JP2009058233A - Method for determining contamination level of bathing facility, method for determining microbicidal effect of microbicide in bathtub water and method for controlling water quality of bathtub water - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining a contamination level of a bathing facility capable of determining a contamination level of a bathing facility accurately, easily and in a short time. <P>SOLUTION: On a sample that is taken from a bathing facility and expected to contain particles, intensities of scattered light and fluorescence which are emitted from the particles are measured by flow cytometry. Based on the measurements, particles within the ranges of intensities of scattered light and fluorescence corresponding to each of microorganisms involved in contamination of the bathing facility are classified and counted as a contaminating microorganism, and the contamination level of the bathing facility is determined based on the number of the counted particles. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for determining the degree of contamination of bathing equipment, a method for determining the microbicidal effect of a microbicide in bath water, and a method for managing the water quality of bath water. More specifically, the present invention relates to a method for judging the degree of contamination of bathing equipment and a device used therefor, a method for judging the microbicidal effect of a microbicide in bath water and a device used therefor, and a method for managing the water quality of bath water and a device used therefor. About.

  Natural water environments and artificial water environments, such as fountains and bathtub water, may contain pathogenic microorganisms, microorganisms that cause contamination, and the like. For example, Legionella bacteria are bacteria that inhabit the water environment. The Legionella bacterium is known as a causative microorganism of Legionella disease, and water, for example, water stored in a cooling tower, hot spring water, circulation bath water, etc. is contaminated with this Legionella bacterium Legionellosis can be caused by human inhalation of aerosols generated from these waters.

  Conventionally, the inspection of microorganisms contained in natural water environments and artificial water environments such as fountains and bathtub waters is conducted by examining the presence of microorganisms that grow by incubating a sample collected from the water environment in a dedicated medium. Has been done. Specifically, for example, the examination of Legionella genus bacteria in a bathing facility is performed by incubating a sample collected from the bathing equipment on a Legionella selective agar medium under a predetermined condition, and the Legionella genus bacteria on the Legionella selective agar medium. This is performed by confirming the formation of colonies (see, for example, Non-Patent Document 1).

  On the other hand, flow cytometers are sometimes used for counting and detecting microorganisms (see Patent Document 1, Patent Document 2, and the like).

JP 2004-89034 A JP-A-2-31892 “New edition of Legionellosis Prevention Guidelines”, supervised by Ministry of Health and Welfare Bureau of Health and Sanitation Planning Division, Building Management Center, November 1999

  However, the method described in Non-Patent Document 1 requires time for culturing Legionella bacteria on a medium and obtaining a test result, so that it is difficult to confirm the test result during the culture period for the test. The safety of the water environment cannot be judged at an early stage, it is slow to take measures to be taken against the test results, and it is difficult to evaluate other than the microorganisms that grow on the medium used. There are drawbacks. In addition, the methods described in Patent Document 1 and Patent Document 2 do not describe at all the method for determining the degree of contamination and the method for determining the microbicidal effect of the microbicide in bathing equipment such as bath water.

  The present invention has been made in view of such circumstances, and it is possible to determine the degree of contamination of a bathing facility with high accuracy and simply in a short time, and a method for determining the degree of contamination of a bathing facility and bathing. One object is to provide an apparatus for determining the degree of contamination of facilities. In addition, the present invention can easily determine the microbicidal effect of a microbicide in a bath water in a short time, a method for determining the microbicidal effect of a microbicide in a bath water, and a microbicide of a bath water It aims at providing the determination apparatus of a microbicidal effect. Furthermore, an object of the present invention is to provide a bathtub water quality management method and a bathtub water quality management apparatus that can easily manage the quality of bathtub water in real time with high accuracy.

The method for determining the degree of contamination of a bathing facility according to the present invention comprises: (A) mixing a sample suspected of containing particles collected from a bathing facility with a reagent containing a fluorescent dye that binds or associates with microorganisms; Preparing a sample;
(B) introducing the measurement sample obtained in step (A) into the flow cell of the flow cytometer and flowing it;
(C) irradiating the flow cell with light in the step (B), and measuring the intensity of scattered light emitted from the particles in the measurement sample flowing in the flow cell and the intensity of fluorescence;
(D) Based on the scattered light intensity and the fluorescence intensity measured in the step (C), the particles included in the range of the predetermined scattered light intensity and the fluorescence intensity are classified as contaminating microorganisms involved in the contamination of the bathing facility. Step,
(E) Counting the number of particles classified as contaminating microorganisms in step (D), and (F) Contamination of bathing equipment based on the number of particles counted as contaminating microorganisms in step (E). Determining the degree,
It is characterized by including.

  In the method for determining the degree of contamination of the bathing facility of the present invention, in step (C), the intensity of scattered light and the intensity of fluorescence emitted from the particles are measured, and in step (D), the scattered light intensity and fluorescence intensity are measured. However, in order to classify particles contained in the range of scattered light intensity and fluorescence intensity corresponding to microorganisms involved in contamination of bathing equipment as contaminating microorganisms, it is easy and quick to participate in contamination of bathing equipment. It is possible to detect microorganisms that perform contamination and to detect contaminating microorganisms with high accuracy. Therefore, according to the determination method of the contamination level of the bathing facility of the present invention, the contamination level of the bathing facility can be easily determined with high accuracy.

  The fluorescent dye is preferably a nucleic acid staining fluorescent dye.

  In the method for determining the degree of contamination of a bathing facility according to the present invention, the bathing facility is preferably a hot water storage tank, a filtration tank, a hair catcher, a pipe, a bathtub, a bathroom wall, a bathroom floor, a bathroom joint, or a shower facility. .

  The reagent is preferably a reagent further containing a surfactant.

  In the determination method of the contamination level of the bathing facility of the present invention, the distribution of particles measured in step (C) is displayed on the distribution map with the scattered light intensity and the fluorescence intensity as coordinate axes, and the distribution map Preferably, the method further includes a step of displaying a range corresponding to the contaminating microorganism.

  The contaminating microorganism is preferably Legionella bacteria.

  In the method for determining the contamination level of the bathing facility of the present invention, it is preferable to determine the contamination level of the bathing facility by comparing the threshold value with the number of particles counted as the contaminating microorganism in the step (F).

The method for determining the microbicidal effect of the microbicide in the bath water of the present invention is as follows: (a) a reagent containing a bath water suspected of containing particles treated with a microbicide and a fluorescent dye that binds or associates with microorganisms To prepare a measurement sample,
(B) introducing the measurement sample obtained in step (a) into the flow cell of the flow cytometer and flowing it;
(C) irradiating the flow cell with light in the step (b), and measuring the intensity of scattered light emitted from particles in the measurement sample flowing in the flow cell and the intensity of fluorescence;
(D) Based on the scattered light intensity and the fluorescence intensity measured in the step (c), the particles included in the range of the predetermined scattered light intensity and the fluorescence intensity are classified as contaminating microorganisms involved in bath water contamination. Step,
(E) counting the number of particles classified as contaminating microorganisms in step (d), and (f) based on the number of particles counted as contaminating microorganisms in step (e), Determining a microbicidal effect;
It is characterized by including.

  In the determination method of the microbicide effect of the microbicide in the bathtub water of the present invention, in the step (d), based on the scattered light intensity and the fluorescence intensity of the particles measured by the flow cytometer in the step (c), Classifying particles included in a predetermined range of scattered light intensity and fluorescence intensity as contaminating microorganisms involved in bath water contamination, and based on the number of particles counted as contaminating microorganisms in step (f), The microbicidal effect of the microbicide is determined. Therefore, according to the determination method of the microbicide effect of the microbicide in the bathtub water of the present invention, the microbicide effect of the microbicide in the bath water can be determined with high accuracy and in a short time.

  The microbicide is preferably an oxidizing biocide. The oxidizing biocide is preferably a compound that generates hypochlorous acid and / or hypobromite in water, a peroxide, or ozone.

The water quality control method for bathtub water according to the present invention comprises (I) mixing each sample suspected of containing particles collected from bath water with time and a reagent containing a fluorescent dye that binds or associates with microorganisms. Preparing the measurement sample,
(II) introducing the measurement sample obtained in step (I) into a flow cell of a flow cytometer and flowing it;
(III) irradiating the flow cell with light in the step (II), and measuring the intensity of scattered light emitted from particles in the measurement sample flowing in the flow cell and the intensity of fluorescence;
(IV) Based on the scattered light intensity and the fluorescence intensity measured in the step (III), the particles included in the range of the predetermined scattered light intensity and the fluorescence intensity are classified as contaminating microorganisms involved in bath water contamination. Step,
(V) counting the number of particles classified as contaminating microorganisms in step (IV), and (VI) based on the number of particles counted as contaminating microorganisms in step (V), contained in the bath water. It is characterized by including the step which outputs the information regarding the time-dependent change of the number of contaminating microorganisms so that display is possible.

  In the water quality management method for bathtub water of the present invention, in step (III), the scattered light intensity of particles in the bathtub water collected over time and the fluorescence intensity based on a fluorescent dye bound to or associated with the particles are obtained. In the step (IV), based on the scattered light intensity and the fluorescence intensity, the particles contained in the predetermined scattered light intensity and the fluorescence intensity range are contaminated microorganisms involved in bath water contamination. Therefore, it is possible to easily detect the contaminating microorganisms in the bath water with time and to detect the contaminating microorganisms with high accuracy. Therefore, according to the determination method of the microbicide effect of the microbicide in the bathtub water of the present invention, the microbicide effect of the microbicide in the bath water can be easily determined with high accuracy over time.

  Information on the change in the number of contaminating microorganisms over time is obtained by displaying the measured particle distribution on a two-dimensional distribution diagram with the scattered light intensity and the fluorescence intensity as coordinate axes, and on the two-dimensional distribution diagram. It is preferable to include a diagram showing a change over time of a two-dimensional distribution diagram in which the range is displayed.

  The bathtub water preferably includes hot spring water.

  The apparatus for determining the degree of contamination of a bathing facility according to the present invention is a flow site for measuring the intensity of scattered light emitted from particles in a sample suspected of containing particles collected from a bathing facility and the intensity of fluorescence. Based on the scattered light intensity and fluorescence intensity of the particles measured by the flow cytometer, the particles included in the range of the predetermined scattered light intensity and fluorescence intensity are classified as contaminated microorganisms involved in bathing equipment contamination. A contamination degree information storage for storing information relating the number of contaminating microorganisms and the degree of contamination of the bathing facility, and a particle counting unit for counting the number of particles classified as contaminating microorganisms in the microorganism classification unit Degree of contamination determination unit for determining the degree of contamination of the bathing facility based on the numerical value of the number of particles counted as contaminated microorganisms by the particle counting unit and the information stored in the contamination degree information storage unit It is characterized in that it comprises a.

