JP2001286296A - Method for measuring microorganism and device for measuring the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and a device for rapidly and sensitively measuring an microorganism being an object of a microorganism test in a detecting device. SOLUTION: The device is composed of 4',6-diamidino-2-phenylindole dihydrochloride 1, propidium iodide 2, attaching plate 3 for microorganisms, plate platform 4, excitation light source 5 irradiating exciting light, fluorescence receiver 6 confirming fluorescence activated by the microorganism and spectroscopic film 7 restricting wavelength of the fluorescence. A solution containing the microorganism is attached on the surface of the attaching plate 3 and a detecting agent is dropped. The excitation light source 5 is lighted from the bottom of the attaching plate 3, the microorganism on the attaching plate 3 is allowed to develop the fluorescence, the fluorescence is received by the fluorescence receiver 6, and the microorganism irradiating the fluorescence is individually lighted by using the spectroscopic film 7 capable of transmitting only light with a specific wavelength. Number of both live cells and dead cells or the number of the dead cells is measured by counting the individual cell, the number of the existing live cell is calculated, and thus the number of the live cell or the dead cell in a specimen is confirmable.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the possibility that a plurality of types of microorganisms (the meaning of which is at least a concept including bacteria and fungi), that is, the possibility that bacteria and fungi, etc. may be attached or contaminated. The present invention relates to a microorganism weighing method and a microorganism weighing apparatus capable of simultaneously coloring live cells, dead cells, and specific microorganisms by coloring them from a certain sample.

[0002]

2. Description of the Related Art Conventionally, this kind of microorganism weighing method and microorganism weighing apparatus are disclosed in Japanese Patent Application Laid-Open No. Hei 10-323197.
Means for detecting a microorganism that has been decomposed by an enzymatic reaction in a microorganism and emits fluorescence are known, and this is recognized as a living microorganism. In addition, the number of living and dead microorganisms is determined by using a reagent for staining dead cells in combination.
Further, as another example, a method for detecting luminescence of a specific microorganism using an antibody as described in JP-A-7-140148 is known, and a specific microorganism is used in combination with a reagent that stains nonspecifically. And what grasps a living and dead microorganism is known.

In the field of food hygiene and the like, there is a demand for rapid detection of specific microorganisms. Specific microorganisms such as Escherichia coli group or specific Escherichia coli and, depending on foods, specific microorganisms such as Staphylococcus aureus are measured. However, when specific microorganisms are mixed with general bacteria or in the presence of impurities, it is necessary to separate or separate culture using separation means or to remove impurities. It could not be weighed at the same time.

[0004]

In such a conventional method for measuring microorganisms and a conventional apparatus for measuring microorganisms, the former method for immediately distinguishing microorganisms comprises a fluorescent sample composed of fluorescein or a derivative thereof, and propidium iodide. Although a fluorescent dye was used under limited conditions,
Since a fluorescent sample composed of fluorescein or a derivative thereof requires a process of emitting light by degradation by esterase expressed by microorganisms, a large part due to the enzymatic activity is large. That is, the temperature of the enzyme activity or the substrate specificity that differs depending on the type of microorganism is cited as an error affecting factor. Therefore, high-sensitivity measurement is expected in an environment where only microorganisms of the same species are present, but under actual use environment, there is a high possibility that underestimation or overestimation will occur. Further, the esterase may be produced outside the cells of the microorganism, and in that case, the background and the microorganism may be integrated, so that accurate detection may not be possible. Therefore, there is a problem in that the number of living and dead microorganisms cannot always be grasped even when used in combination with the propidium iodide or when double staining is performed.

[0005] On the other hand, the latter method of counting microorganisms uses a method of specifically detecting microorganisms using an antibody and a staining reagent that nonspecifically binds to microorganisms.
Although it is essential to use an antibody, the antibody is highly sensitive because it specifically binds, but cannot be used to determine whether it is dead or not. Furthermore, in the technique using this technique, it is not possible to determine the viability of a microorganism stained with a staining reagent that non-specifically stains contained in a specimen.

There is also a need to measure specific microorganisms and general bacteria simultaneously and quickly.

An object of the present invention is to solve such a conventional problem and to provide a microorganism weighing method for accurately detecting the amount of microorganisms by eliminating an error-influencing factor due to the enzyme activity of the microorganisms.

It is another object of the present invention to provide a method for measuring microorganisms that prevents the integration of microorganisms with the background to be measured and enables simultaneous measurement of general bacteria and specific microorganisms.

