JP2003139704A - Device and method for dyeing microbe - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device and a method for dyeing microbes, capable of dyeing the microbes contained in a specimen forming an object to be inspected by safely and speedily capturing them. SOLUTION: After mounting a filtering container 2 and a suction part 13 to a filter unit 3 at a position C, microbes are captured on a membrane filter by injecting a prescribed quantity of liquid to be inspected in the filtering container 2 to suck and filter it. Subsequently, the filtering container 2 and the suction part 13 are detached from the filter unit 3, the filter unit 3 is moved to a position B, a cap 14 for dyeing and a dyeing part 4 are mounted to the filter unit 3, the captured microbes are dyed under a prescribed condition by injecting dyeing liquid while preventing the dyeing liquid from evaporating under a sealed condition. After dyeing, residual dyeing liquid is removed without adhering to the microbes and the surface of the membrane filter. By the above treatment, the microbes contained in the liquid to be inspected can be safely and accurately caught and the only captured microbes can be selectively dyed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a dyeing apparatus and a dyeing method for a microorganism, for example, for capturing a microorganism contained in a liquid to be inspected on a membrane filter and fluorescently staining the captured microorganism for fluorescence microscope observation. The present invention relates to a dyeing device and a dyeing method for a microorganism as a sample.

[0002]

2. Description of the Related Art Conventionally, inspection of microorganisms in manufacturing process control, product quality control, etc. of beverages such as beer, foods, pharmaceuticals, cosmetics, etc. has been carried out by a culture test requiring a variety of culture media. Took a considerable number of days to complete. For this reason, the number and types of microorganisms contained in the liquid to be inspected have been lagging behind, and there have been great restrictions at the stage of research, development, manufacturing or product shipment in various fields.

From such a background, there has been proposed a method of reliably capturing the microorganisms contained in the liquid to be inspected, staining the captured microorganisms, and rapidly detecting the microorganisms by observing with a fluorescence microscope. For example, as a method for staining microorganisms, DAPI (dibumidinophenylindole) or P
I (propidium iodide) etc. are used to stain microbial nucleic acid, 6CFDA (6-carboxyfluorescein diacetate) etc. are used to stain microbial enzyme activity, FITC and other fluorescent substances are used to label antibodies. There are a fluorescent antibody method for staining and then a FISH method for labeling and staining a nucleic acid probe with a fluorescent substance such as FITC. The microorganisms stained by the above staining method are detected using a fluorescence microscope or the like.

However, in order to detect the microorganisms with high accuracy by the above-mentioned fluorescence microscope observation, a treatment for surely capturing the microorganisms contained in the test liquid and an appropriate fluorescence staining of the captured microorganisms for fluorescence microscope observation. It is necessary to perform a dyeing process as a sample for use.

By the way, microorganisms are surely captured from a liquid to be inspected suspected of being contaminated with microorganisms, such as raw water from a water purification plant, drinking water such as mineral water and beer, and appropriately fluorescently stained to be used as a sample for fluorescence microscope observation. To do so, various complicated processes are required.

[0006] As an example of the above-mentioned treatment, for example, a treatment for filtering the liquid to be inspected with a filtration container to capture microorganisms on the membrane filter, a washing for washing the liquid to be inspected adhering to the wall of the filtration container, etc. Processing, processing to remove the membrane filter from the filtration container and attach it to the dyeing container,
The treatment includes staining the microorganisms with a stain and removing the stain remaining after staining.

However, when the above-mentioned processing is manually performed by an operator, filtration processing, washing solution injection processing, fixing solution injection processing for pre-staining processing, membrane filter removal and installation processing, and dyeing are performed. It is necessary to perform a complicated process such as a liquid injection process and a cleaning liquid injection process, each of which requires a considerable amount of time. Further, when the worker manually performs the above processing, it is necessary to perform processing for preventing infection by microorganisms such as bacteria. Further, in order to prevent the occurrence of human error during the complicated processing, the operator is forced to make a great effort. Therefore, there is a problem that the above-described manual work by the operator is not suitable for dyeing a large number of liquids to be inspected.

When dyeing the captured microorganisms with a dyeing solution containing a dyeing agent, the membrane filter has to be in contact with the dyeing solution for a predetermined time. This dyeing treatment is carried out under the condition that light is present. There was a problem that the dyeing solution was gradually deteriorated by the light.

Furthermore, if the dyeing solution evaporates during the dyeing process and the dyeing agent adheres to the membrane filter, it becomes difficult to remove the adhering dyeing agent, and the accuracy of detection of microorganisms by a fluorescence microscope deteriorates. There was also a problem.