  In the apparatus for determining the degree of contamination of a bathing facility of the present invention, the measured value of the scattered light intensity of the particles and the measured value of the fluorescence intensity measured by the flow cytometer are respectively scattered light corresponding to the microorganisms involved in the contamination of the bathing facility. It is determined whether or not it is included in the range of intensity and the range of fluorescence intensity, and the particle determined by the microorganism classification unit that the measured value of scattered light intensity and the measured value of fluorescence intensity are included in the range, Classified as a contaminating microorganism. Therefore, according to the determination apparatus for the contamination level of the bathing facility of the present invention, the contaminating microorganisms present in the bathing facility can be measured with high accuracy and in a short time, so the bathing facility with high accuracy and simply in a short time. The degree of contamination can be determined.

The apparatus for determining the microbicidal effect of the microbicide in the bath water of the present invention is the intensity of scattered light emitted from the particle in the bath water suspected of containing particles treated with the microbicide and the particle fluorescence. A flow cytometer for measuring the intensity of the water, and based on the scattered light intensity and the fluorescence intensity of the particles measured by the flow cytometer, particles contained in a predetermined range of the scattered light intensity and the fluorescence intensity, A microbe classifying unit for classifying as a contaminating microbe involved in contamination, a particle counting unit for counting the number of particles classified as a contaminating microbe in the microbe classifying unit,
A microbicidal effect information storage unit that stores information relating the number of contaminating microorganisms and the microbicidal effect of the microbicide, and information stored in the microbicidal effect information storage unit and counted as a contaminating microorganism by the particle counting unit And a microbicidal effect determination unit that determines the microbicidal effect of the microbicide based on the number of particles formed.

  In the determination apparatus of the microbicide effect of the microbicide in the bathtub water of the present invention, the microorganism classification unit is involved in the contamination of the bathtub water based on the scattered light intensity and the fluorescence intensity of the particles measured by the flow cytometer, respectively. It is determined whether or not the particles are included in a predetermined scattered light intensity range and fluorescence intensity range corresponding to the microorganism, and particles included in the predetermined range are classified as contaminating microorganisms. Based on the information relating the microbicidal effect of the microbiological agent and the number of particles classified and counted as microorganisms involved in bath water contamination, the microbicidal effect determining unit determines the microbicidal effect of the microbicidal agent. Determined. Therefore, according to the determination device for the microbicidal effect of the microbicide in the bathtub water of the present invention, the contaminating microorganisms present in the bath water treated with the microbicide can be easily measured. The microbicidal effect of the microbial agent can be determined.

  The water quality management device for bathtub water of the present invention is a flow cytometer that measures the intensity of scattered light emitted from the particles in the bathtub water that is suspected to contain particles collected over time, and the intensity of fluorescence. Based on the scattered light intensity and fluorescence intensity of the particles measured with a flow cytometer, a microorganism classification unit that classifies particles included in a predetermined range of scattered light intensity and fluorescence intensity as contaminated microorganisms involved in bath water contamination And a particle counting unit that counts the number of particles classified as contaminating microorganisms in the microorganism classifying unit, and the number of contaminating microorganisms contained in the bath water based on the number of particles counted as contaminating microorganisms in the particle counting unit. The water quality information output part which outputs the information regarding a time-dependent change of this so that display is possible is provided.

  The water quality management device for bathtub water according to the present invention is such that, for the particles in the bath water collected over time, the measured values of the scattered light intensity and the fluorescence intensity of the particles measured by the flow cytometer are measured by the microorganism classification unit. , It is determined whether or not it is included in a predetermined scattered light intensity range and fluorescent intensity range corresponding to microorganisms involved in the contamination of bath water, respectively, and particles included in the predetermined range are classified as contaminating microorganisms In addition, based on the number of particles classified and counted as contaminated microorganisms and information relating the number of microorganisms involved in the contamination of bathtub water and the water quality, the water quality determination unit determines the water quality of the bathtub water. Is determined. Therefore, according to the water quality management apparatus for bathtub water of the present invention, it is possible to easily detect contaminating microorganisms in the bathtub water over time and to detect contaminating microorganisms with high accuracy. Therefore, according to the water quality management device for bathtub water of the present invention, the water quality of bathtub water can be managed with high accuracy over time, in a simple manner and in a short time in real time.

  According to the method for determining the degree of contamination of a bathing facility and the apparatus for determining the degree of contamination of a bathing facility according to the present invention, the degree of contamination of a bathing facility can be determined easily and in a short time with high accuracy. Moreover, the determination method of the microbicidal effect of the microbicide of the bathtub water of the present invention and the determination device of the microbicidal effect of the microbicide of the bath water are highly accurate, simply, and in a short time, the microbicide in the bath water. The microbicidal effect of can be determined. Furthermore, according to the water quality management method and the water quality management device for bathtub water of the present invention, the water quality of the bathtub water can be managed in real time with high accuracy.

[1] Determination method of contamination degree of bathing equipment and determination device of contamination degree of bathing equipment The determination method of the contamination degree of bathing equipment according to the present invention is (A) a sample that is suspected to contain particles taken from the bathing equipment. And a step of preparing a measurement sample by mixing a reagent containing a fluorescent dye that binds or associates with a microorganism ("measurement sample preparation step" in FIG. 1 (step S11)),
(B) a step of introducing and flowing the measurement sample obtained in the step (A) into the flow cell of the flow cytometer [in FIG. 1, “measurement sample introduction step” (step S12)],
(C) The flow cell is irradiated with light in the step (B), the intensity of the scattered light emitted from the particles in the measurement sample flowing in the flow cell, and the fluorescence based on the fluorescent dye bound to or associated with the particles Step of measuring the intensity ["Measurement step" in FIG. 1 (step S13)],
(D) Based on the scattered light intensity and the fluorescence intensity measured in the step (C), the particles included in the range of the predetermined scattered light intensity and the fluorescence intensity are classified as contaminating microorganisms involved in the contamination of the bathing facility. Step [in FIG. 1, "microorganism classification step" (step S14)],
(E) Counting the number of particles classified as contaminating microorganisms in step (D) [in FIG. 1, “microorganism counting step” (step S15)], and (F) contaminating microorganisms in step (E) Step of determining the contamination level of the bathing facility based on the number of particles counted as [in FIG. 1, "contamination level determination step" (step S16)],
It is a method including.

  The determination method of the contamination degree of the bathing facility of the present invention is that the sample collected from the bathing facility has a measured value of scattered light intensity and a measured value of fluorescence intensity measured by flow cytometry in the step (C). In the step (D), the particles included in the scattered light intensity range and the fluorescence intensity range corresponding to the microorganisms involved in the contamination of the bathing facility are classified as contaminating microorganisms, and the number of particles classified as contaminating microorganisms. In step (F), the degree of contamination is determined. Thus, according to the determination method of the contamination level of the bathing facility of the present invention, since the step (C) and the step (D) are performed, it is possible to easily detect contaminating microorganisms in a short time, Contaminating microorganisms can be detected with high accuracy. Moreover, according to the determination method of the contamination degree of the bathing facility of the present invention, in order to perform the step (F) in addition to the step (C) and the step (D), based on the number of counted particles, The degree of contamination of bathing facilities can be determined with high accuracy. Therefore, according to the determination method of the contamination level of the bathing facility of the present invention, the contamination level of the bathing facility can be easily determined with high accuracy.

  In the present specification, the microorganism is not particularly limited as long as it is a minute organism present in the water environment. For example, bacteria, amoeba and the like are included.

  The microorganism involved in the contamination of the bathing facility, that is, the contaminating microorganism is not particularly limited as long as it is a water-borne infectious gram-negative spore-free bacilli. For example, Acinetobacter calcoaceticus, Aeromonas hydrophila , Aeromonas sobria, Aeromonas caviae, Campylobacter jejuni, Campylobacter coli, Chromobacterium violaceum, Candob reb (Citrobacter sp.), Enterobacter sp., Escherichia coli serotypes, Flavobacterium meni Gosepticum (Flavobacterium meningosepticum), Francisella tularensis, Fusobacterium necrophorum, Klebsiella pneumione, Legionella pneumion, Legionella pneumion Legionella santicrucis, Legionella israelensis, Legionella parisiensis, Legionella spiritensis, Legionella hackione, Legionella helelia Legionella anisa, Legionella Legionella jamestowniensis, Morganella morganii, Plesiomonas shigelloides, Pseudomonas aeruginos, Pseudomonas Promtete, Pseudomonas promte vulgaris), Salmonella enteritidis, Salmonella sp., Salmonella paratyphi, Salmonella typhi, Salmonella cenphiratium, Salmonella typhimurium , Shigella dysenteriae, Shigella flexneri, Shigella sonnei Vibrio alginolyticus, Vibrio cholerae, Vibrio fluvialis, Vibrio mimicus, Vibrio parahaemolyticus, Vibrio vulni Examples thereof include Colissica (Yersinaia enterocolitica). In the present invention, Legionella bacteria are preferred.

  The bathing facility is not particularly limited, and examples thereof include a hot water storage tank, a filtration tank, a hair catcher, piping, a bathtub, a bathroom wall, a bathroom floor, a bathroom joint, and a shower facility. The method for determining the degree of contamination of bathing facilities of the present invention is suitable for determining the degree of contamination of these bathing facilities.

  In the method for determining the degree of contamination of a bathing facility of the present invention, a reagent containing a fluorescent dye that binds or associates with a microorganism is used. Therefore, in the method for determining the contamination level of the bathing facility according to the present invention, when a microorganism is present in the sample collected from the bathing facility, the fluorescent dye of the reagent binds or associates with the microorganism. Microorganisms can be detected based on the fluorescence from the fluorescent dye. Therefore, according to the determination method of the contamination degree of the bathing facility of the present invention, microorganisms involved in the contamination of the bathing facility can be detected with high accuracy.

  Examples of the fluorescent dye that binds to or associates with the microorganism include, for example, a nucleic acid-staining fluorescent dye, and a detectable substance that binds to a marker specific to the microorganism (for example, a polypeptide, a sugar chain, etc.) (for example, a labeled antibody, Labeling ligand, etc.). In the method for determining the degree of contamination of a bathing facility of the present invention, a nucleic acid-staining fluorescent dye is preferably used from the viewpoint of improving the ease of operation and improving the accuracy of detection of viable cells.

  In the present invention, from the viewpoint of incorporating the fluorescent dye into a microorganism and binding or associating with the microorganism more efficiently, the reagent preferably further contains a surfactant.

  In the present specification, “binding or associating with a microorganism” means binding or associating with a nucleic acid or the like present in the microorganism in addition to binding or association with the microorganism itself (for example, a cell membrane of the microorganism). To do.

  In the method for determining the contamination level of the bathing facility of the present invention, since a flow cytometer is used, it is possible to easily detect microorganisms in a short time in combination with the use of the reagent containing the fluorescent dye. . Therefore, according to the determination method of the contamination level of the bathing facility of the present invention, the contamination level of the bathing facility can be easily determined in a short time.

  The determination method of the contamination level of the bathing facility of the present invention is preferably performed by using, for example, the contamination level determination apparatus A of the bathing facility shown in FIGS.

  The determination device A is composed of a flow cytometer (measurement device) 1 and an information processing device 2 as shown in FIG.