[0009] In addition, by making the color development fluorescent, it is possible to control the wavelength of the excitation light and the fluorescence having a specific wavelength to be colored by the excitation light, and to accurately measure the cells to be detected. The aim is to provide a method.

[0010] It is another object of the present invention to provide a microorganism measuring method capable of measuring the number of living and dead cells up to a single cell level by using a compound that binds to nucleic acids of living and dead cells.

It is another object of the present invention to provide a microorganism measuring method capable of measuring the number of dead cells down to a single cell level by using a compound that binds to nucleic acids of dead cells.

It is another object of the present invention to provide a microorganism measuring method capable of measuring only the number of living microorganisms by converting a compound capable of coloring a living cell into a compound which forms a color by reacting with a substance derived from a microorganism. And

It is another object of the present invention to provide a method for measuring microorganisms which can determine the presence or absence of living microorganisms with higher sensitivity by using a compound having a microorganism-derived substance as an enzyme protein.

It is another object of the present invention to provide a microorganism weighing method that can determine the presence or absence of a specific microorganism with higher sensitivity by using a compound derived from a specific microorganism-derived substance as an enzyme protein.

Further, there is provided a microorganism measuring method capable of detecting the amount of a specific microorganism by contacting a compound which forms a color by reacting with a substance derived from a specific microorganism with a sample, and detecting the wavelength and the amount of color development. The purpose is to:

It is another object of the present invention to provide a microorganism weighing device capable of automatically and rapidly measuring the amount of microorganisms by providing a light source and light receiving means.

[0017]

According to the present invention, there is provided a microorganism weighing method and a microorganism weighing apparatus, wherein a first compound for coloring viable cells and a second compound for coloring dead cells at a wavelength different from the above-mentioned color are provided to achieve the above object. And contacting the sample with a sample, and simultaneously detecting both live cells and dead cells from the difference in wavelength and the difference in the amount of color development.

According to the present invention, the substance can be measured with high sensitivity even in an environment where various kinds of microorganisms are present, and can be evaluated even in an actual environment. Since the number of living and dead cells can be accurately grasped because it is not affected by this, the present technology is non-specifically binding and will detect all cells. In addition, the life or death of the cell can be determined. In addition, a compound for coloring live and dead cells and a compound for coloring dead cells have high stability, are easy to store, and provide a method for measuring microorganisms that can be used even in an environment such as a low temperature.

Further, a first compound for coloring live cells and dead cells and a second compound for coloring dead cells at a wavelength different from the above color development and a third compound for coloring live cells at a wavelength different from the above color development.
One or more kinds of the compounds, and at least one or more kinds of fourth compounds, which are colored at a wavelength different from the color development by reacting with a specific microorganism-derived substance, are brought into contact with the sample, and the wavelength difference and the color development The method is characterized in that live cells and / or dead cells and a specific microorganism are simultaneously detected from the amount.

According to the present invention, a specific microorganism can be measured from a variety of cells and microorganisms with high sensitivity and can be evaluated even in an actual environment. It is not affected by substances inside cells and microorganisms and substances discharged outside microorganism cells, so that the presence of microorganisms can be confirmed, and furthermore, there is no separation and washing means as pretreatment, and microorganism measurement can be performed quickly. A method is obtained.

Further, the color is fluorescent.

According to the present invention, there is provided a microorganism weighing method capable of measuring only fluorescent microorganisms with high sensitivity.

Further, the present invention is characterized in that the first compound is a nucleic acid-binding compound.

According to the present invention, it is possible to measure microorganisms down to the level of living and dead cells alone, and to obtain a microorganism measuring method capable of performing highly sensitive measurement.

Further, the present invention is characterized in that the second compound is a nucleic acid binding compound.

According to the present invention, it is possible to measure a microorganism to a single dead cell level, and to obtain a microorganism measuring method capable of performing highly sensitive measurement.

Further, the third compound is characterized in that it is a compound which develops a color by reacting with a substance derived from a microorganism in a specimen. Furthermore, the present invention is characterized in that the microorganism-derived substance is an enzyme protein.

According to these inventions, the number of living microorganisms alone can be measured. further,
By using a microorganism-derived substance as an enzyme protein, the presence or absence of a living microorganism can be determined with higher sensitivity.

Further, the specific microorganism-derived substance is an enzyme protein.