As a method of solving these problems, for example, in Japanese Patent Publication No. 4-7466, a dye is added in advance to the liquid to be inspected in order to facilitate the treatment described above, and A method for dyeing microorganisms present in the above and then collecting by filtration is disclosed. However, in Japanese Examined Patent Publication No. 4-7466, since the dye is diluted with a large amount of the test liquid, in order to dye the target microorganism,
For example, since expensive dyes such as labeled probes must be used in large amounts, they are not suitable for dyeing a large amount of test liquid. Further, it cannot be used when the stain reacts with the test liquid and decomposes.

Further, in Japanese Patent Laid-Open No. 62-138185, a sample sampled by a sampling pump is filtered by a membrane filter to capture microorganisms contained in the sample on the membrane filter, and then this microorganism is captured. There is disclosed a microorganism counter in which the above-mentioned membrane filter is conveyed by a roller and brought into contact with a dyeing solution, and the dyed membrane filter is conveyed by a roller and observed by a fluorescence microscope.

However, the above-mentioned microorganism counter is
When dyeing a microorganism with a fluorescent dye, it is impossible to prevent the dye from evaporating and sticking the dye on the membrane filter, and it is also impossible to remove an unnecessary dye after fluorescent dyeing the microorganism. For this reason, in addition to the dyed microorganisms, there is also a dyeing agent that should be removed on the membrane filter. Therefore, in the fluorescence microscope observation performed with the above-mentioned microorganism counter, when detecting a microorganism, the detection accuracy of the microorganism is lowered due to the influence of the remaining stain.

Further, in Japanese Patent Application Laid-Open No. 7-177875, in order to facilitate the dyeing process with a membrane filter, a filter cassette equipped with a membrane filter having nucleic acid fixed thereon is mechanically stretched with a roller on a plastic film. Push it in to form a dyeing container with the filter cassette and film, cover the opening of the container with a narrowing and heating mechanism, inject the dyeing solution, further heat reaction, and release the tension of the film after dyeing to release the dyeing container There is disclosed a nucleic acid hybridization device for breaking a filter and removing a filter.

However, the above-mentioned hybridization device does not have a filtering section for filtering the liquid to be inspected containing microorganisms on the membrane filter, and after washing the unnecessary dye with the fluorescent dye, the membrane filter is brought into contact with the fluorescent dye. There is no cleaning part to remove. For this reason, it is not possible to process the test liquid containing the microorganisms on the membrane filter from the filtration to the dyeing in a unified and rapid and safe manner, and the stains remaining on the membrane filter other than the dyed microorganisms. Also, since the presence of the above-mentioned presence also causes a problem such as a decrease in the detection accuracy of the microorganism when detecting the microorganism with a fluorescence microscope.

[0015]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems of the prior art, and its object is, for example, a membrane containing microorganisms contained in a test liquid such as beer. Reliable capture on the filter,
It is another object of the present invention to provide an inspection apparatus and an inspection method for a microorganism, which can prepare a sample in which the captured microorganism can be fluorescently stained to detect the microorganism with a fluorescence microscope with high accuracy.

[0016]

An inspection apparatus for inspecting microorganisms according to an embodiment of the present invention for achieving the above object has the following configuration. That is, a dyeing device for dyeing microorganisms contained in a liquid to be inspected, a capturing means for filtering the microorganisms contained in the liquid to be inspected and capturing them on a filter, and a dyeing liquid on the filter capturing the microorganisms. It is characterized by comprising a dyeing means for introducing and dyeing a microorganism with the dyeing solution, and a sealing means for shielding the periphery of the filter into which the dyeing solution is introduced to form a sealed space.

Here, for example, it is preferable that the sealing means prevents the dyeing liquid from being evaporated during dyeing to have a high concentration and being fixed to the surfaces of the microorganisms and the filter so that they cannot be removed.

Here, for example, the volume of the closed space formed by the sealing means is preferably 350 cm ³ or less.

Here, for example, the capturing means has a filtration container including a membrane filter, and the liquid to be inspected is filtered by the filter container to capture the microorganisms contained in the liquid to be inspected on the membrane filter. Preferably.

Here, for example, it is preferable that the capturing means has an accelerating means for accelerating the speed of the filtration.

Here, for example, it is preferable that the sealing means holds the dyeing solution so that a predetermined amount of the dyeing liquid remains in the sealed space during the dyeing period.

Here, for example, it is preferable that the sealing means is formed of a light shielding material that does not transmit light.

Here, for example, it further comprises a transport means for transporting the capturing means to preset first and second positions, and a transport control means for controlling the transport means, and the capturing means is It is preferable that the transport means transports the microorganisms to the first position to capture the microorganisms, and then the transport means transports the microorganisms to the second position to stain the microorganisms.