  In FIG. 3, the schematic explanatory drawing of the optical detection part of the flow cytometer 1 is shown. The optical detection unit introduces a sample into the flow cell 101, generates a liquid flow of the sample in the flow cell 101, and measures by irradiating the semiconductor laser light to particles contained in the liquid flow passing through the flow cell 101. . The optical detection unit includes a sheath flow system 100, a beam spot forming system 110, a forward scattered light receiving system 120, a side scattered light receiving system 130, and a side fluorescent light receiving system 140. The sheath flow system 100 improves the accuracy and reproducibility of particle counting by flowing in the flow cell 101 in a state where the sample is wrapped in the sheath liquid and in a state where the particles are arranged in a line. The beam spot system 110 is configured such that the light irradiated from the semiconductor laser 111 is irradiated to the flow cell 101 through the collimator lens 112 and the condenser lens 113. The beam spot system 110 also includes a beam stopper 114. The forward scattered light receiving system 120 is configured to condense forward scattered light by the front condensing lens 121 and receive the light passing through the pinhole 122 by the forward scattered light receiving unit 123. The side scattered light receiving system 130 collects the scattered light to the side by the side condenser lens 131, reflects part of the light by the dichroic mirror 132, and receives the light by the side scattered light receiving unit 133. It is configured. The side fluorescent light receiving system 140 is configured such that the light transmitted through the dichroic mirror 132 is further passed through the spectral filter 141 and received by the fluorescent light receiving unit 142. When the light receiving units 123, 133, and 142 receive light, the light receiving units 123, 133, and 142 output electrical pulse signals. Measurement data is created from this electrical pulse signal. The measurement data is transmitted from the flow cytometer 1 to the information processing apparatus 2, and the information processing apparatus 2 performs processing / analysis.

  Scattered light is generated when particles exist as obstacles in the traveling direction of light and the light changes its traveling direction. By detecting the scattered light, information on the size and material of the particles can be obtained, and in particular, information on the size of the particles can be obtained from the forward scattered light. Moreover, the information inside the particles can be obtained from the side scattered light.

  In the present specification, the scattered light is not particularly limited, and includes both forward scattered light and side scattered light, but forward scattered light is preferable.

  In FIG. 6, the functional block diagram of the determination apparatus A of the contamination degree of bathing equipment is shown. A determination apparatus A shown in FIG. 6 includes a flow cytometer 1 and an information processing apparatus 2. The flow cytometer 1 measures the intensity of scattered light emitted from particles in bath water treated with a microbicide and the intensity of fluorescence based on a fluorescent dye bound to or associated with the particles. The information processing apparatus 2 includes a microorganism classification unit 12, a particle counting unit 13, a contamination degree information storage unit 14, a contamination degree determination unit 15, a display unit 16, and a storage unit 23.

  The flow cytometer 1 measures the intensity of scattered light emitted from particles in a sample collected from a bathing facility and the intensity of fluorescence based on a fluorescent dye bound to or associated with the particles.

  In the information processing apparatus 2, the microorganism classification unit 12 is configured such that the scattered light intensity measurement value and the fluorescence intensity measurement value of the particles measured by the flow cytometer 1 respectively correspond to the microorganisms involved in the bath water contamination. It is determined whether it is included in the range of intensity and the range of fluorescence intensity, and the particles determined to be included in the range are classified as contaminating microorganisms. Further, the particle counting unit 13 counts the number of particles classified as contaminating microorganisms by the microorganism classifying unit 12. The contamination level information storage unit 14 stores a predetermined threshold value as information relating the number of contaminating microorganisms and the contamination level of the bathing facility. The contamination degree determination unit 15 determines the contamination degree of the bathing facility based on the numerical value of the number of particles counted as the contaminating microorganism by the particle counting unit 13 and the threshold value stored in the contamination degree information storage unit 14. The display unit 16 displays the result of determination by the contamination degree determination unit 15. The storage unit 23 stores information on the range of scattered light intensity and the range of fluorescence intensity of the contaminating microorganisms used in the microorganism classification unit 12.

  FIG. 22 is a block diagram illustrating a hardware configuration of the information processing apparatus 2. The information processing apparatus 2 is composed of a computer mainly composed of a main body S110, an output device S120 (display unit 16), and an input device S130. The main body S110 mainly includes a CPU S110a, a ROM S110b, a RAM S110c, a hard disk S110d, a reading device S110e, an input / output interface S110f, a communication interface S110g, and an image output interface S110h. The ROM S110b, the RAM S110c, the hard disk S110d, the reading device S110e, the input / output interface S110f, the communication interface S110g, and the image output interface S110h are connected by a bus S110i so that data communication is possible.

The CPU S110a can execute a computer program stored in the ROM S110b and a computer program loaded in the RAM S110c. When the CPU S110a executes the application program S140a, the above functional blocks are realized, and the computer functions as the information processing apparatus 2.
The ROM S110b is configured by a mask ROM, PROM, EPROM, EEPROM, or the like, in which computer programs executed by the CPU S110a, data used for the same, and the like are recorded.

The RAM S110c is configured by SRAM, DRAM, or the like. The RAM S110c is used for reading computer programs recorded in the ROM S110b and the hard disk S110d. Further, when these computer programs are executed, they are used as a work area of the CPU S110a.
The hard disk S110d is installed with various computer programs to be executed by the CPU S110a, such as an operating system and application programs, and data used for executing the computer programs. The application program S140a is also installed in the hard disk S110d.

  The reading device S110e is configured by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, or the like, and can read a computer program or data recorded on the portable recording medium S140. The portable recording medium S140 stores an application program S140a for causing the computer to function as the system of the present invention. The computer reads the application program S140a according to the present invention from the portable recording medium S140, and The application program S140a can be installed in the hard disk S110d.

  Note that the application program S140a is not only provided by the portable recording medium S140, but also from an external device that is communicably connected to a computer via an electric communication line (whether wired or wireless) through the electric communication line. It is also possible to provide. For example, the application program is stored in a hard disk of an application program providing server computer on the Internet. The server computer can be accessed to download the computer program and install it on the hard disk S110d. .

  In addition, an operating system that provides a graphical user interface environment such as Windows (registered trademark) manufactured and sold by US Microsoft Co. is installed in the hard disk S110d. In the following description, it is assumed that the application program S140a operates on the operating system.

  The input / output interface S110f includes, for example, a serial interface such as USB, IEEE 1394, and RS-232C, a parallel interface such as SCSI, IDE, and IEEE1284, and an analog interface including a D / A converter and an A / D converter. ing. An input device S130 including a keyboard and a mouse is connected to the input / output interface S110f, and the user can input data to the computer by using the input device S130.

  The communication interface S110g is, for example, an Ethernet (registered trademark) interface. The computer can transmit and receive data to and from the flow cytometer 1 using a predetermined communication protocol through the communication interface S110g.

  The image output interface S110h is connected to a display S120 constituted by an LCD, a CRT, or the like, and outputs a video signal corresponding to the image data given from the CPU S110a to the output device S120. The output device S120 displays an image (screen) according to the input video signal.

  The hard disk S110d has a storage area corresponding to the contamination level information storage unit 14 and the storage unit 23, and further has a storage area in which various computer programs are installed. The computer program for determining the degree of contamination of the bathing facility according to the present embodiment stores the measured value of the scattered light intensity and the measured fluorescence intensity of the particles measured by the flow cytometer 1 in the storage unit 23. Determining whether it is included in the range of scattered light intensity and fluorescence intensity corresponding to the microorganisms involved in contamination of the bathing facility, and classifying the particles included in the range as contaminating microorganisms, and The particle counting unit 13 that counts the number of particles classified as contaminating microorganisms by the microorganism classifying unit 12, the numerical value of the number of particles counted as contaminating microorganisms by the particle counting unit 14, and the pollution degree information storage unit 14 are stored. Based on the received information, it is made to function as the contamination degree determination unit 15 that determines the contamination degree of the bathing facility.

  In the method for determining the contamination level of a bathing facility according to the present invention, in step (A), a sample collected from the bathing facility and a reagent containing a fluorescent dye that binds or associates with microorganisms are mixed to prepare a measurement sample. .

  Thereafter, the prepared measurement sample is introduced into the flow cell of the flow cytometer 1 in step (A), and is flowed into the flow cell.

  Thereafter, in step (C), the flow cell is irradiated with light in the flow cytometer 1. As a result, the flow cytometer 1 measures the intensity of the scattered light emitted from the particles in the measurement sample flowing in the flow cell and the intensity of the fluorescence based on the fluorescent dye bound to or associated with the particles.

  Next, in step (D), the microorganism classification unit 12 uses the scattered light intensity and the fluorescence intensity to cause the particles included in the predetermined scattered light intensity and fluorescence intensity ranges to be contaminated microorganisms that are involved in the contamination of the bathing facility. Classify as Specifically, the measurement value of the scattered light intensity and the measurement value of the fluorescence intensity measured by the flow cytometer 1 are transmitted to the microorganism classification unit 12 of the information processing apparatus 2. Thereafter, the measured value of the scattered light intensity and the measured fluorescence intensity of the particles measured by the flow cytometer 1 are included in the scattered light intensity range and the fluorescent intensity range corresponding to the microorganisms involved in the contamination of the bathing facility. If it is classified as a contaminating microorganism.

  Thereafter, in step (E), the particle counter 13 counts the number of particles classified as contaminating microorganisms.

  Next, in step (F), the contamination degree determination unit 15 determines the contamination degree of the bathing facility based on the number of particles counted as contaminating microorganisms. In this step (F), from the viewpoint of determining the degree of contamination with higher accuracy, the number of particles counted as microorganisms involved in the contamination of the bathing facility is compared with a threshold value (contamination level information) to thereby obtain the bathing facility. The degree of pollution is judged. Note that a table indicating the relationship between the contamination degree and the number of contaminating microorganisms may be stored in the contamination degree information storage unit 14 instead of the threshold value.

  In the method for determining the contamination level of the bathing facility of the present invention, from the viewpoint of more easily and visually determining the contamination level, (G) a step (C) on the distribution map in which the scattered light intensity and the fluorescence intensity are coordinate axes. It is preferable that the method further includes a step of displaying the distribution of the particles measured in step 1) and displaying the range corresponding to the microorganisms involved in the contamination of the bathing facility on the distribution map. Examples of the distribution map include a diagram shown in a schematic diagram of a scattergram of bacteria based on scattered light and fluorescence shown in FIG. In FIG. 10, a specific region I201 is a range corresponding to the water-borne infectious gram-negative spore-free bacilli, and a specific region II203 particularly corresponds to the Legionella bacterium in the water-based infectious gram-negative spore-free bacilli. It is the range to do. In FIG. 10, the non-living area 202 is a range corresponding to dead microorganisms, inorganic particles, and the like.