According to the present invention, there is provided a microorganism weighing method capable of recognizing the presence or absence of a specific microorganism by detecting a substance derived from the specific microorganism that is expressed in or outside the cells of the specific microorganism. By limiting the specific microorganism-derived substance to an enzyme protein, a microorganism weighing method capable of recognizing the presence or absence of a specific microorganism with higher sensitivity can be obtained.

Further, the present invention is characterized in that a compound which forms a color by reacting with a substance derived from a specific microorganism is brought into contact with a sample, and the specific microorganism is detected from the wavelength and the amount of color development.

According to the present invention, the specific microorganism is surely recognized by specifically binding to the specific microorganism, and the number of the specific microorganism is accurately grasped from the amount of fluorescent color different from the color of the reagent. And a method for measuring microorganisms that can be performed.

Further, the present invention is characterized in that a microorganism light measuring device is provided which includes a light source and a light receiving means, and measures cells based on a wavelength difference of a specimen and an amount of fluorescent color.

According to the present invention, there is provided a microorganism weighing apparatus capable of measuring microorganisms quickly and accurately.

[0035]

BEST MODE FOR CARRYING OUT THE INVENTION A first compound for coloring live and dead cells and a second compound for coloring dead cells at a wavelength different from the above-mentioned color are brought into contact with a sample, and the live cells are determined from the difference in wavelength and the amount of coloring. And the simultaneous detection of both dead cells and dead cells.They can be measured with high sensitivity even in an environment where many types of microorganisms are present, without depending on the enzyme activity expressed by the microorganisms. It can be evaluated under the use environment.

In addition, even when an enzyme expressed by a microorganism is produced outside the cells of the microorganism, the enzyme does not react with the enzyme, so that the integration of the microorganism with the background can be prevented and detected.

Also, unlike antibody detection, it specifically binds to various kinds of cells, so that the sensitivity is high and it is possible to discriminate between life and death. For example, a reagent in which 4 ', 6-diamidino-2-phenylindole dihydrochloride which binds to nucleic acid contained in living and dead cells and propidium iodide which binds to nucleic acid contained in dead cells are simultaneously sampled. The number of living cells can be measured from the number of living and dead cells by measuring the number of living and dead cells simultaneously. Furthermore, since the color development reacts quickly with individual cells, it has the effect that the presence of living and dead cells can be confirmed in a shorter time than in the conventional culture method or antibody method.

One of a first compound for coloring live and dead cells, a second compound for coloring dead cells at a wavelength different from the above-described color, and a third compound for coloring live cells at a wavelength different from the above-described color Or a plurality of types, at least one or more types of fourth compounds that develop a color at a wavelength different from the color development by reacting with a specific microorganism-derived substance, and contact the sample,
It is characterized by simultaneous detection of live cells and / or dead cells and specific microorganisms from the wavelength difference and the amount of color development, and capable of simultaneously measuring live and dead cells, dead cells and live cells, and specific microorganisms. Thus, measurement of microbial contamination in medical and food hygiene and monitoring of microorganisms in the environment can be performed quickly. For example, the number of general bacteria can be measured in the food field by viable cell count, dead cell count, and live cell count, and the state after sterilization can be ascertained by measuring dead cells. In addition, since the coliform group excretes a specific enzyme protein called β-galactosidase, the presence of the coliform group is clearly determined by using 4-methylumbelliferyl-β-D-galactoside which reacts with the enzyme protein. In other words, it has the effect of being able to inspect microbial contamination in medical care, food hygiene, etc.

By making the color fluorescent, it is possible to prevent interference of the wavelength of the light to be colored, to easily recognize the color visually, and to measure the number of cells accurately.

The first compound is a compound capable of binding to a nucleic acid. By binding to the nucleic acid, it can be measured at the level of a single living or dead cell, and can be used to remove impurities such as metabolites, dust, and inorganic substances from microorganisms. It has the effect that viable and dead cells can be accurately measured with high sensitivity even under mixed conditions.

The second compound is a compound capable of binding to a nucleic acid. By binding to the nucleic acid, it can be measured at the level of a single dead cell, and can be used to remove impurities such as metabolites, dust, and inorganic substances from microorganisms. It has the effect that dead cells can be accurately measured with high sensitivity even when mixed.