Here, for example, the dyeing means may be arranged such that the dyeing agent is applied from the primary side of the filter, which is a side for injecting the test liquid into the filter, or the secondary side of the filter facing the primary side. It is preferable to inject a solution containing

Here, for example, the dyeing means has a constant temperature means for keeping the closed space closed by the sealing means at a predetermined temperature and a dyeing time control means for controlling the dyeing time to be a predetermined time. Is preferred.

Here, for example, it is preferable to further include a removing means for removing the dyeing solution after dyeing the microorganism with the dyeing solution.

Here, for example, it is preferable to further include a supply means for supplying the inspection target liquid, the pretreatment liquid for dyeing, the sterilizing liquid and the cleaning liquid to the capturing means.

Here, for example, the sterilizing solution and / or the washing solution is preferably supplied after the microorganisms are dyed with the dyeing agent.

Here, for example, the capturing means has a filtration container for holding the inspection target liquid, the supply means has a rotary nozzle for spraying the cleaning liquid and a nozzle drive section, and the supply means is It is preferable to remove the liquid to be inspected attached to and remaining on the inner wall of the filtration container by using the rotary nozzle.

Here, for example, the pretreatment liquid and / or the cleaning liquid is preferably supplied after the liquid to be inspected is passed through the filter.

Here, for example, it is preferable that the filter has a predetermined filter diameter and is processed into a shape that can be installed in the capturing means.

Here, for example, it is preferable that the filter is a flat plate membrane filter, and the smoothness of the flat surface has a maximum error of 100 μm or less.

Here, for example, the pore diameter of the filter is 5
It is preferably smaller than 0 μm.

A dyeing method according to an embodiment of the present invention for achieving the above object has the following constitution. That is, a staining method for staining microorganisms contained in the inspection target liquid, a capturing step of filtering the microorganisms contained in the inspection target liquid on a filter, and the periphery of the filter into which the staining liquid is introduced. And a sealing step of forming a closed space by shielding the same, and a staining step of introducing the staining solution into the filter that has captured the microorganisms and staining the microorganisms with the staining solution.

Here, for example, it is preferable that the sealing step prevents the dyeing liquid from evaporating to a high concentration during dyeing and sticking to the surface of the microorganisms and the filter so that they cannot be removed.

Here, for example, the volume of the closed space formed by the closing step is preferably 350 cm ³ or less.

Here, for example, in the capturing step, it is preferable that the inspection target liquid is filtered with a filtration container including a membrane filter and the microorganisms contained in the inspection target liquid are captured on the membrane filter.

Here, for example, in the capturing step, it is preferable to use a pressurizing or depressurizing pump to accelerate the filtration rate.

Here, for example, in the sealing step, it is preferable to hold the dyeing solution so that a predetermined amount remains in the sealed space during the dyeing period.

Here, for example, it is preferable that the closed space is closed by a light blocking material that does not transmit light.

Here, for example, it further comprises a carrying step for carrying the filter to preset first and second positions, and a carrying control step for controlling the carrying step, wherein the filter carries the carrying step. Is transported to the first position by the capturing step, the microorganisms are captured on the filter by the capturing step, the filter is transported to the second position by the transporting step, and the microorganisms are stained by the staining step. Preferably.

Here, for example, in the dyeing step, the dyeing solution is applied from the primary side of the filter, which is the side for injecting the test liquid into the filter, or the secondary side of the filter facing the primary side. Is preferably injected.

Here, for example, in the dyeing step, a temperature controller that keeps the closed small space at a predetermined temperature and a dyeing time controller that controls the dyeing time to a predetermined time are used. preferable.

Here, for example, it is preferable to further include a removing step of removing the dyeing solution after dyeing the microorganism with the dyeing solution.

Here, for example, it is preferable to further include a supply step of supplying the inspection target liquid, the pretreatment liquid for dyeing, the sterilizing liquid and the cleaning liquid to the filter.

Here, for example, the sterilizing solution and / or the washing solution is preferably supplied after the microorganisms are dyed with the dyeing agent.

Here, for example, it is preferable that the pretreatment liquid and / or the cleaning liquid is supplied after the inspection target liquid is passed through the filter.

Here, for example, a filtration container holding the inspection target liquid is used in the capturing step, and the cleaning liquid is applied to the inspection target liquid remaining on the inner wall of the filtration container in the supplying step. It is preferable to spray and remove using a rotary nozzle.

Here, for example, it is preferable that the filter has a predetermined filter diameter and is processed into a shape that can be installed in a filtration container including the filter.

Here, for example, the filter is a flat plate membrane filter, and the smoothness of the flat surface preferably has a maximum error of 100 μm or less.

Here, for example, the pore size of the filter is 5
It is preferably smaller than 0 μm.

