​the 1.0 μL drop on the glass surface, and the viable bacteria concentration of the sample was calculated from the dilution of the sample. The viable bacteria concentration was 7.5 x 10 ７ cells / mL.

[0077] The culture method used a sheet medium for general viable count (3M (registered trademark) Petrifilm (AC) for measuring viable count). The general viable count contained in 1.0 mL of sample was 6.0 x 10 ７ CFU / mL.

[0078] (Example 6: Evaluation of viable cell count) The viable cell count contained in pond water was calculated and compared with the culture method. Example 6 was carried out under the following conditions. Soluble substance: MTT Deposition time: 37°C, 15 minutes (constant temperature incubator) Centrifugation time: 10,000 x G, 10 minutes Dark field microscope: manufactured by Nikon Corporation Light scattering spectrum analyzer: manufactured by Ocean Optics

[0079] <Operation Procedure 1> (1) One liter of water was collected from a university pond. (2) After repeated filtering, centrifugal concentration, and decantation, the water was concentrated to a final volume of 500 μL (sample). (3) The general viable bacterial count in the sample was evaluated using Petrifilm (AC). (4) MTT was added to NB medium containing 0.1% (w / v) glucose, and the concentration was adjusted to 0.5 mM. (5) 100 μL of the sample was added to the medium prepared in (3) above, and allowed to stand at 37°C for 15 minutes. (6) Centrifugal washing was repeated twice, and the sample was dispersed in 1.0 mL of sterilized water. (7) 1.0 μL of the dispersion liquid from (5) above was dropped onto a glass slide and dried in a refrigerator at 4°C for 5 hours. (8) The sample was observed using a dark-field microscope (x100, exposure time 100 ms).

[0080] The number of cells forming MTT formazan was counted in multiple fields of view by visual inspection and image processing. The total number of bacteria was estimated taking into account the multiple fields of view. An example of scattered light image data is shown in Figure 9B. The number of cells was counted in multiple fields of view 1 to 4, and the average was 2.25. The area of ​​the field of view was calculated using the scale bar, and the cell number (viable bacteria number) was converted taking into account the ratio to the area of ​​the 1.0 μL drop on the glass surface, and the viable bacteria concentration of the sample was calculated from the dilution of the sample. The viable bacteria concentration was 2.67 x 10 ７ cells / mL.

[0081] The culture method used a sheet medium for general viable count (3M (registered trademark) Petrifilm (AC) for viable count measurement). The general viable count contained in 300 μL of sample was 8.1 × 10 ６ Therefore, the viable cell concentration of this sample was 2.7 x 10 ７ CFU / mL was calculated.

[0082] Table 2 shows the counting results for Examples 5 and 6. It was confirmed that the accuracy was equivalent to that of the culture method.

[0083] (Example of Immobilization Carrier) Figure 10B shows an example in which agar was used as the immobilization carrier. An agar film (0.3 mm thick) containing 0.5 mM MTT and 0.1% (v / w) glucose was formed on the surface of a glass slide, and 2 μL of E. coli dispersion was dropped onto it and observed under a dark-field microscope. Scattered light images from 0 to 20 minutes and their corresponding spectral images are shown.

[0084] Figure 10C shows an example in which the immobilization carrier was agarose gel. Eight milliliters of dissolved agarose gel and 2 milliliters of E. coli dispersion were mixed, and 10 μL of the mixture was dropped onto a glass surface. A film was then formed using a spin coater (8,000 rpm, 30 seconds). One milliliter of a 0.5 mM MTT aqueous solution was added, and the bacteria were allowed to come into contact with the MTT aqueous solution for 15 minutes. The top image is a bright-field microscope image, and the bottom image is a dark-field microscope image.

[0085] Figure 10D shows an example in which polypyrrole (PPy) was used as the immobilization carrier. A PPy film containing bacteria was formed on the glass surface. The glass was immersed in a liquid medium containing 0.1 mM MTT and observed under a dark-field microscope. Scattered light images from 0 to 90 minutes are shown.