The third compound is a compound that develops a color by reacting with a substance derived from a microorganism in a specimen.
By reacting with a microorganism-derived substance such as a metabolite of a microorganism, it has an effect that the presence or absence of a living microorganism can be confirmed. In addition, by using a microorganism-derived substance as an enzyme protein, the presence or absence of a living microorganism can be determined with higher sensitivity.

The substance derived from the specific microorganism is used as an enzyme protein. Generally, the enzyme protein is a substance metabolized by living cells. Has the effect that can be measured.

A compound which forms a color by reacting with a substance derived from a specific microorganism is brought into contact with a sample, and the specific microorganism is detected from the wavelength and the amount of color development.
An appropriate amount of the detection reagent can be supplied to the sample as appropriate, and there is an effect that an operator does not come into direct or indirect contact with the sample, and highly sensitive and highly sensitive measurement can be performed.

This device is provided with a light source and a light-receiving means, and measures cells based on the wavelength difference of the specimen and the amount of fluorescent color. This makes it possible to easily measure the cells that emit fluorescent light, thereby shortening the examination time. Has the effect of being able to

[0046]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiment 1 of the present invention will be described below.

First, the microorganism quantification method is based on the following method. A first compound, 4 ', 6-diamidino-2-phenylindole dihydrochloride, and a second compound, propidium iodide, are attached to a specimen to which cells or microorganisms are attached. For each solution or mixed reagent. 4 ', 6-diamidino-2-phenylindole dihydrochloride passes through the cell membrane of the specimen and binds to the nucleic acid. In addition, since the molecular weight of propidium iodide is large, it cannot pass through the cell membrane of living cells and binds to nucleic acids of dead cells.

The 4 ', 6-diamidino-2-phenylindole dihydrochloride bound to the nucleic acid of living and dead cells absorbs the light of the excitation wavelength at the excitation wavelength of 359 nm and absorbs the light at the excitation wavelength of 461 nm.
And emits viable cells. Further, the propidium iodide bound to the nucleic acid of the dead cell absorbs the excitation wavelength at the excitation wavelength of 535 nm to 617 nm.
And emits dead cells. The developed cells are weighed visually or with a weighing instrument to check the number of viable and dead cells.

Next, a method for measuring microorganisms will be described with reference to FIG. The method for measuring microorganisms is as follows: 4 ′, 6-diamidino-2-phenylindole dihydrochloride 1 and propidium iodide 2 are used as detection reagents, and a glass attachment plate 3 and an attachment plate 3 on which cells and microorganisms of a sample are attached. And a fluorescent light receiving unit 6 provided with an excitation light source 5 for irradiating the attached plate 3 with excitation light, and a magnifying glass for confirming fluorescence of cells and microorganisms of the specimen. And a spectral film 7 for limiting the fluorescence wavelength on the upper part of the fluorescent light receiving section 6.

The weighing method uses a mixture prepared by mixing 4 ', 6-diamidino-2-phenylindole dihydrochloride 1 and propidium iodide 2 as a detection reagent, and includes cells and microorganisms obtained from a specimen. The solution is attached or dropped on the surface of the attachment plate 3, the attachment plate 3 is placed on the plate base 4, and the detection reagent is dropped on the surface of the attachment plate 3. The excitation light source 5 provided below the attachment plate 3
Is turned on to cause cells and microorganisms on the attachment plate 3 to emit fluorescent light, and the fluorescent light receiving section 6 expands the fluorescent light emission. Further, the cells and microorganisms that emit fluorescence are turned on individually by the spectral film 7 that transmits only a specific wavelength. The number of living and dead cells and the number of dead cells are measured by counting the number of simple substances that are turned on for each specific wavelength, and the existing living cells are calculated. And
The number of live cells and dead cells in the specimen can be confirmed.

Further, 200 to 1
It has a 000x lens.

Hereinafter, a second embodiment of the present invention will be described.

FIG. 2 is a schematic view of an apparatus for automatically weighing. As shown in FIG. 2, the microorganism weighing device 8 includes a light source unit 9 having a lamp that emits excitation light and 4 ′, 6-diamidino-2 that is a detection reagent for coloring microorganisms taken from a specimen and viable cells of the microorganisms. A sample reagent containing portion 10 made of glass containing a solution containing phenylindole dihydrochloride 1 and a propidium iodide 2 which is a detection reagent for coloring dead cells of microorganisms, and a light receiving portion 11 for receiving light to be colored And a wavelength spectroscopic unit 12 for dispersing the light of the light receiving unit 11, a light amount measuring unit 13 for measuring the light amount, an arithmetic processing unit 14 for calculating the number of living cells and dead cells, and a display unit 15 for displaying the calculated cell number. It is configured.