BEST MODE FOR CARRYING OUT THE INVENTION A microbial dyeing apparatus 100 and a microbial dyeing method using this microbial dyeing apparatus, which are preferred embodiments of the present invention, will be described below in detail with reference to the drawings.

In the following description, the FISH method is used as an example of a method for dyeing microorganisms using the apparatus 100 for staining microorganisms for the sake of simplicity, but the present invention is limited to the described examples. Not a thing. For example, a fluorescent antibody method, a nucleic acid staining method, or the like may be used as a method for staining a microorganism.

In the following description, a case where the operator directly injects a predetermined amount of the liquid to be inspected into the filtration container 2 will be described as an example. However, the liquid to be inspected is stored in the solution holder 8 and the liquid feed pump 9 is used. May be used to automatically inject into the filtration container 2 via the injection part 1.

[Outline of Microorganism Staining Apparatus] First, an outline of the microorganism staining apparatus 100 will be described. In order to rapidly detect whether or not a microorganism that is not originally contained in a beverage such as beer (liquid to be inspected) is mixed in a manufacturing process or the like, a method for detecting a microorganism by a fluorescence microscope is used. In order to carry out the inspection of microorganisms by this fluorescence microscope quickly and accurately, in advance a capture process that reliably captures the microorganisms contained in the test liquid and dye the captured microorganisms so that they can be accurately detected by the fluorescence microscope. Therefore, it is necessary to perform a dyeing treatment for removing unnecessary stains from the surface of microorganisms.

The microorganism staining apparatus 100 is capable of promptly providing a sample to be used for detecting and detecting a microorganism by a fluorescence microscope, by safely and appropriately performing the above-mentioned microorganism-trapping treatment and the captured microorganism staining treatment. is there. Therefore, the microorganism staining apparatus 100 described below reliably captures the microorganisms on the membrane filter by filtering the test liquid with a filtration container equipped with a membrane filter.

After that, the filter unit containing the membrane filter that has captured the microorganisms is sealed with a lid or the like, and the volume of the sealed space is set to a predetermined volume or less, while the evaporation of the dyeing solution is restricted, the membrane filter is covered with a fluorescent substance ( Alternatively, it is contacted with a fluorescent labeling substance) to stain the microorganism.
By limiting the evaporation of the dyeing solution, the dyeing solution is prevented from evaporating at the time of dyeing and sticking to the surfaces of the microorganisms and the membrane filter so that they cannot be removed. Also, after the dyeing, the microorganisms and the dyeing liquid remaining on the surface of the membrane filter are surely removed by suction filtration or the like. It should be noted that most of the series of processes described above can be performed automatically.

As described above, the microbial dyeing apparatus 1
00 is a dye for dyeing at the time of dyeing so that microorganisms can be reliably captured, only a predetermined part of the captured microorganisms is dyed with a predetermined dyeing solution, and excessively supplied dyeing solution does not adhere to the surfaces of the microorganisms and the membrane filter. A sample suitable for microscopic observation can be prepared by surely limiting the evaporation of the liquid and removing the staining liquid.

[Inspection Liquid] Next, the inspection liquid will be described. The inspection target liquid that stains microorganisms with the microorganism staining device 100 is, for example, a beverage such as beer, which originally does not contain microorganisms. However, microorganisms may be mixed in the beverage during the manufacturing process. Examples of microorganisms in beverages include bacteria and yeast. As an example, Pectinatus, which is a bacterium harmful to beer, has a width of 0.5 to 2 μm and a curve length of 1.
It is 5 to 10 μm. As another example, yeast has a width as well as a curve length of 3 to 10 μm.

[Membrane Filter] As described above, various sizes can be considered as the size of the microorganisms mixed in the test liquid. Therefore, the pore size of the membrane filter 23 used in the microorganism staining device 100 can be appropriately changed and used according to the size of an object that may be mixed so that it can be completely captured. As an example, for example, a membrane filter has a flat plate shape such as a disc having many holes and a filter diameter of 10 to 10.
The filter pore size is, for example, 0.2 to
It is 50 μm, and the number of holes can be arbitrarily designed as needed. Therefore, it is possible to capture microorganisms larger than the filter pore diameter using the membrane filter. Further, since the membrane fill is a flat plate membrane filter, and the flatness of the flat surface is designed to have a maximum error of 100 μm or less, the microorganisms captured and stained by the membrane filter are focused in fluorescence microscope observation. It is easy to align and is suitable for fluorescence microscope observation.

[Staining with Fluorescent Substance] Here, a method for staining the microorganisms captured on the above-described membrane filter with a fluorescent substance will be described in detail. As a method for staining a microorganism with a fluorescent substance, there are a nucleic acid staining method, an esterase method, a FISH method and a fluorescent antibody method.