[2] Determination method of microbicide effect of microbicide in bathtub water and determination device of microbicide effect of microbicide in bathtub water ) A step of preparing a measurement sample by mixing a bath water suspected of containing particles treated with a microbicide and a reagent containing a fluorescent dye that binds or associates with microorganisms. Preparation step "(step S21)],
(B) a step of introducing and flowing the measurement sample obtained in step (a) into the flow cell of the flow cytometer [in FIG. 4, “measurement sample introduction step” (step S22)],
(C) The flow cell is irradiated with light in the step (b), the intensity of the scattered light emitted from the particles in the measurement sample flowing in the flow cell, and the fluorescence based on the fluorescent dye bound to or associated with the particles A step of measuring the intensity ["Measurement step" in FIG. 4 (step S23)],
(D) Based on the scattered light intensity and the fluorescence intensity measured in the step (c), the particles included in the range of the predetermined scattered light intensity and the fluorescence intensity are classified as contaminating microorganisms involved in bath water contamination. Step [in FIG. 4, "microorganism classification step" (step S24)],
(E) a step of counting the number of particles classified as contaminating microorganisms in step (d) [in FIG. 4, “microbe counting step” (step S25)], and (f) a contaminating microorganism in step (e). A step of determining the microbicidal effect of the microbicide based on the number of particles counted as [in FIG. 4, "microbicidal effect determination step" (step S26)],
It is a method including.

  In the determination method of the microbicide effect of the microbicide in the bathtub water of the present invention, the scattered light intensity and the fluorescence intensity of the particles measured by the flow cytometer in the step (c) are obtained for the bath water treated with the microbicide. Based on the above, in the step (d), particles included in the range of the predetermined scattered light intensity and fluorescence intensity are classified as contaminating microorganisms involved in bath water contamination, and classified and counted as contaminating microorganisms. In step (f), the microbicidal effect of the microbicide is determined on the basis of the number of. Therefore, according to the determination method of the microbicide effect of the microbicide in the bathtub water of the present invention, the microbicide effect of the microbicide in the bath water can be determined with high accuracy and in a short time.

  In the method for determining the microbicidal effect of the microbicide in the bathtub water of the present invention, the microbicidal effect is determined based on the number of particles counted as contaminating microorganisms. Specifically, it is executed by comparing the number of contaminating microorganisms with a predetermined threshold value.

  In the method for determining the microbicidal effect of the microbicide in the bathtub water of the present invention, particles are present in the sample obtained by mixing the bathtub water and a reagent containing a fluorescent dye that binds or associates with microorganisms in the step (f). The number of particles classified as microorganisms involved in bath water contamination based on the intensity of scattered light emitted from the particles and the intensity of fluorescence based on fluorescent dyes bound to or associated with the particles is used as a control. Then, the microbicidal effect of the microbicide may be determined by comparing the number of the control particles with the number of particles counted as the contaminating microorganism in the step (e). In this case, depending on the degree of difference between the number of control particles and the number of particles counted as contaminating microorganisms in step (e), the degree of microbicidal effect of the microbicide can be evaluated. There is an advantage.

  The microbicide is not particularly limited as long as it reduces living microorganisms present in the water environment, but an oxidizing biocide is preferable. Although it does not specifically limit as said oxidizing biosite, For example, the compound, peroxide, ozone etc. which generate | occur | produce hypochlorous acid and / or hypobromite in water are mentioned.

  The microorganisms involved in the contamination of the bath water are the same as the microorganisms involved in the contamination of the bathing facility.

  The method for determining the microbicidal effect of the microbicide is preferably performed by using, for example, the apparatus B for determining the microbicidal effect of the microbicide in the bath water shown in FIG.

  As shown in FIG. 7, the determination apparatus B includes a flow cytometer 1 and an information processing apparatus 200. The flow cytometer 1 measures the intensity of scattered light emitted from particles in bath water treated with a microbicide and the intensity of fluorescence based on a fluorescent dye bound to or associated with the particles. The information processing apparatus 200 includes a microorganism classification unit 12, a particle counting unit 13, a microbicidal effect information storage unit 17, a microbicidal effect determination unit 18, a display unit 16, and a storage unit 23.

  In the information processing apparatus 200, the microorganism classification unit 12 is configured to contaminate bathtub water in which the measurement values of the scattered light intensity and the fluorescence intensity of the particles measured by the flow cytometer 1 are information stored in the storage unit 23. It is determined whether or not it is included in the range of scattered light intensity and the range of fluorescence intensity corresponding to the microorganisms involved in the above, and the particles determined to be included in the range are classified as contaminating microorganisms. Further, the particle counting unit 13 counts the number of particles classified as contaminating microorganisms by the microorganism classifying unit 12. The microbicidal effect information storage unit 17 stores a threshold value that is information that associates the number of contaminating microorganisms with the microbicidal effect of the microbicide. The microbicidal effect determination unit 18 determines the microbicidal effect of the microbicide based on the information stored in the microbicidal effect information storage unit 17 and the number of particles counted as contaminating microorganisms by the particle counting unit 13. . The display unit 16 displays the result of determination by the microbicidal effect determination unit 18. The storage unit 23 stores information on the range of scattered light intensity and the range of fluorescence intensity of the microorganism used in the microorganism classification unit 12.

  In the determination apparatus B, the scattered light intensity corresponding to the microorganisms involved in the contamination of the bath water are respectively measured by the microbe classification unit 12 and the measured values of the scattered light intensity and the fluorescence intensity of the particles measured by the flow cytometer 1. The particles determined to be included in the range and the fluorescence intensity range are classified as contaminating microorganisms, the number of contaminating microorganisms and the microbicidal effect of the microbicide The microbicidal effect of the microbicide is determined by the microbicidal effect determination unit 18 based on the information associated with the above and the number of particles classified and counted as contaminating microorganisms. Therefore, according to the determination apparatus B, since the contaminating microorganisms present in the bath water treated with the microbicide can be easily measured, the microbicidal effect of the microbicide in the bath water can be easily determined.

  The hardware configuration of the information processing apparatus 200 is the same as that of the information processing apparatus 2 as shown in FIG.

  The hard disk S110d has storage areas corresponding to the microbicidal effect information storage unit 17 and the storage unit 23, and further has storage areas in which various computer programs are installed. The computer program for determining the microbicidal effect of the microbicide in the bathtub water of the present embodiment stores the CPU S110a in which the measured value of the scattered light intensity and the measured value of the fluorescence intensity of the particles measured by the flow cytometer 1 are stored. Microorganisms that determine whether they are included in the range of scattered light intensity and the range of fluorescence intensity corresponding to the microorganisms involved in the contamination of bath water stored in the unit 23, and classify particles included in the above range as contaminating microorganisms The classification unit 12, the particle counting unit 13 that counts the number of particles classified as contaminating microorganisms by the microorganism classification unit 12, and the information stored in the microbicidal effect information storage unit 17 and counted as contaminating microorganisms by the particle counting unit 13 It is made to function as the microbicidal effect determination part 18 which determines the microbicidal effect of the said microbicide based on the number of particles which were made.

  In the determination method of the microbicide effect of the microbicide in the bathtub water of the present invention, the step (a), step (b), step (c), step (d), and step (e) are treated with the microbicide. A measurement sample in which a bath water suspected of containing particles and a reagent containing a fluorescent reagent that binds or associates with microorganisms are mixed, and the scattered light intensity corresponding to the microorganisms involved in bath water contamination is Step (A), Step (B), Step (C), Step (D) and Step (E) of the method for determining the contamination level of the bathing facility, except that the range and the range of fluorescence intensity are used for the determination. Substantially similar operations are performed.

  In the determination method of the microbicide effect of the microbicide in the bathtub water of the present invention, the microbicide is determined based on the number of particles counted as the contaminating microorganism by the microbicide effect determination unit 18 in the step (f). The microbicidal effect is determined. Specifically, for example, information that associates the number of contaminating microorganisms stored in the microbicidal effect information storage unit 17 with the microbicidal effect of the microbicide, and the number of particles classified and counted as contaminating microorganisms, Based on the above, the microbicidal effect determination unit 18 determines the microbicidal effect of the microbicide. Therefore, the microorganisms in the bath water treated with the microbicide can be easily detected, and the contaminating microorganisms can be detected with high accuracy. Therefore, according to the determination method of the microbicide effect of the microbicide in the bathtub water of the present invention, the microbicide effect of the microbicide in the bath water can be determined with high accuracy and in a short time.

  In the determination method of the microbicidal effect of the microbicide in the bathtub water of the present invention, the display unit displays the determination result of the microbicidal effect (step (g)).

  The microbicidal effect information storage unit 17 binds or associates with the intensity of scattered light emitted from particles in a sample in which bath water and a reagent containing a fluorescent dye that binds or associates with microorganisms are mixed. The number of particles classified as contaminating microorganisms from the intensity of fluorescence based on the fluorescent dye that is present may be stored as a control. In this case, the microbicidal effect determination unit 18 can determine the degree of the microbicidal effect of the microbicide based on the degree of difference between the number of control particles and the counted number of particles, for example. .

[3] Bath Water Quality Management Method and Bath Water Quality Management Device The bathtub water quality management method according to the present invention includes: (I) each sample suspected of containing particles separated from bathtub water over time; , A step of preparing a measurement sample by mixing with a reagent containing a fluorescent dye that binds to or associates with a microorganism ("measurement sample preparation step" in FIG. 5 (step S31)),
(II) a step of introducing and flowing the measurement sample obtained in the step (I) into the flow cell of the flow cytometer [in FIG. 5, “measurement sample introduction step” (step S32)],
(III) The flow cell is irradiated with light in the step (II), and the intensity of the scattered light emitted from the particles in the measurement sample flowing in the flow cell and the fluorescence based on the fluorescent dye bound to or associated with the particles Step of measuring the intensity ("Measurement step" in FIG. 5 (step S33)),
(IV) Based on the scattered light intensity and the fluorescence intensity measured in the step (III), the particles included in the range of the predetermined scattered light intensity and the fluorescence intensity are classified as contaminating microorganisms involved in bath water contamination. Step [in FIG. 5, “microorganism classification step” (step S34)],
(V) a step of counting the number of particles classified as contaminating microorganisms in step (IV) [in FIG. 5, “microbe counting step” (step S35)], and (VI) a contamination microorganism in step (V). Based on the number of counted particles, a step of outputting information relating to a change over time in the number of contaminating microorganisms contained in the bath water [display step in FIG. 5 (step S36)]
It is a method including.

  In the water quality management method of the bathtub water of the present invention, for the sample collected from the bathtub water over time, in the step (III), based on the scattered light intensity and fluorescence intensity of the particles measured by the flow cytometer, In the step (IV), the particles included in the predetermined scattered light intensity and fluorescence intensity ranges are classified as contaminating microorganisms involved in bath water contamination, and the number of particles classified and counted as contaminating microorganisms is counted. Based on the numerical value and the threshold value that is information relating the number of contaminating microorganisms and the water quality, the water quality of the bathtub water is determined in the step (VI). Therefore, according to the water quality management method of the bathtub water of the present invention, it is possible to easily detect contaminating microorganisms in the bathtub water over time and to detect the contaminating microorganisms with high accuracy. Therefore, according to the water quality management device for bathtub water of the present invention, the water quality of bathtub water can be managed with high accuracy over time, in a simple manner and in a short time in real time.