An example of using the microorganism weighing device 8 will be described below.

As a method of using the microorganism weighing device 8, there are
5 g of vegetables were immersed in 9.5 ml of physiological saline to prepare a suspension, and then 1 ml of the suspension and 4 ′, 6-diamidino-2 were added.
Mixing phenylindole dihydrochloride 1 and propidium iodide 2 into the sample reagent containing part 10. After the mixing, the detection reagent and the suspension contained in the sample reagent containing section 10 are automatically stirred, so that the detection reagent and the microorganism cells in the suspension are combined. Then, by illuminating a lamp of the light source unit 9 that emits excitation light and automatically irradiating the sample with the excitation light, the nucleic acid of the dead cell and the nucleic acid of the dead cell combined with the detection reagent develops a color and irradiates the light receiving unit 11. Is done. The light applied to the light receiving unit 11 is split by the wavelength spectroscopy unit 12 into 450 to 480 nm as the fluorescence wavelength of living and dead cells and 500 to 620 nm as the fluorescence wavelength of dead cells, and the light amount measurement unit 13 converts the light amount into an electric signal. Is done. The converted electrical signal is
The arithmetic processing unit 14 calculates each cell number from the calibration curve of each cell number and the electric signal, and subtracts the number of dead cells from the number of viable and dead cells,
Calculate viable cell count. Then, the number of live cells and the number of dead cells in 1 ml of the suspension mixed in the sample reagent containing section 10 can be automatically displayed on the display section 15.

FIG. 3 shows a calibration curve graph of the dead cells and the electric signal output, and FIG. 4 shows a calibration curve graph of the dead cells and the electric signal output.

The amount of 4 ', 6-diamidino-2-phenylindole dihydrochloride 1 added dropwise or added to the suspension was 10 nmol as the final concentration based on the amount of the sample.
The optimum concentration is 100 n
mol / ml, and the dropping amount of propidium iodide 2 or the amount added to the suspension is 10 nmol to 1 μmol / ml as the final concentration with respect to the amount of the sample, and the optimum concentration is 100 nmol / ml.

The excitation light source 5 has an excitation wavelength
A device that emits light having a wavelength of 400 to 700 nm is used.

According to the present invention, the visual level and the amount of microorganisms of cells and microorganisms in a sample can be detected quickly and accurately by using fluorescence.

Although the attachment plate 3 is made of glass in the first embodiment, a material such as acrylic or poly film which transmits the excitation light may be used.

In the first and second embodiments, the number of cells is measured by converting the amount of light into an electric signal. However, a method of taking an image and measuring the number of cells based on the lighting amount of the image may be used.

In the second embodiment, although the sample reagent containing portion 10 made of glass is used, a material that transmits the excitation wavelength and the fluorescence wavelength may be used.

Hereinafter, a third embodiment of the present invention will be described.

First, the microorganism quantification method is based on the following method. The first compound 4 ′, 6-diamidino-2-phenylindole dihydrochloride and the second compound propidium iodide And a third compound, 6-carboxyfluorescein diacetate, and a fourth compound.
4-methylumbelliferyl-β-D-
The reagent in which galactoside is mixed is brought into contact. 4 ', 6-diamidino-2-phenylindole dihydrochloride bound to nucleic acids of living and dead cells absorbs the excitation wavelength at an excitation wavelength of 359 nm, emits a fluorescence wavelength of 461 nm, and causes the coloring of the living and dead cells. Further, at the excitation wavelength of 535 nm, the propidium iodide bound to the nucleic acid of the dead cells absorbs the light of the excitation wavelength and changes to the fluorescence wavelength of 617 nm to develop only the dead cells. Also, 6-carboxyfluorescein diacetate reacted with ester of live cells is
It is decomposed by esterase to form fluorescein, and when the excitation wavelength is 474 nm, it absorbs the excitation wavelength to 541 nm.
And emits only viable cells. In addition, 4-methylumbelliferyl-β-D-galactoside reacted with β-galactosidase which is metabolized by Escherichia coli group is 4-methylumbelliferyl-β-D-galactoside.
It becomes methylumbelliferone, and when the excitation wavelength is 394 nm, it absorbs the excitation wavelength and emits a fluorescence wavelength of 489 nm,
The coliform group develops color. The number of viable and dead cells is weighed from the number of cells that have developed color, and the number of viable and dead cells is calculated.
Further, the presence or absence of living cells and living coliforms as specific microorganisms can be confirmed.