The nucleic acid staining method is a staining method for staining the nucleic acid of a bacterium, the esterase method is a staining method utilizing the action of an enzyme possessed by a live bacterium, and these staining methods are one-step staining treatments. Although it can be stained with, it is characterized by nonspecific staining of microorganisms. On the other hand, the FISH method is a staining method that targets a unique sequence of an organism, and the fluorescent antibody method is a staining method that targets a unique structure of an organism. Although it can stain microorganisms, it is characterized in that it requires multi-stage staining treatment.

The FISH method and the fluorescent antibody method will be described in detail below. First, the FISH method will be described. The FISH method is a method of detecting a microorganism by fluorescent staining with a nucleic acid probe using a nucleic acid probe as a target. This method does not require a step of extracting a nucleic acid from a microorganism and is a method in which a fluorescently labeled nucleic acid probe is directly added to a pretreated microorganism to hybridize with rRNA or chromosomal DNA among nucleic acids in a microorganism cell.

Generally, among nucleic acids in microbial cells, r
Target RNA to the probe. Since there are thousands to hundreds of thousands of copies of rRNA in living microbial cells, the target of the probe is present by the number of copies of rRNA. For this reason, a large amount of the fluorescent substance bound to the nucleic acid probe remains in the target microbial cell, and when the fluorescent substance used is irradiated with appropriate excitation light, only the target microbial cell fluoresces while maintaining its morphology under a fluorescence microscope. You can observe and emit. It is also possible to use a complementary sequence of a bacterial species-specific region of the chromosomal DNA as a probe, and similarly, a bacterial species-specific microbial cell can be fluorescently stained.

Next, the fluorescent antibody method will be described. The fluorescent antibody method selectively detects a target microorganism by using an antibody that specifically recognizes an antigen composed of a protein, a saccharide, or a lipid of a target microorganism cell. Is the way. This method generally uses an antibody that recognizes an antigen present on the cell surface. Either directly fluorescently label the antibody, or
By fluorescently labeling the secondary antibody that binds to the primary antibody, a microorganism having a surface antigen recognized by the primary antibody is specifically fluorescent-stained and detected.

FIG. 6 shows an example of the fluorescent substance used when the microorganisms captured on the membrane filter described above are stained by the FISH method or the fluorescent antibody method.

In FIG. 6, when excitation light having a specific wavelength corresponding to each fluorescent substance is irradiated, fluorescence having a specific wavelength corresponding to each fluorescent substance is emitted. Therefore, by using FIG. 6, it is possible to select the fluorescent substance and the wavelengths of the excitation light and the fluorescence.

[Structure of Microorganism Staining Device: FIG. 1A, 1
B] Next, the microorganism dyeing apparatus will be described in detail.

First, a microorganism staining apparatus 100 according to an embodiment of the present invention will be described with reference to FIGS. 1A, 1B and 2.

1A and 1B show a microbial dyeing apparatus 1
00 is an external view showing the overall configuration of the device 00, and FIG. 2 is an external view showing the control unit 110 of the microorganism staining device 100.

In FIGS. 1A and 1B, the injection part 1 is
A solution such as a liquid to be inspected, a pretreatment liquid for dyeing, a sterilizing liquid and a cleaning liquid is injected into the filtration container 2 as necessary. The filtration container 2 holds a solution such as a liquid to be inspected, and supplies this solution to the filter unit 3. The filtration container holding part 5 holds the filtration container 2, and further, the dyeing cap 14 (see FIG. 3).
A) is also kept flush with the filtration container 2.

The filter unit 3 filters the test liquid with a membrane filter to capture the microorganisms contained in the test liquid on the membrane filter. The dyeing unit 4 dyes the microorganisms captured on the membrane filter. The filtration container holder 5 holds the filtration container 2. The filter unit holder 6 holds the filter unit 3.

The dyeing liquid holding unit 7 holds the dyeing unit 4.
The solution holder 8 stores a solution such as a cleaning liquid, and the liquid feed pump 9 is used to feed the filtration container 2 from the solution holder 8 via the injection part 1.
Inject various solutions into it. The temperature controller 11 holds the solution holder at a predetermined temperature, and the temperature controller 12 holds the filtration container 2, the filter unit 3, and the dyeing section 4 at a predetermined temperature.

The drive unit (not shown) housed in the box 10 moves the filtration container holding unit 5, the filter unit holding unit 6, and the dyeing liquid holding unit 7 to predetermined positions to move the filtration container. 2, the dyeing cap 14, the filter unit 3, the suction unit 13, and the dyeing unit 4 are moved to predetermined positions suitable for the capture process and the dyeing process described later.