[0086] Figure 10E shows an example in which the immobilization carrier was silane coupling. Glass was immersed in 1% 3-aminopropyltrimethoxysilane for 1 hour and then washed with water. 1 mL of E. coli dispersion was added dropwise and allowed to stand for 1 hour. The surface was rinsed with water and observed under a microscope. 1 mL of 0.5 mM MTT aqueous solution was added dropwise, allowed to stand for 15 minutes, and observed under a microscope. The top image is taken under a bright-field microscope, and the bottom image is taken under a dark-field microscope.

[0087] The immobilization carrier allows the cells to bind or adhere to the glass slide, facilitating focusing during microscopic observation. Furthermore, comparing the timing of adding MTT to the immobilization carrier beforehand, dissolving the MTT in a bacterial suspension, and mixing it with the bacteria, with the timing of adding the MTT solution dropwise after immobilizing the bacteria on the immobilization carrier, the timing of adding the MTT did not affect the measurement of bacterial activity, and the degree of activity could be measured in both cases. Furthermore, the immobilization carrier prevented the aqueous solvent in the suspension from drying out, allowing for relatively long-term observation and measurement (up to 180 minutes).

[0088] (Evaluation of injured bacteria) Escherichia coli K12 dispersion (about 10 ６ cells / mL) were exposed to UV (W) for 0, 25, 50, 100, 200, 300, 1000, 2000, 3000, 400, and 5000 seconds (0, 25, 50, 100, 200, 300, 1000, 2000, 3000, 400, and 5000 mJ / cm ２ ) was irradiated. 100 μL of the irradiated E. coli dispersion was added to 1.0 mL of liquid medium containing MTT (0.50 mM) and glucose (0.1% (v / w)) and cultured for 15 minutes. After culture, the cells were centrifuged (×10,000 G, 3 minutes) to obtain a precipitate, which was dispersed in 100 μL of sterile water and dropped onto a glass slide. After air-drying at 4°C, the cells were observed with a dark-field microscope and the scattered light spectrum was obtained using a spectroscope. The ratios obtained by dividing the peak area at a wavelength of 670 nm by the peak area at a wavelength of 600 nm in the scattered light spectrum of a single cell are shown in Table 3 and Figure 11.

[0089]

[0090] When UV irradiation time was short, MTT formazan particle formation was observed within the cells. As the irradiation time increased, the MTT formazan particles became smaller, and finally, when the irradiation time was extended, the MTT formazan particles disappeared. From these results, it was possible to distinguish between healthy bacteria, injured bacteria (small, large), and dead bacteria by setting a threshold value in proportion to the amount of UV irradiation. The degree of damage in injured bacteria could be evaluated by setting a threshold value.

[0091] REFERENCE SIGNS LIST 1 Cell activity measuring device 7 Dark-field microscope 8 Imaging device 21 Viable cell count calculation unit 22 Cell activity determination unit 23 Spectral cell activity determination unit

Claims

1. A deposition step involves placing the cells to be measured into a measurement solution containing a soluble substance that is permeable to the cell membrane and capable of forming an insoluble reducing substance that is reduced within the active cells and deposited within the active cells, thereby allowing the soluble substance to be taken up into the active cells, forming and depositing the insoluble reducing substance; The process includes, after the deposition step, an optical determination step in which the cellular activity of individual cells is determined based on a specific color caused by the scattered light of the insoluble reducing substance within the deposited cells, The optical determination step is, An optical observation step of observing the scattered light using an optical observation means that generates scattered light, A step of counting the number of active cells, indicated by the specific color, in a predetermined area by image analysis or visual inspection, The process of counting the number of active cells includes counting the number of cells that show crystallization at each elapsed time, calculating the rate of active cells, and determining the degree of cell activity from the fluctuations thereof. The aforementioned active cell rate is determined by the following formula (1), which is a method for measuring cell activity. Activated cell rate (%) = Number of cells showing crystallization / Total number of cells (1) Total number of cells = number of active cells + number of inactive cells + number of other cells Number of cells showing crystallization = Number of active cells

2. The method for measuring cell activity according to claim 1, wherein the optical determination step includes a viability determination step in which the viability of individual cells is determined based on the presence or absence or brightness of the specific color by image analysis or visual inspection.