Next, the microorganism weighing method of the present invention shown in FIG. 5 will be described. The method for measuring microorganisms is as follows: 4 ', 6-diamidino-2-phenylindole dihydrochloride 1, propidium iodide 2, 6-carboxyfluorescein diacetate 16, and 4-methylumbelliferyl-β-D-
Using galactoside 17 as a detection reagent, a glass attachment plate 3 to which cells and microorganisms are attached, a plate base 4 on which the attachment plate 3 is placed, and an adhesion plate 3 below the plate base 4
A light source 5 for irradiating the sample with excitation light, a fluorescent light receiving unit 6 provided with a magnifying glass on the upper part of the attachment plate 4 for capturing cells and microorganisms in the specimen, and a wavelength of the fluorescent light emitted on the fluorescent light receiving unit 6 is limited. It is composed of a spectral film 7.

As a measuring method, 4 ′, 6
-Diamidino-2-phenylindole dihydrochloride 1, propidium iodide 2, 6-carboxyfluorescein diacetate 16 and 4-methylumbelliferyl-
Using a mixture prepared by mixing β-D-galactoside 17, the attachment plate 3 with a solution containing cells and microorganisms taken from the specimen is placed on the plate base 4, and the detection reagent is dropped onto the surface of the attachment plate 3. Then, the excitation light source 5 provided at the lower portion of the attachment plate 3 is turned on, and the cells and microorganisms on the attachment plate 3 are fluoresced. The fluorescent light is enlarged and transmitted by the fluorescent light receiving unit 7, and cells and microorganisms are turned on individually by the spectral film 7 that transmits only a specific wavelength. Viable and dead cells can be visually confirmed, and the presence or absence of highly active viable cells and coliforms can be visually confirmed.

Further, the spectroscopic film 7 is a film that transmits a fluorescence wavelength of 450 to 480 nm when measuring living and dead cells, and a film that transmits a fluorescence wavelength of 500 to 620 nm when measuring dead cells. 45 when weighing
One that transmits a fluorescent wavelength of 0 to 560 nm is used, and one that transmits a fluorescent wavelength of 400 to 550 nm is used for weighing coliform bacteria.

Hereinafter, a fourth embodiment of the present invention will be described.

An apparatus for automatically weighing will be described below with reference to FIG. As shown in FIG. 2, the microorganism weighing device 8 includes a light source unit 9 having a lamp that emits excitation light and 4 ′, 6-diamidino-2 that is a detection reagent for coloring microorganisms taken from a specimen and viable cells of the microorganisms. Phenylindole dihydrochloride 1 and propidium iodide 2 which is a detection reagent for coloring dead cells of microorganisms and 6- is a detection reagent for coloring live cells of microorganisms
The sample reagent containing portion 10 made of glass mixed with a solution containing carboxyfluorescein diacetate 16 and 4-methylumbelliferyl-β-D-galactoside 17 which is a detection reagent for coloring Escherichia coli group, and receives light to be colored. A light receiving unit 11 and a wavelength splitting unit 12 that splits light of the light receiving unit 11
It comprises a light quantity measuring unit 13 for measuring the light quantity, an arithmetic processing unit 14 for calculating the number of living cells and dead cells or the number of coliform bacteria, and a display unit 15 for displaying the calculated number of cells.

An example of use of the microorganism weighing device 8 will be described below.