[Control Unit: FIG. 2] In the control unit 110 of FIG. 2, the display unit 13 displays various operating conditions and the state of each processing step during automatic operation, and the input unit 14 displays various operating conditions. input. Although not shown in FIG. 2, the control unit 110 has a CPU, a RAM, a ROM, an HDD (hard disk drive), and the like. Where the CPU
Is a controller that controls the entire system,
M is a system work memory for the CPU to operate, and is also a memory for temporarily storing print data. Further, the ROM stores a system program, and the HDD stores system software, a control program for controlling a microorganism capturing process and a staining process, recorded data, and the like. Further, the CPU loads the control program stored in the HDD into the RAM and executes the microorganism capturing process and the staining process based on the control program.

[Structure and Arrangement of Each Part: FIGS. 3A to 3C] FIG.
A to 3C are a filter container 2, a filter unit 3, a suction unit 13, and a dyeing cap in a capturing process for capturing microorganisms on a membrane filter in the microorganism staining device 100 and a staining process for staining the captured microorganisms with a fluorescent substance. 14 is a diagram illustrating in detail the arrangement of the dyeing unit 4. FIG.

FIG. 3A shows the relationship between the position where the filter unit 3 is first installed (position A), the position where the microorganisms are captured (position C) and the position where the captured microorganisms are stained (position B) in the microorganism staining device 100. It is a figure explaining.

Position C in FIG. 3A (position for capturing microorganisms)
The filter container 2 and the suction unit 13 are the filter unit 3
It shows a state in which it is placed in a fixed position before being attached to the.

Similarly, position B (position for dyeing captured microorganisms) in FIG. 3A shows a state in which the dyeing cap 14 and the dyeing part 4 are arranged at fixed positions before being attached to the filter unit 3. ing. In addition, the filter unit 3 when the filter unit 3 that has captured the microorganisms is transferred from the position C by the drive unit and is arranged at the fixed position of the position B is shown in the position B by an imaginary line.

Similarly, at position A, a fixed position where the filter unit 3 is first installed in the microorganism staining device 100 is indicated by a virtual line.

FIG. 3B shows a position for capturing microorganisms (FIG. 3A).
The position C) of the filter container 2 and the suction part 13 is moved and attached to the filter unit 3 in the position C).
FIG. 3C shows an arrangement in which the dyeing cap 14 and the dyeing unit 4 are moved and attached to the filter unit 3 at the position where the captured microorganisms are dyed (position B in FIG. 3A).

Hereinafter, with reference to FIGS. 3A to 3C, details of the configuration of the filtration container 2, the filter unit 3, the suction unit 13, the dyeing cap 14, and the dyeing unit 4 in the process of capturing the microorganisms and the dyeing process of the captured microorganisms. And their movements will be described in detail.

[Structure of Each Part: FIG. 3A] First, the details of the structure of each part of the microorganism staining device 100 will be described.

In FIG. 3A, the filtration container 2 includes a rotary nozzle 15, a nozzle drive unit 16, a clamp lever 17,
It is composed of a connecting portion 18 and the like. The filtration container 2 is held at a predetermined position by the filtration container holder 5. The filtration container holding unit 5 also holds the dyeing cap 14 at a predetermined position. Therefore, when the drive unit moves the filtration container holding unit 5 to a predetermined position in the downward direction indicated by Y in FIG. 3A, the filtration container 2 and the dyeing cap 14 can be simultaneously moved to the predetermined position.

When the liquid to be inspected containing bubbles such as beer is filtered through the filtration container 2, the liquid to be inspected as foam adheres to the inner wall of the filtration container 2 and remains. Therefore, when filtering such an inspection target liquid, the cleaning liquid is sprayed (discharged) from the nozzle 15 onto the inner wall of the filtration container 2, and the inspection target liquid attached as bubbles is surely filtered and removed. To do.

The filter unit 3 includes the filter holder 2
1, a glass filter 22 and the like. Further, the filter unit 3 is held at a predetermined position by the filter unit holding portion 6 including the heater 19.

The suction part 13 is composed of a connecting part 18-2, a waste liquid manifold 20, and the like. The dyeing section 4 is
The nozzle 27 and the like for injecting the dyeing solution for injecting the dyeing solution from below the filter unit 3 are configured.

The dyeing cap 14 for preventing evaporation of the dyeing solution injected into the filter unit 3 at the time of dyeing has a nozzle 26 for injecting the dyeing solution for injecting the dyeing solution from above the filter unit 3.

The dyeing cap 14 is not limited to the structure shown in FIG. 3A. For example, the dyeing cap 14-1 having the structure shown in FIG.
Can be used. In addition, the dyeing cap 14-
1 is a dyeing unit 4 of FIG.
Injection from below). The dyeing unit 4 is not limited to the structure shown in FIG. 3A. The dyeing solution injection nozzle is arranged in the dyeing cap 14-1 shown in FIG. 5A, the waste liquid manifold is arranged below the dyeing unit 4, and the waste liquid manifold is provided. The structure may be connected to a suction pump.