  Information on changes in the number of contaminating microorganisms over time is based on a two-dimensional distribution map with coordinate axes of scattered light intensity and fluorescence intensity, from the viewpoint that water quality management can be easily performed visually. It is preferable to include a diagram showing a change over time in the two-dimensional distribution diagram displaying the distribution and displaying the range of contaminating microorganisms on the two-dimensional distribution diagram.

  The water quality management method of the bathtub water of the present invention is suitable when the bathtub water is bathtub water containing hot spring water.

  The water quality management method for bathtub water of the present invention is suitably implemented by the water quality management apparatus C for bathtub water shown in FIG. 8 and the water quality management apparatus D for bathtub water shown in FIG.

  The water quality management device C for bathtub water shown in FIG. 8 includes a flow cytometer 1 and an information processing device 300. The flow cytometer 1 measures the intensity of scattered light emitted from particles in a sample collected from a bathing facility and the intensity of fluorescence based on a fluorescent dye bound to or associated with the particles. In the water quality management device C, the information processing device 300 of the water quality management device C includes a microorganism classification unit 12, a particle counting unit 13, a water quality information output unit 19, and a display unit 16.

  In the information processing apparatus 300, the microorganism classification unit 12 is configured to contaminate bathtub water in which the measurement values of the scattered light intensity and the fluorescence intensity of the particles measured by the flow cytometer 1 are information stored in the storage unit 23. It is determined whether or not it is included in the range of scattered light intensity and the range of fluorescence intensity corresponding to the microorganisms involved in the above, and the particles determined to be included in the range are classified as contaminating microorganisms. The particle counting unit 13 counts the number of particles classified as contaminating microorganisms by the microorganism classifying unit 12. Based on the number of particles counted as contaminated microorganisms by the particle counting unit 13, the water quality information output unit 19 outputs information related to changes over time in the number of contaminated microorganisms contained in the bath water. The display unit 16 displays information output from the water quality information output unit 19. The storage unit 23 stores information on the range of scattered light intensity and the range of fluorescence intensity of the contaminating microorganisms used in the microorganism classification unit 12.

  The hardware configuration of the information processing apparatus 300 is the same as that of the information processing apparatus 2 as shown in FIG.

  The hard disk S110d has storage areas corresponding to the microbicidal effect information storage unit 17 and the storage unit 23, and further has storage areas in which various computer programs are installed. The computer program for determining the microbicidal effect of the microbicide in the bathtub water of the present embodiment stores the CPU S110a in which the measured value of the scattered light intensity and the measured value of the fluorescence intensity of the particles measured by the flow cytometer 1 are stored. Microorganisms that determine whether they are included in the range of scattered light intensity and the range of fluorescence intensity corresponding to the microorganisms involved in the contamination of bath water stored in the unit 23, and classify particles included in the above range as contaminating microorganisms The classification unit 12, the particle counting unit 13 that counts the number of particles classified as contaminating microorganisms involved in the contamination of the bathing facility by the microorganism classification unit 12, and the number of particles counted as contaminating microorganisms by the particle counting unit 13 Based on this, it is made to function as the water quality information output unit 19 that outputs information on the change over time of the number of contaminating microorganisms contained in the bath water so that it can be displayed.

  In the water quality management method for bathtub water of the present invention, the step (I), step (II), step (III), step (IV) and step (V) are particles separated from the bathtub water over time. A sample containing a sample containing a fluorescent reagent that binds or associates with microorganisms and a range of scattered light intensity and fluorescence corresponding to microorganisms involved in bath water contamination Step (A), step (B), step (C), step (D) and step (E) of the method for determining the contamination level of the bathing facility are substantially the same except that the range of intensity is used for the determination. Similar processing is performed.

  In step (VI), the water quality information output unit 19 can display information on the change over time of the number of contaminating microorganisms contained in the bath water based on the number of particles counted as contaminating microorganisms by the particle counting unit 13. Is output.

  In the water quality management method for bathtub water according to the present invention, the display unit 16 displays information on the change over time in the number of contaminating microorganisms contained in the bathtub water (step (VII)).

  A water quality management device D shown in FIG. 9 includes a flow cytometer 1 and an information processing device 400. The flow cytometer 1 measures the intensity of scattered light emitted from particles in a sample collected from a bathing facility and the intensity of fluorescence based on a fluorescent dye bound to or associated with the particles. In the water quality management device C, the information processing device 400 includes a microorganism classification unit 12, a particle counting unit 13, a water quality information storage unit 20, a water quality determination unit 21, a water quality information output unit 22, a display unit 16, and a storage unit. 23.

  In the information processing device 400 of the water quality management device D, the microorganism classifying unit 12 is a microorganism in which the measured value of the scattered light intensity and the measured value of the fluorescence intensity of the particles measured by the flow cytometer 1 are involved in the contamination of the bath water, respectively. Are determined to be included in the range of scattered light intensity and the range of fluorescence intensity corresponding to, and the particles determined to be included in the range are classified as contaminating microorganisms. The particle counting unit 13 counts the number of particles classified as contaminating microorganisms by the microorganism classifying unit 12. The water quality information storage unit 20 stores a threshold value that is information in which the number of contaminating microorganisms is associated with the water quality. The water quality determination unit 21 determines the water quality based on the numerical value of the number of particles counted as contaminating microorganisms by the particle counting unit 13 and the information stored in the water quality information storage unit 20. The water quality information output unit 22 outputs information on the water quality determined by the water quality determination unit 21 and information on the temporal change in the number of contaminating microorganisms contained in the bath water so as to be displayed. The display unit 16 displays information output from the water quality information output unit 22. The storage unit 23 stores information on the range of scattered light intensity and the range of fluorescence intensity of the contaminating microorganisms used in the microorganism classification unit 12.

  According to the water quality management device D, in step (VI), based on the number of particles counted by the water quality determination unit 21 as the contamination microorganisms in the particle counting unit 13, the contamination microorganisms stored in the water quality information storage unit 20 are stored. The information relating the number and the water quality is referred to, the water quality is determined, the water quality information output unit 22 determines the water quality information, and the information on the temporal change in the number of contaminating microorganisms contained in the bath water Will be output in a displayable manner.

  The hardware configuration of the information processing apparatus 400 is the same as that of the information apparatus 2 as shown in FIG.

  The hard disk S110d has storage areas corresponding to the water quality information storage unit 20 and the storage unit 23, and further has storage areas in which various computer programs are installed. The computer program for managing the water quality of the bathtub water of the present embodiment stores the measured value of the scattered light intensity and the measured fluorescence intensity of the particles measured by the flow cytometer 1 in the storage unit 23. Determining whether it is included in the range of scattered light intensity and fluorescence intensity corresponding to the microorganisms involved in contamination of the bath water, and classifying the particles included in the above range as contaminating microorganisms, and A particle counting unit 13 that counts the number of particles classified as contaminating microorganisms by the microorganism classifying unit 12, a numerical value of the number of particles counted as contaminating microorganisms by the particle counting unit 13, and information stored in the water quality information storage unit 20 Based on the above, the water quality determination unit 21 for determining the water quality, the information on the water quality determined by the water quality determination unit 21 and the information on the temporal change in the number of contaminating microorganisms contained in the bath water Function as water quality information output unit 22 for outputting viewable to.

  In the water quality management method for bathtub water according to the present invention, the display unit 16 displays information on the change over time in the number of contaminating microorganisms contained in the bathtub water (step (VII)).

  The water quality management device C and the water quality management device D include a water quality adjustment unit that includes a water quality adjustment unit that adjusts a predetermined water quality to a predetermined standard water quality, and a water quality adjustment unit information storage unit that stores information on the water quality adjustment unit. And a water quality adjusting unit selecting unit that selects a water quality adjusting unit provided in the water quality adjusting unit based on the result of the water quality determined by the water quality determining unit and the information stored in the water quality adjusting unit information storage unit And a water quality adjusting means control unit that controls the water quality adjusting means selected by the water quality adjusting means selecting unit.

  EXAMPLES Hereinafter, although an Example etc. demonstrate this invention in detail, this invention is not limited by this Example.

  Number of general and heterotrophic bacteria in 225 samples (121 bath water samples, 37 spring water samples, 31 hot water tank water samples, 24 hair catcher water samples, 8 pipe water samples) collected from a total of 23 facilities in public baths and inns And the number of bacteria by flow cytometry. Of the 23 facilities, 12 are hot spring facilities, one simple acidic spring facility, three sulfate spring facilities, five chloride spring facilities, and five bicarbonate spring facilities. The remaining 11 facilities are non-hot spring facilities.

  General bacteria counts and heterotrophic bacteria counts are based on the method of Inoue et al. (Research Grant for Health and Welfare Sciences, Health Sciences Research Project “Study on Development of Appropriate Hygiene Management Techniques in Floating Hot Springs, etc.” 2005 Summary / Shared Report In accordance with the hand-drawn hot spring survey Bacteria Test Manual, pages 115 to 119, database of welfare science research results), measurements were made as follows. The specimen was appropriately diluted with sterile physiological saline (0.85% aqueous sodium chloride solution) to obtain 0.1 ml of a sample. After autoclaving at 121 ° C. for 20 minutes in advance, a standard agar medium (manufactured by Eiken Chemical Co., Ltd.) or R2A agar medium (manufactured by Becton Dickinson) was hardened on a flat plate. On each of the obtained flat plates, 0.1 ml of the specimen was applied with a sterilized congeal rod. Samples coated on standard agar plates were incubated at 35 ° C. for 48 hours. Moreover, the sample apply | coated on the flat plate of R2A agar culture medium was incubated at 42 degreeC for 7 days. Thereafter, colonies were counted on the flat plate on which 30 to 300 CFU (Colony Forming Unit) counting target colonies were formed per flat plate. Correlation analysis was performed only on flat plates exceeding 30 CFU.

  On the other hand, the number of bacteria by flow cytometry was measured using a determination apparatus A shown in FIG. 120 μl ml of the sample was placed in a sample tube (trade name: SU-40, manufactured by Sysmex Corporation) and set together with the empty tube on the sample table of the determination apparatus A shown in FIG. A sample of 50 μl and a diluent (trade name: Bactoquick Diluent) 340 μl are automatically quantified and mixed, and a nucleic acid-staining fluorescent dye (trade name: Bactoquik Stain) is further mixed. The mixed measurement sample is guided to the detection unit. Scattered light and fluorescence generated when the bacterium dyed the nucleic acid with the fluorescent dye crosses the laser beam is detected, and the scattered light intensity and fluorescence intensity are measured. Based on the scattered light and fluorescence, the detected bacteria A distribution map (scattergram) was prepared. FIG. 11 shows a scattergram of bacteria based on scattered light and fluorescence. Note that the scattergram of FIG. 11 is composed of two signals of scattered light intensity and fluorescence intensity obtained by measurement. The scattered light intensity is proportional to the size of bacteria, and the fluorescence intensity is proportional to the intensity of nucleic acid staining (nucleic acid amount). As the scattered light intensity and fluorescence intensity increase, the bacterial distribution position in the scattergram moves in the upper right direction. The number of bacteria is first detected based on the specific area 1 (microbe appearance area) preset in the scattergram corresponding to the water-borne infectious gram-negative sporeless bacilli that are microorganisms involved in contamination of bathing facilities. Obtained by reanalyzing the bacterial particles. The region of the scattergram in FIG. 11 is defined by dividing into a specific region 1, a non-living region 2 that is not stained with nucleic acid, and another region 3 existing between the specific region 1 and the non-living region 2.