The method of using the microorganism weighing device 8 is as follows.
5 g of vegetables were immersed in 9.5 ml of physiological saline to prepare a suspension, and then 1 ml of the suspension and 4 ′, 6-diamidino-2 were added.
-Phenylindole dihydrochloride 1, propidium iodide 2, 6-carboxyfluorescein diacetate 16 and 4-methylumbelliferyl-β-D-galactoside 17 are mixed into the sample reagent-containing part 10. After the mixing, the detection reagent and the suspension contained in the sample reagent containing section 10 are automatically stirred, so that the detection reagent and the microorganism cells in the suspension are combined. Then, by illuminating a lamp that emits excitation light of the light source unit 9 and automatically irradiating the sample with excitation light, the nucleic acid of the dead cell and the nucleic acid of the dead cell combined with the detection reagent are colored, and the esterase of the live cell And fluorescein and 4-methylumbelliferone are colored by the action of β-galactosidase which is metabolized by
1 is irradiated. The light applied to the light receiving unit 11 is emitted from the wavelength spectroscopy unit 12 as 461 nm as the fluorescence wavelength of the dead cells, 617 nm as the fluorescence wavelength of the dead cells, 541 nm as the fluorescence wavelength of the living cells, and 489 nm as the fluorescence wavelength of the coliform group.
m, and the light quantity is converted into an electric signal by the light quantity measuring unit 13. In the converted electric signal, the number of cells is calculated by the arithmetic processing unit 14 from the calibration curve of the electric signal, and the number of living cells is calculated from the difference between the number of living cells and the number of dead cells. Then, 1 ml mixed with the sample reagent containing section 10 is displayed on the display section 15.
The number of live and dead cells, the number of dead cells, the number of viable cells with strong activity, and the number of coliforms in the suspension can be automatically displayed.

FIG. 6 shows a calibration curve graph of live cells having strong activity and electric signal output, and FIG. 7 shows a calibration curve graph of E. coli group and electric signal output.

According to the present invention, the number of living cells and dead cells is measured by measuring the amount of living cells and dead cells by measuring the amount of viable cells and dead cells based on the fluorescent wavelength at which the cells and microorganisms in the specimen are emitted. Detection can be easily performed, and the amount and presence or absence of microorganisms such as coliforms and viable active cells can be confirmed.

[0074]

As is apparent from the above examples, according to the present invention, a sample is brought into contact with a first compound for coloring viable cells and a second compound for coloring dead cells at a wavelength different from the color. By simultaneously detecting both live and dead cells based on the wavelength difference and the difference in the amount of color development, it is possible to accurately measure live and dead cells, and understand the environmental pollution status of cells. It is possible to quickly take measures to prevent contamination. In addition, it is possible to provide a microorganism weighing method that has an effect of confirming toxicity to cells, bactericidal effect of microorganisms, and the like.

Further, the first compound for coloring live and dead cells and the second compound for coloring dead cells at a wavelength different from the above-mentioned color development and the third compound for coloring live cells at a wavelength different from the above-mentioned color development.
One or more kinds of the compounds, and at least one or more kinds of fourth compounds, which are colored at a wavelength different from the color development by reacting with a specific microorganism-derived substance, are brought into contact with the sample, and the wavelength difference and the color development Simultaneous detection of living cells and / or dead cells and specific microorganisms from the amount enables real-time environmental monitoring of food, water, etc. stains, as well as in the medical and food hygiene fields. It is possible to provide a microorganism weighing method that can be applied and that has an effect of being able to quickly ascertain the presence of specific microorganisms such as Escherichia coli and the like, and to quickly prevent the influence of microbial contamination on market distribution.

In addition, by using fluorescent color, it is possible to provide a microorganism measuring method which has an effect of preventing erroneous recognition at the time of light reception by a work measurer and enabling accurate and accurate measurement of the number of cells.

In addition, since the first compound is a nucleic acid-binding compound, accurate measurement can be performed even in the presence of cell metabolites and contaminants. Since the cells can be measured, it is possible to provide a microorganism weighing method that has an effect that the test time can be reduced and the measurement can be performed quickly.

In addition, since the second compound is a nucleic acid-binding compound, accurate measurement can be performed even in the presence of cell metabolites and contaminants, so that samples such as food and sludge can be directly killed. Since only cells can be measured, it is possible to provide a microorganism weighing method that has an effect of reducing test time and measuring more quickly.

Further, the third compound is a compound which develops a color by reacting with a microorganism-derived substance in a sample, so that the presence or absence of living microorganisms can be confirmed. Can be provided. Also,
By using a microorganism-derived substance as an enzyme protein, it is possible to provide a microorganism measuring method that has an effect of enabling the presence or absence of a living microorganism to be determined with higher sensitivity.

In addition, by using the specific microorganism-derived substance as an enzyme protein, the presence or absence of a special microorganism can be confirmed, and a microorganism measuring method having an effect of monitoring the microorganism can be provided. Further, it is possible to provide a microorganism weighing method having an effect of eliminating the necessity of a pretreatment or the like and further reducing the inspection time.