In FIG. 3A, the dyeing unit 4 and the suction unit 13 have an integrated structure, and the dyeing holding unit 7 described later is used.
Is moved in the vertical direction indicated by Y, the dyeing section 4 and the suction section 1 are also moved.
It is structured to move at the same time as 3.

[Movement during Microbial Capture Processing: FIGS. 3A and 3B] Next, the filtration container 2 during the capture processing of microorganisms,
The movements of the filter unit 3 and the suction unit 13 will be described.

First, in FIG. 3A, when a worker installs the filter unit 3 at a predetermined position (the position of the filter unit 3 indicated by a two-dot chain line at the position A in FIG. 3A) of the microbial staining apparatus 100, it is not shown. The driving unit moves the filter unit holding unit 6 in the horizontal direction and moves the filter unit 3 to the installation position of the filter unit 3 (from the position of the filter unit 3 indicated by the two-dot chain line at the position A in FIG. 3A to the position in FIG. 3A). The position of the filter unit 3 shown by the solid line at position C).

Next, at a position C in FIG. 3A, a driving unit (not shown) moves the filtration container holding unit 5 to a predetermined downward position and the dyeing unit holding unit 7 to a predetermined upward position to perform filtration. The container 2 and the suction unit 13 are attached to the filter unit 3 as shown in FIG. 3B.

Subsequently, in the state of FIG. 3B, the filter unit 3 is brought to a predetermined temperature, and then the liquid to be inspected is injected into the filtration container 2, and this liquid is suction-filtered to capture the microorganisms on the membrane filter. If necessary, a cleaning solution or the like is poured into the filtration container 2 and suction filtered.

Next, from the arrangement shown in FIG. 3B, a drive unit (not shown) moves the filtration container holding unit 5 to a predetermined upward position and the dyeing unit holding unit 7 to a predetermined downward position to thereby filter the filtration container. 2 and the suction part 13 are detached from the filter unit 3 as shown in the position C of FIG. 3A.

Next, from the arrangement shown in FIG. 3A, a driving unit (not shown) moves the filter unit holding unit 6 in the horizontal direction, so that the filter unit 3 is moved from the capture processing position (position C in FIG. 3A) to the staining processing position (position C). The position B shown in FIG. 3A is moved to the position indicated by the two-dot chain line).

Next, at a position B in FIG. 3A, a driving unit (not shown) moves the filtration container holding unit 5 to a predetermined downward position and the dyeing unit holding unit 7 to a predetermined upward position, whereby dyeing is performed. The cap 14 and the dyeing part 4 are attached to the filter unit 3 as shown in FIG. 3C. Subsequently, in the state of FIG. 3C, the dyeing unit 4 and the filter unit 3 are brought to a predetermined temperature, then the dyeing solution is injected into the filter unit 3 and held for a predetermined time, and then the remaining dyeing solution is sucked. Filter to remove stains from microorganisms and membrane filter surfaces. Further, subsequently, if necessary, a cleaning liquid is injected into the filter unit 3 for cleaning.

Next, at a position B in FIG. 3C, a driving unit (not shown) moves the filtration container holding unit 5 to a predetermined upward position and the dyeing unit holding unit 7 to a predetermined downward position, thereby dyeing. The cap 14 for the dye and the dyeing part 4 are placed at the position B in FIG. 3A.
As shown in FIG.

Next, a driving unit (not shown) moves the filter unit holding unit 6 in the horizontal direction, so that the filter unit 3 is installed at the installation position of the filter unit 3 (the filter unit 3 indicated by a two-dot chain line at the position A in FIG. 3A). Position).

In this way, the microbial dyeing apparatus 100 is used.
Is capable of automatically performing a microbial capture process and a dyeing process of the captured microbial organism.

[Capture of Microorganisms: FIGS. 4A and 4B] FIG. 4A is a diagram for explaining the details of the arrangement of the filtration container 2, the filter unit 3, and the suction unit 13 in the capture of microorganisms. The membrane filter 23 is the glass filter 22.
The filter holder 21 fixes the filter. The solution to be inspected, the cleaning solution or the like injected into the filtration container 2 is connected to the suction unit 13 by the suction pump 2
4 (FIG. 4B), the membrane filter 2 installed in the filter unit 3 by being suction-filtered.
Microorganisms filtered in 3 and contained in the test liquid are captured on the membrane filter 23.