  Further, FIG. 12 shows a correlation diagram between the number of general bacteria obtained from the colonies formed on the standard agar medium and the number of bacteria by flow cytometry. Further, FIG. 13 shows a correlation diagram between the number of heterotrophic bacteria determined from the colonies formed on the R2A agar medium and the number of bacteria by flow cytometry.

  From the results shown in FIG. 12, it can be seen that, in 123 samples, there is a relatively high positive correlation with a correlation coefficient R = 0.5361 between the number of general bacteria and the number of bacteria by flow cytometry. In addition, the results shown in FIG. 13 indicate that in 123 samples, there is a very high positive correlation between the number of heterotrophic bacteria and the number of bacteria by flow cytometry, with a correlation coefficient R = 0.8006. . From these results, it can be seen that the number of bacteria by flow cytometry has a quantitative property equivalent to the number of general bacteria and heterotrophic bacteria conventionally used as an indicator of the amount of bacteria present in the water environment.

  On the other hand, the measurement of the number of heterotrophic bacteria generally requires 7 days, but the measurement of the number of bacteria by flow cytometry can be evaluated in 2 minutes. It is suggested that this method is useful as a method that can easily and easily evaluate the degree of contamination with high accuracy.

  In two hot water storage tanks of one hot spring facility, changes in bacterial contamination such as inner walls before and after cleaning were observed. The hot water storage tanks have a capacity of 10 tons and 30 tons, respectively. In the hot spring facility, a 10-ton tank is provided at the outlet of the pump, and the hot water is primarily disinfected. In addition, after the hot spring water is stored in a 30 ton tank connected to the 10 ton tank by piping and the temperature is adjusted, the hot spring water is supplied to the bathtub.

  The hot water storage tank was washed by removing the contaminated components from the hot water storage tank by high-pressure washing after draining, and then spraying a 12% by mass aqueous sodium hypochlorite solution onto the hot water storage tank. A commercially available wiping sampling kit (trade name) for each of the three locations of the wall, floor, and upper surface of the water (upper boundary where hot springs are stored) before and after high pressure washing before spraying the sodium hypochlorite aqueous solution. Sampling was performed by wiping a range of about 100 square centimeters of the target wall or floor or a range of an area corresponding to the target using a wiping material of wiping check II, Eiken Chemical Co., Ltd.). The obtained sample was suspended in 10 ml of sterile phosphate buffered saline to obtain a sample. The number of general bacteria, the number of heterotrophic bacteria, and the number of bacteria by flow cytometry were measured, and the average value, the removal rate of contamination, and the residual rate of contamination were calculated. The results are shown in Table 1.

  From the results shown in Table 1, the number of bacteria by flow cytometry is almost the same as that measured for both the general and heterotrophic bacteria counts before and after washing. It can be seen that it can be measured with the same accuracy as the method for measuring the number of general bacteria and the method for measuring the number of heterotrophic bacteria.

  Next, in the hot water storage tank after washing, the upper surface of the water surface (301 in FIG. 14) where the tank material surface remains, the wall portion (302 in FIG. 14) on which the scale of raw water is attached and blackened, and the floor portion The effect of the high-pressure washing used for washing was verified by comparing the scattergrams of various Frostometry and the results of various culture methods. The results are shown in Table 2 and FIG. (A)-(C) of FIG. 15 shows the result of a water surface upper part, a wall part, and a floor part, respectively.

  Flow cytometry was performed according to the method of Example 1.

  Moreover, the measurement of the Legionella genus bacteria number was performed as follows based on the method as described in the said new edition Legionella disease prevention guideline. 500 ml of test water was collected in a sterile polypropylene bottle (manufactured by ASONE) containing 1 ml of 25% by mass sodium thiosulfate aqueous solution to obtain a sample. The obtained sample was suction filtered with a membrane filter (trade name: A045H047A, manufactured by Advantech) having a diameter of 47 mm and a pore diameter of 0.45 μm, the obtained filter was suspended in 5 ml of sterile distilled water, and the filter was pulverized. The obtained mixture was heated at 50 ° C. for 20 minutes, and 0.1 ml of the supernatant was added to GVPC medium (manufactured by Biomeryu Japan). The GVPC medium to which the supernatant was added was aerobically incubated at 35 ° C. for several days. Thereafter, when a colony suspected of being a Legionella bacterium was detected, whether or not the bacterium forming the colony was a Legionella bacterium was examined by a serotype test and a PCR test. Finally, incubation was performed for up to 10 days, and a sample that did not form a colony was determined to be a “below detection limit” sample.

The amoeba test was also carried out as follows in accordance with the method described in the new edition of Legionellosis Prevention Guidelines. The E. coli DH1 strain distributed from the National Institute of Infectious Diseases was used as a test E. coli strain used for amoeba testing. The Escherichia coli DH1 strain was transferred from a storage medium (trade name: Dorsett Egg Medium, manufactured by Nissui Co., Ltd.) to a normal agar medium and cultured at 35 ° C. for 48 hours. E. coli in sterile distilled water. D. = Suspension was prepared so as to be about 3.0 to 5.0. The resulting suspension was warmed at 60 ° C. for 1 hour and then quenched. The amount of E. coli in the resulting suspension was about 10 ⁹ CFU / ml. The suspension was diluted to 1/10 concentration with sterile distilled water. 0.5 ml of the obtained suspension was evenly applied to the surface of a 1.5% by mass agar plate (trade name: Bacto-Agar, manufactured by BBL), sufficiently dried and then dried with E. coli. Medium).

  The specimen was collected in a 50 ml sterilized centrifugal polypropylene bottle (Iwaki Glass Co., Ltd.). 1 ml of the obtained specimen was uniformly applied to an amoeba medium, and the residue obtained by centrifugal concentration at 1000 × g (3000 rpm) at 25 ° C. for 5 minutes was applied to another amoeba medium. After drying the surface of the amoeba medium after application, the amoeba medium was transferred to an incubator maintained at 30 ° C. and maintained for 2 weeks, and plaques derived from amoeba were observed. The observation was carried out daily with the naked eye and an inverted microscope, and those with confirmed plaques were morphologically identified with an optical microscope.

  Since the surface of the wiping material after wiping the vicinity of the upper surface of the water was smooth and the material surface was preserved, it was expected that the vicinity of the upper surface of the water surface was highly clean visually. However, from the results shown in Table 2 and FIG. 15, it can be seen that there are particles that are suspected to be bacteria in the specific region I by the number of bacteria by flow cytometry. From this wipe, both general bacteria and heterotrophic bacteria were detected, and Legionella bacteria were not detected. However, an amoeba species (Acansoameba) closely related to contamination by Legionella bacteria was detected.

  On the other hand, the wiping material of the wall and floor surface, which has a spring-derived scale attached to the surface and has a rough appearance with a black surface, was not confirmed by the flow cytometry, and bacteria in the specific region I were not confirmed. It can be seen that all the results are below the detection limit value. From these results, according to the number of bacteria by flow cytometry, bacterial components remain on the upper surface of the hot water tank after washing with high-pressure washing, but most of the bacterial components on the wall and floor are removed. It can be confirmed that Moreover, it turns out that the result of the number of bacteria by flow cytometry is supported also from the result by the conventional culture method.

  From these results, it can be seen that the conventional count of bacteria is confused by the cleanliness of the appearance and may be insufficiently washed. On the other hand, according to the method for measuring the number of bacteria by flow cytometry, it is possible to verify the effect of high-pressure washing with the same degree of accuracy as the culture method in a short time without being confused by the appearance. I understand that.

Ten kinds of Legionella bacteria standard strains shown in Table 3 were suspended in sterile phosphate buffered saline to prepare a bacterial suspension of about 10 ⁷ CFU / ml. The obtained bacterial suspension was measured by flow cytometry and analyzed with an analysis profile based on the specific region II shown in FIG. The result is shown in FIG. In FIG. 16B, 1 to 10 correspond to 1 to 10 in Table 3.

  From the results of 1 to 10 in FIG. 16B, it can be seen that most of the Legionella bacteria particles prepared here can be measured in the specific region II.

By examining the number of bacteria by flow cytometry and the number of bacteria by a conventional culture method for a total of 102 samples of hot water storage tank water, bathtub water, and piping water of one hot spring facility, Legionella The genus bacteria was examined. In this example, the detection limit of flow cytometry is 10 ⁴ counts / ml and the detection limit of the number of Legionella bacteria is 10 CFU / 100 ml. Correlation analysis is performed only for samples that exceed these values. Targeted. The results are shown in Table 4.

  There is no high correlation between the number of bacteria measured by flow cytometry and the number of bacteria measured by the conventional culture method. A significant difference was observed between the two test methods (chi-square test, P> 0.01). However, from the results shown in Table 4, it can be seen that about 70% of the total number of bacteria measured by flow cytometry agrees with the culture method. It can also be seen that only one sample is negative in the method for measuring the number of bacteria by flow cytometry and positive in the culture method. About the said example, since the result by a culture method is 10 CFU / 100ml which is the limit of detection by a culture method originally, there exists a possibility that the positive determination by a culture method is an error. In addition, the number of bacteria by flow cytometry can be measured in approximately 2 minutes compared to the culture method that requires 5-7 days for the test. Therefore, the number of bacteria by flow cytometry can be measured in Legionella in the water environment. It turns out that it is useful for the detection of bacteria.

  The state of bacteria before and after contacting Legionella pneumophila strain (Legionella pneumophila ATCC (American Type Culture Collection) 33152), which is a kind of Legionella bacteria, with sodium hypochlorite, which is a kind of oxidizing biocide, is as follows. As described above, the cells were observed by flow cytometry, fluorescent staining, genetic testing and culture.

The Legionella pneumophila strain stored frozen at −80 ° C. was restored, and 10 ml of sterile phosphate buffered saline (pH 7.2) prepared using a commercially available phosphate buffer (manufactured by Sigma) was contained. The Legionella pneumophila strain was added to an L-shaped test tube so that the initial value was about 10 ⁵ CFU / ml to prepare a Legionella bacterium suspension. An appropriate amount of 0.12% by mass sodium hypochlorite aqueous solution is added to the Legionella bacterium suspension so as to maintain an effective chlorine concentration of 10 mg / l, which is a lethal concentration for most microorganisms. The resulting mixture was incubated at 30 ° C. for 5 minutes. The state before and after the incubation was observed by flow cytometry, fluorescent staining method, genetic test method and culture method.