Further, by contacting a compound that forms a color by reacting with a substance derived from a specific microorganism with a specimen and detecting the specific microorganism from its wavelength and the amount of color development, only the specific microorganism can be quickly weighed. In addition, it is possible to provide a microorganism weighing method that has an effect of grasping the state of food contamination and preventing contamination of a specific microorganism with a human body.

Further, by providing a light source and a light receiving means and featuring a microorganism weighing device for weighing cells based on the wavelength difference of the specimen and the amount of fluorescence, the human error can be minimized, and even if there is no inspection work experience, it can be easily performed. It is possible to provide a microorganism weighing device having an effect of being able to perform measurement in a short time.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of a method for measuring living and dead cells and dead cells according to a first embodiment of the present invention.

FIG. 2 is a schematic view of a microorganism weighing device according to the second embodiment.

FIG. 3 is a calibration curve graph of living and dead cells and electric signal output in Examples 1 and 2;

FIG. 4 is a calibration curve graph of dead cells and electric signal output in Examples 1 and 2;

FIG. 5 is a configuration diagram of a method for measuring living cells, dead cells, living cells, and coliform bacteria in Example 3;

FIG. 6 is a calibration curve graph of live cells with strong activity and electric signal output in Examples 3 and 4.

FIG. 7 is a calibration curve graph of coliform bacteria and electric signal output in Examples 3 and 4;

[Explanation of symbols]

Reference Signs List 1 4 ', 6-diamidino-2-phenylindole dihydrochloride 2 Propide iodide 3 Attachment plate 4 Plate base 5 Excitation light source 6 Fluorescence light receiving unit 7 Spectral film 8 Microorganism measuring device 9 Light source unit 10 Sample reagent containing unit DESCRIPTION OF SYMBOLS 11 Light receiving part 12 Wavelength spectroscopy part 13 Light quantity measurement part 14 Operation processing part 15 Display part 16 6-carboxyfluorescein diacetate 17 4-methylumbelliferyl- (beta) -D-galactoside

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/48 G01N 33/48 MP (72) Inventor Kazuo Nashimoto 6-chome Imafuku Nishi, Joto-ku, Osaka-shi, Osaka No. 2 No. 61 Matsushita Seiko Co., Ltd. F-term (reference) 2G045 AA28 BB24 CB01 CB21 DA12 DA36 FA11 FA29 FB12 GC15 2G054 AA08 CA20 CE02 EA01 EB02 JA06 4B029 AA07 BB01 BB02 FA03 FA09 4B063 QA01 QA18 QQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ QQ QR QGQGQQQQQQQQQQQQQQQQQ requires Attra At At Asa at Asa at Asa at Asa at Asa at Asa at Asa at Asa at Att. QX02

Claims (10)

[Claims] 1. A test compound is contacted with a first compound for coloring live and dead cells and a second compound for coloring dead cells at a wavelength different from the color development. A method for measuring microorganisms, comprising simultaneously detecting both cells. 2. A method according to claim 1, wherein the first compound for coloring live and dead cells and the second compound for coloring dead cells at a wavelength different from the coloring and the third compound for coloring living cells at a wavelength different from the coloring. At least one or more kinds of fourth compounds that develop a color at a wavelength different from the color development by reacting one or more kinds with a substance derived from a specific microorganism are brought into contact with the specimen, and the living cells and the dead cells are determined from the wavelength difference and the color development amount. A method for measuring microorganisms, comprising simultaneously detecting a specific microorganism with both or one of cells. 3. The method according to claim 1, wherein the color is fluorescent. 4. The method according to claim 1, wherein the first compound is a nucleic acid-binding compound. 5. The method according to claim 1, wherein the second compound is a nucleic acid-binding compound. 6. The method according to claim 2, wherein the third compound is a compound that develops a color by reacting with a substance derived from a microorganism in a sample. 7. The method according to claim 6, wherein the substance derived from the microorganism is an enzyme protein. 8. The method according to claim 2, wherein the substance derived from the specific microorganism is an enzyme protein. 9. A method for measuring microorganisms, comprising contacting a compound that forms a color by reacting with a substance derived from a specific microorganism with a sample, and detecting the specific microorganism from the wavelength and the amount of color development. 10. A test compound is contacted with a first compound for coloring live and dead cells and a second compound for coloring dead cells at a wavelength different from the color development. A device for simultaneous detection of both cells,
A microorganism weighing device provided with a light source and a light receiving means, and weighs a cell based on a wavelength difference of a specimen and an amount of fluorescent color.