3. A deposition step involves placing the cells to be measured into a measurement solution containing a soluble substance that is permeable to the cell membrane and capable of forming an insoluble reducing substance that is reduced within the active cells and deposited within the active cells, thereby allowing the soluble substance to be taken up into the active cells, forming and depositing the insoluble reducing substance; The process includes, after the deposition step, an optical determination step in which the cellular activity of individual cells is determined based on a specific color caused by the scattered light of the insoluble reducing substance within the deposited cells, The optical determination step is, The imaging process involves acquiring a scattered light image using an imaging device and an optical observation means that generates scattered light, and A step of calculating the number of active cells, which involves image analysis of the scattered light image and calculating at least the number of active cells based on the number of insoluble reducing substances crystallized within the active cells, and / or, A method for measuring cell activity, comprising: an image analysis step of the scattered light image and a cell activity determination step of determining the cell activity in individual cells based on the size of the crystallized insoluble reducing substance within individual cells or the size of crystallization relative to a predetermined time in the deposition step.

4. A deposition step involves placing the cells to be measured into a measurement solution containing a soluble substance that is permeable to the cell membrane and capable of forming an insoluble reducing substance that is reduced within the active cells and deposited within the active cells, thereby allowing the soluble substance to be taken up into the active cells, forming and depositing the insoluble reducing substance; The process includes, after the deposition step, an optical determination step in which the cellular activity of individual cells is determined based on a specific color caused by the scattered light of the insoluble reducing substance within the deposited cells, The optical determination step is, A light scattering spectrum measurement step involves irradiating the cells to be measured with light using an optical observation means that generates scattered light, and measuring the spectrum using the resulting scattered light. A method for measuring cell activity, comprising: a spectral cell activity determination step, which determines the degree of cell activity in individual cells based on the intensity of wavelengths of specific colors indicating deposited crystals.

5. A cell count calculation unit calculates the number of active cells by placing the target cells in a measurement solution containing a soluble substance that is permeable to the cell membrane and capable of forming an insoluble reducing substance that is reduced within the active cells and deposited therein, allowing the soluble substance to be taken up into the active cells, forming and depositing the insoluble reducing substance, irradiating the cells with light using an optical observation means that generates scattered light, imaging the resulting scattered light, performing image analysis on the obtained scattered light image, and calculating at least the number of active cells based on the number of insoluble reducing substances crystallized within each cell, and / or A cell activity measuring device comprising: an image analysis unit that analyzes scattered light images obtained by the aforementioned imaging device and determines the cell activity level of individual cells based on the size of crystallized insoluble reducing substances within the cells or the size of crystallization relative to a predetermined time; and a cell activity measuring device.

6. An optical observation means that irradiates the deposited cells with light to generate scattered light, The system further comprises an imaging device that captures scattered light generated by the optical observation means and generates a scattered light image, The active cell count calculation unit uses the scattered light image generated by the imaging device, The cell activity measurement device according to claim 5, wherein the cell activity determination unit uses a scattered light image generated by the imaging device.

7. An optical observation means comprising: placing cells to be measured into a measurement solution containing a soluble substance that is permeable to the cell membrane and capable of forming an insoluble reducing substance that is reduced within the active cell and deposited within the active cell; allowing the soluble substance to be taken up into the active cell, forming and depositing the insoluble reducing substance; irradiating the cells in this state with light to generate scattered light; A light scattering spectrum measuring device that measures a spectrum using the scattered light, A cell activity measuring device comprising a spectral cell activity determination unit that determines the cell activity level in individual cells based on the intensity or change in intensity of a specific color wavelength obtained from a light scattering spectrum measuring device.