  In the method by flow cytometry, measurement was performed by the determination apparatus B shown in FIG. 7 using 120 μl of Legionella suspension before incubation with chlorine and 120 μl of the mixture after incubation with chlorine. These samples are automatically mixed with 50 μl of sample, 340 μl of diluent (trade name: Bactoquick Diluent), and further mixed with 10 μl of fluorescent dye for staining nucleic acid (trade name: Bactoquik Stain). The The mixed measurement sample is guided to the detection unit.

  In the fluorescent staining method, 1 ml of Legionella suspension before incubation with chlorine and 1 ml of the mixture after incubation with chlorine were each trapped on a 25 mm black polycarbonate membrane filter (pore size 0.2 μm, manufactured by Advantech). Product name: LIVE / DEAD BacLight Bacterial Viability Kit (manufactured by Invitrogen), according to the attached instruction manual, 1000 on the filter by B excitation of an epifluorescence microscope (trade name: Eclipse E800, manufactured by Nikon Corporation) Fluorescence was observed at double. The LIVE / DEAD BacLight Bacterial Viability Kit can dye a living cell to a red color with a special fluorescent dye.

  In the culture method, before incubation with chlorine, 0.1 ml of a solution obtained by appropriately diluting Legionella spp. Suspension is applied to BCYEα medium (Nihon Biomelieu Co., Ltd.) and cultured at 35 ° C for 72 hours. The number of viable bacteria per ml was calculated. On the other hand, after incubation with chlorine, 200 ml of the mixture after incubation with chlorine was concentrated by filtration with a polycarbonate membrane filter, and the resulting concentrate was suspended in 2 ml of sterilized distilled water. The suspension was heated at 50 ° C. for 20 minutes, and then 0.1 ml of the suspension was smeared on the BCYEα medium and cultured at 35 ° C. for 72 hours, and the viable cell count per 100 ml was calculated.

Genetic testing was performed by the LAMP method. Before incubation with chlorine, nucleic acid was extracted from Legionella bacteria using 1 ml of Legionella suspension and trade name: QIAamp DNA mini Kit (Qiagen). In addition, after incubation with chlorine, 200 ml of the mixture after incubation with chlorine was concentrated by filtration using a polycarbonate membrane filter, and 1 ml of the resulting suspension and product name: QIAamp DNA mini Kit (manufactured by Qiagen) were used. The nucleic acid was extracted from Legionella bacteria. Using the obtained nucleic acid and Legionella detection reagent kit E (Eiken Chemical Co., Ltd.), a gene sequence specific to Legionella bacteria is amplified, and a real-time turbidity measuring device (trade name: LA320C, Eiken) The turbidity was measured by Chemical Co., Ltd. The Legionella detection reagent kit E increases in turbidity only when a gene sequence specific for Legionella bacteria is present in the nucleic acid to be tested. The results are shown in Table 5 and FIG. (A) to (D) of FIG. 17 are respectively a scattag tam before incubation with sodium hypochlorite, a scattag tam after incubation with sodium hypochlorite, and hypochlorous acid. The observation photograph figure of the shape of Legionella genus bacteria before the incubation with sodium and the observation photograph figure of the shape of Legionella bacterium in the case after incubation with sodium hypochlorite are shown. Moreover, in Table 5, it prepares so that it may become initial value 10 < ⁵ > CFU / ml before chlorination. Moreover, * 2 in the table | surface is the value which measured what was concentrated and filtered 100 times.

  From the results shown in Table 5, according to the measurement of the number of bacteria by flow cytometry, a clear change in the scattergram by flow cytometry was confirmed before and after incubation with sodium hypochlorite, which is an oxidizing biocide. It can be seen that a state that is not detected by any of the fluorescent staining method, the genetic test method, and the culture method can be discriminated by a characteristic image. From the results shown in (A) and (B) of FIG. 17, in the scattergram of Legionella pneumophila strain, before incubation with sodium hypochlorite, which is the oxidizing biocide ((A) of FIG. 17), It can be seen that what was detected in the specific region I has shifted to the non-living region after incubation with sodium hypochlorite (FIG. 17A). In addition, from the results shown in FIGS. 17C and 17D, from the scattergram of Legionella pneumophila strain, before incubation with sodium hypochlorite, which is the oxidizing biocide (FIG. 17C) Indicates that most of the cells of Legionella bacteria are stained green and are alive. On the other hand, after incubation with sodium hypochlorite ((D) of FIG. 17), the cells are in a shell state and are not stained in green or red, and most cells are denatured. I understand that. In the genetic test method, 200 ml of a mixture of Legionella bacteria and chlorine after incubation is filtered and concentrated with a polycarbonate membrane filter, and a gene sequence specific to Legionella bacteria is extracted from nucleic acid extracted from 1 ml of the obtained suspension. Was not amplified. In the culture method, 200 ml of a mixture of Legionella bacteria and chlorine after incubation was filtered and concentrated with a polycarbonate membrane filter, and 0.1 ml of the resulting suspension was applied to the BCYEα medium, and Legionella bacteria were detected. There wasn't. From these results, the bacterial cell detected in the non-living region by flow cytometry has the structure of the gene destroyed so that it cannot be fluorescently stained by any of the fluorescent staining method, genetic test method and culture method, It became clear that it could not be detected by genetic testing or culture methods involving filtration and concentration.

  Therefore, according to the measurement of the number of bacteria by flow cytometry, it is possible to easily measure the living bacteria with high accuracy. It is suggested that it can be determined. Moreover, the measurement of the number of bacteria by flow cytometry suggests that the water quality can be easily managed with high accuracy.

In a building facility with the 5th floor as the top floor, roof water storage tank water, 1st floor pump water, and 3rd to 5th floor bathroom shower water were each collected in a 500 ml sterilized polypropylene container. About the obtained specimen, effective chlorine concentration, the number of general bacteria, the number of heterotrophic bacteria, and the number of bacteria by flow cytometry were measured, respectively. Flow cytometry was performed using the determination apparatus B shown in FIG. The effective chlorine concentration was measured by the free residual chlorine concentration by the diethyl-p-phenylenediamine method (DPD method). The number of general bacteria, heterotrophic bacteria, and bacteria by flow cytometry were measured by the same method as in Example 1, respectively. The detection limit of the number of general bacteria and heterotrophic bacteria was calculated as 30 CFU / ml, and the detection limit of flow cytometry was calculated as 10 ⁴ counts / ml, and the numerical value below that was ND (Not Detected). The results are shown in Table 6 and FIG. In the table, * indicates a flow cytometry detection limit of less than 10 ⁴ counts / ml.

  From the results in Table 6, roof water storage tank water having an effective chlorine concentration of 0.05 to 0.2 mg / l, 1st floor pumping tank water, 4th floor shower water, and 5th floor shower water are specific areas in flow cytometry. It can be seen that no bacteria are found in I, and no bacteria are detected even in the case of the number of general bacteria and heterotrophic bacteria. The 3F shower water, in which no effective chlorine concentration was observed, was found to have bacteria in the specific region I as shown in the boxed area of FIG. 18, for example, both in the case of the number of general bacteria and the number of heterotrophic bacteria. It can be seen that it is detected. From these results, even at a low chlorine concentration of 0.05 to 0.2 mg / l, the number of bacteria by flow cytometry clearly shows the sample in which chlorine disinfection is effective and the sample ineffective. It can be discriminated and is consistent with the results of the conventional culture method. Therefore, the measurement of the number of bacteria by flow cytometry suggests that the microbicidal effect of the microbicide can be determined in a short time with high accuracy. Moreover, the measurement of the number of bacteria by flow cytometry suggests that the water quality can be easily managed with high accuracy.

  Effective water concentration, general bacterial count, heterotrophic bacterial count, and bacterial count by flow cytometry for bath water in public baths using water sources that do not contain substances that relatively inhibit chlorine, such as tap water and well water. Was measured. The effective chlorine concentration was measured by the free residual chlorine concentration by the DPD method. The number of general bacteria, heterotrophic bacteria, and bacterial counts by flow cytometry were each measured by a water quality management device C shown in FIG. The results are shown in Table 7. A corresponding scattergram is shown in FIG. In the table, * indicates a minimum value of 1.48 log CFU / ml or less for each of general bacterial counts and heterotrophic bacteria counts, and ** indicates a flow cytometry detection limit of 4.0 log Counts / ml or less.

  From the results of Table 7 and FIG. 19, two samples (Sample Nos. 1 and 2) in which no effective chlorine concentration was observed clearly showed the presence of bacteria in the specific region I by flow cytometry. General bacterial counts and heterotrophic bacterial counts were detected. Bath water (sample numbers 3 to 5) having an effective chlorine concentration of 0.25 to 1.0 mg / l was a low numerical value, but bacteria were observed in the specific region I by flow cytometry, and the culture method was relatively High general bacterial counts and heterotrophic bacterial counts were detected. In bath water (sample numbers 6 to 10) with an effective chlorine concentration of 0.9 to 1.5 mg / l, no bacteria were observed in the specific region I of flow cytometry, and the number of general bacteria and heterotrophic bacteria In some cases, few bacteria were detected. From these results, it can be seen that by measuring the number of bacteria by flow cytometry, the samples in which chlorine is effective and the samples ineffective are clearly distinguished and are consistent with the results of the conventional culture method.

  As shown in Example 5, in vitro, for example, sodium hypochlorite, which is an oxidizing biocide, is brought into contact with a specimen for 3 to 5 minutes so that the residual chlorine concentration is 10 mg / l. In such a case, the scattergram as shown in FIG. 20A becomes a scattergram as shown in FIG. The residual chlorine concentration of 10 mg / l is the final concentration detected when oxidizing biocide is reacted with the sample. For hot spring water containing abundant inhibitors of chlorine such as iron (II) ions, ammonium ions, various organic substances, and microbial components, effective chlorine disappears or denatures, and the effectiveness decreases. Therefore, it is necessary to sufficiently act chlorine until the inhibitory action by the inhibitory substance disappears. In Example 5 above, the bacteria detected in the specific region I were shifted to the non-living region of the scattergram after incubation with chlorine at a residual chlorine concentration of 10 mg / l before incubation with chlorine. Bacterial nucleic acids are destroyed. In addition, nothing is detected after incubation with chlorine at a residual chlorine concentration of 10 mg / l, even by the conventional culture method.

  In order to evaluate whether or not the same method as in Example 5 is effective even in a specimen in an actual bathtub facility, ammonium ion is 1.04 ± 1.43 mg / l (mean ± standard deviation), maximum value 8.52; A total of 171 samples of 52 specimens of hot water storage tank water, 85 specimens of bathtub water, and 34 specimens of piping water of hot spring facilities using the hot spring raw water showing a minimum value of 0.051 mg / l were collected in 52 times over one year. Then, using the water quality control device C shown in FIG. 7, the number of bacteria was measured by flow cytometry. The obtained scattergram was compared with the result of the conventional culture method. Measurement of the number of general bacteria and heterotrophic bacteria by the culture method was performed in the same manner as in Example 1 and Example 2.

As a result, among the samples tested, the samples that showed the same scattergram as in FIG. 20B were, as shown in FIG. 21, an example of 16 samples of hot water tank water, 33 samples of bathtub water, and piping water. There were 5 samples. Tables 8 to 10 show the effective chlorine concentration, the number of general bacteria, the number of heterotrophic bacteria, and the number of bacteria by flow cytometry for these specimens. In the table, * 1 indicates a flow cytometry detection limit of less than 10 ⁴ Counts / ml. * 2 indicates less than 10 CFU / 100 ml, which is the detection limit for the number of Legionella bacteria, and * 3 indicates 2 PFU / 100 ml, which is the detection limit for the number of amoeba.

  From the results shown in Tables 8 to 10, general bacteria were detected in 5 samples out of samples showing a scattergram similar to (B) in FIG. Although vegetative bacteria are detected, it can be seen that neither Legionella bacteria nor amoeba have been detected from all specimens. From these results, it can be seen that most microorganisms are not detected from the sample showing a scattergram similar to FIG. 20B, and the hygienic state is good. Thus, according to the measurement of the number of bacteria by flow cytometry, it can be seen that the number of bacteria can be measured with high accuracy even in the case of an actually operating bathtub facility. Therefore, the measurement of the number of bacteria by flow cytometry suggests that the microbicidal effect of the microbicide can be determined in a short time with high accuracy. Moreover, the measurement of the number of bacteria by flow cytometry suggests that the water quality can be easily managed with high accuracy.

It is a schematic explanatory drawing of the step of the determination method of the contamination degree of the bathing equipment of this invention. It is a schematic explanatory drawing of the one aspect | mode of the determination apparatus of the contamination degree of the bathing equipment of this invention. Schematic explanatory drawing of the optical detection part of a flow cytometer. Schematic explanatory drawing of the step of the determination method of the microbicidal effect in the bathtub water of this invention. Schematic explanatory drawing of the step of the water quality management method of bathtub water of the present invention. The functional block diagram of the one embodiment of the determination apparatus of the contamination degree of the bathing equipment of this invention. The functional block diagram of the one embodiment of the determination apparatus of the microbicidal effect in the bathtub water of this invention. The functional block diagram of the one embodiment of the water quality management apparatus of the bathtub water of this invention. The functional block diagram of the one embodiment of the water quality management apparatus of the bathtub water of this invention. Schematic diagram of bacterial scattergram based on scattered light and fluorescence. Bacterial scattergram based on scattered light and fluorescence. Correlation diagram between the number of general bacteria obtained from colonies formed on standard agar and the number of bacteria by flow cytometry Correlation diagram of the number of heterotrophic bacteria determined from colonies formed on R2A agar and the number of bacteria by flow cytometry The drawing substitute photograph which shows a part of hot water storage tank used for Example 2. FIG. Scattergram by flossatemetry of samples collected from the upper surface, wall and floor of a hot water storage tank. Scattergram of Legionella spp. The scattergram and microscope observation photograph figure of the sample before and behind incubation (chlorine treatment) with sodium hypochlorite which is an oxidizing biocide. Scattergram of rooftop water storage tank water, water pumping water on the 1st floor, and bathroom shower water on each floor on the 3rd to 5th floors in building facilities with the 5th floor as the top floor. Scattergram of bath water in public baths with various effective chlorine concentrations. 10 is a scattergram of a typical example in the fifth embodiment. Scattergram of bath facility specimens (hot water tank water, bathtub water, piping water). It is a block diagram which shows the hardware constitutions of the information processing apparatus 2 (200,300,400).

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Flow cytometer 2 Information processing apparatus A Pollution degree judgment apparatus B Microbicidal effect judgment apparatus C Water quality management apparatus D Water quality management apparatus 12 Microorganism classification part 13 Particle counting part 14 Pollution degree information storage part 15 Pollution degree judgment part 16 Display Unit 17 Microbicidal effect information storage unit 18 Microbicidal effect determination unit 19 Water quality information output unit 20 Water quality information storage unit 21 Water quality determination unit 22 Water quality information output unit 23 Storage unit 101 Flow cell 200 Information processing device 201 Singular region I
202 Non-living region 203 Singular region II
300 Information Processing Device 301 Upper Water Surface 302 Wall 400 Information Processing Device

Claims (16)

(A) preparing a measurement sample by mixing a sample suspected of containing particles collected from a bathing facility with a reagent containing a fluorescent dye that binds or associates with microorganisms;
(B) introducing the measurement sample obtained in step (A) into the flow cell of the flow cytometer and flowing it;
(C) irradiating the flow cell with light in the step (B), and measuring the intensity of scattered light and the intensity of fluorescence emitted from particles in the measurement sample flowing in the flow cell;
(D) Based on the scattered light intensity and the fluorescence intensity measured in the step (C), the particles included in the range of the predetermined scattered light intensity and the fluorescence intensity are classified as contaminating microorganisms involved in the contamination of the bathing facility. Step,
(E) Counting the number of particles classified as contaminating microorganisms in step (D), and (F) Contamination of bathing equipment based on the number of particles counted as contaminating microorganisms in step (E). Determining the degree,
A method for determining the degree of contamination of bathing equipment.   The determination method according to claim 1, wherein the fluorescent dye is a nucleic acid-staining fluorescent dye.   The determination method according to claim 1, wherein the reagent further contains a surfactant.   The determination method according to any one of claims 1 to 3, wherein the bathing facility is a hot water storage tank, a filtration tank, a hair catcher, piping, a bathtub, a bathroom wall, a bathroom floor, a bathroom joint, or a shower facility.   A step of displaying the distribution of the particles measured in step (C) on the distribution map having the scattered light intensity and the fluorescence intensity as coordinate axes, and displaying a range corresponding to the contaminating microorganism on the distribution map; The determination method of any one of Claims 1-4 containing.   The determination method according to claim 1, wherein the contaminating microorganism is a Legionella bacterium.   The determination method according to any one of claims 1 to 6, wherein in the step (F), the degree of contamination of the bathing facility is determined by comparing the number of particles counted as contaminating microorganisms with a threshold value. (A) mixing a bath water suspected of containing particles treated with a microbicide and a reagent containing a fluorescent dye that binds or associates with microorganisms to prepare a measurement sample;
(B) introducing the measurement sample obtained in step (a) into the flow cell of the flow cytometer and flowing it;
(C) irradiating the flow cell with light in the step (b), and measuring the intensity of scattered light and the intensity of fluorescence emitted from particles in the measurement sample flowing in the flow cell;
(D) Based on the scattered light intensity and the fluorescence intensity measured in the step (c), the particles included in the range of the predetermined scattered light intensity and the fluorescence intensity are classified as contaminating microorganisms involved in bath water contamination. Step,
(E) counting the number of particles classified as contaminating microorganisms in step (d), and (f) based on the number of particles counted as contaminating microorganisms in step (e), Determining a microbicidal effect;
A method for determining the microbicidal effect of a microbicide in bathtub water.   The determination method according to claim 8, wherein the microbicide is an oxidizing biocide.   The determination method according to claim 9, wherein the oxidizing biocide is a compound that generates hypochlorous acid and / or hypobromite in water, a peroxide, or ozone. (I) mixing each sample suspected of containing particles collected from bath water over time with a reagent containing a fluorescent dye that binds or associates with microorganisms to prepare a measurement sample;
(II) introducing the measurement sample obtained in step (I) into a flow cell of a flow cytometer and flowing it;
(III) irradiating the flow cell with light in the step (II), and measuring the intensity of scattered light and the intensity of fluorescence emitted from particles in the measurement sample flowing in the flow cell;
(IV) Based on the scattered light intensity and the fluorescence intensity measured in the step (III), the particles included in the range of the predetermined scattered light intensity and the fluorescence intensity are classified as contaminating microorganisms involved in bath water contamination. Step,
(V) counting the number of particles classified as contaminating microorganisms in step (IV), and (VI) based on the number of particles counted as contaminating microorganisms in step (V), contained in the bath water. A method for managing the water quality of bathtub water, comprising the step of outputting information on a change in the number of contaminating microorganisms over time in a displayable manner.   Information on changes in the number of contaminating microorganisms over time displays the measured particle distribution on a two-dimensional distribution map with the scattered light intensity and fluorescence intensity as coordinate axes, and contamination on the two-dimensional distribution map. The water quality management method according to claim 11, including a diagram showing a change over time of a two-dimensional distribution diagram displaying a range of microorganisms.   The water quality management method according to claim 11 or 12, wherein the bath water includes hot spring water. A device for determining the contamination level of bathing equipment,
A flow cytometer that measures the intensity of scattered light emitted from the particles in a sample suspected of containing particles collected from a bathing facility and the intensity of fluorescence;
Based on the scattered light intensity and fluorescence intensity of the particles measured with the flow cytometer, the microorganism classification that classifies particles included in the range of the predetermined scattered light intensity and fluorescence intensity as contaminating microorganisms involved in contamination of bathing facilities And
A particle counter for counting the number of particles classified as contaminating microorganisms in the microorganism classification unit;
A pollution degree information storage unit for storing information relating the number of contaminating microorganisms and the degree of contamination of bathing facilities;
Based on the numerical value of the number of particles counted as contaminating microorganisms in the particle counting unit and the information stored in the contamination level information storage unit, a contamination level determination unit that determines the contamination level of the bathing facility,
An apparatus for determining the degree of contamination of a bathing facility, comprising: An apparatus for determining the microbicidal effect of a microbicide in bath water,
A flow cytometer that measures the intensity of scattered light emitted from the particles and the intensity of fluorescence in a bath water suspected of containing particles, treated with a microbicide;
Based on the scattered light intensity and fluorescence intensity of the particles measured with the flow cytometer, the microorganism classification that classifies particles included in the range of the predetermined scattered light intensity and fluorescence intensity as contaminating microorganisms involved in contamination of bathing facilities And
A particle counter for counting the number of particles classified as contaminating microorganisms in the microorganism classification unit;
A microbicidal effect information storage unit for storing information relating the number of contaminating microorganisms and the microbicidal effect of the microbicide,
Based on the information stored in the microbicidal effect information storage unit and the number of particles counted as contaminating microorganisms in the particle counting unit, a microbicidal effect determination unit that determines the microbicidal effect of the microbicide,
An apparatus for determining the microbicidal effect of a microbicide in bath water. A flow cytometer that measures the intensity of scattered light emitted from the particles and the intensity of fluorescence collected in the bath water suspected of containing the particles collected over time;
Based on the scattered light intensity and fluorescence intensity of the particles measured by the flow cytometer, the microorganism classification for classifying particles included in the range of the predetermined scattered light intensity and fluorescence intensity as contaminating microorganisms involved in bath water contamination And
A particle counter for counting the number of particles classified as contaminating microorganisms in the microorganism classification unit;
A water quality information output unit that outputs information on change over time of the number of contaminating microorganisms contained in the bath water based on the number of particles counted as contaminating microorganisms in the particle counting unit. Water quality management device for bathtub water.