FIG. 4A shows a filter container 2, a filter unit 3 and a suction unit 1 immediately before (or immediately after) the process of capturing microorganisms.
3 is a diagram showing the arrangement of FIG. 3 and the arrangement when a cleaning liquid or the like is injected into the filtration container 2. FIG. The cleaning liquid is injected from the solution holder 8 into the filtration container 2 by the liquid feed pump 9 via the injection portion 1, and then suction-filtered by the suction pump 24, and the liquid that has passed through the filter unit 3 is discharged as waste liquid.

[Microbial Staining Process: FIGS. 5A and 5B] FIG. 5A shows a dyeing cap 14-1, a filter unit 3, which is used for preventing evaporation of a dyeing solution in the microbial dyeing process.
It is a figure explaining the arrangement of the dyeing part 4 in detail. The membrane filter 23 is arranged on the glass filter 22,
It is fixed by the filter holder 21. Dyeing part 4
The dyeing solution injected further comes into contact with the lower surface of the membrane filter 23 through the glass filter 22 to keep the microorganisms captured by the membrane filter 23 for a predetermined time, for example, several minutes to several tens of hours, a predetermined temperature, for example, 30-40
Stain under conditions of ° C.

A closed space 25 when the membrane filter 23 that has captured the microorganisms is sealed with the dyeing cap 14
When contacting the membrane filter with the fluorescent substance for a predetermined time while limiting the evaporation of the staining solution,
The evaporation of the dyeing solution is limited to prevent the dyeing solution from evaporating during the dyeing and sticking to the surfaces of the microorganisms and the membrane filter so that the dyeing solution cannot be removed. Therefore, it is preferable that the volume be less than or equal to the predetermined volume. For example, the volume of the closed space 25 is preferably 350 cm ³ or less, more preferably 5～25Cm ^3, more preferably, 0.
It is 1 to 5 cm ³ .

The dyeing conditions such as the dyeing time and the dyeing temperature during the dyeing process described above are merely examples, and the conditions can be changed to those suitable for the dyeing agent and the dyeing method. Further, the membrane filter 23 includes the dyeing part 4 during dyeing and the dyeing cap 14
Since the dyeing solution is prevented from evaporating by being sealed by the membrane filter 23, the membrane filter 23 is maintained at a predetermined temperature and a predetermined humidity.

In FIG. 5A, the dyeing solution injected from the dyeing section 4 is in contact with the lower surface of the membrane filter 23 through the glass filter 22, but the dyeing solution injected from the dyeing section 4 is shown. May be injected from the upper surface of the membrane filter 23 using an injection nozzle 26 provided in the dyeing cap 14 shown in FIG. 3C, for example.

FIG. 5B shows a dyeing cap 14, a filter unit 3, immediately before (or immediately after) the dyeing treatment of microorganisms.
It is a figure which shows arrangement | positioning of the dyeing | staining part 4, and arrangement | positioning at the time of injecting a dyeing liquid. The dyeing solution is injected into the nozzle 27 for injecting the dyeing solution by the liquid sending pump 9.

After the dyeing process is finished, suction filtration of the dyeing solution is performed to remove unnecessary dyeing solution. In addition, a cleaning liquid is injected into the membrane filter 23 as needed to be cleaned. The treatment at this time is the same as the washing treatment using the washing liquid performed in the microorganism trapping treatment described above with reference to FIG. 4A. Therefore, the description of the washing and removing process of the dyeing liquid is redundant and will not be repeated.

[Flow of Microbial Capture Processing and Staining Processing: FIG. 7] Next, the microorganism staining apparatus 100 described above.
The flow of the microorganism capturing process and the dyeing process in the above will be described with reference to FIG. The process shown in FIG. 7 can be all performed automatically, but a part of the process can also be performed manually. For example, only the process of injecting the liquid to be inspected into the filtration container 2 in step S120 may be a manual process.

When the processing in FIG. 7 is automatically performed, based on a preset control program,
It is executed by the control unit 110. Further, when a part of the processing in FIG. 7 is manually performed, it is sufficient to perform the manual processing in advance and then automatically perform the processing based on the control program.

In FIG. 7, steps S110 to S140 are the steps for capturing microorganisms, and step S1
Steps 50 to S190 are a microbial dyeing process.

First, in step S110, at the position C in FIG. 3B, the filtration container 2, the filter unit 3, the suction unit 1 are placed.
3 is moved by using the drive unit, and the filter container 2 and the suction unit 13 are attached to the filter unit 3.

Next, in step S120, a predetermined amount of liquid to be inspected is injected into the filtration container 2, and then step S
At 130, the test liquid is suction-filtered using the suction pump 24 to capture the microorganisms contained in the test liquid on the membrane filter.

Next, in step S140, a predetermined amount of cleaning liquid or culture liquid is injected into the filter container 2 as necessary, and suction filtration is performed using the suction pump 24.

Next, in step S150, the filter container 2, the filter unit 3, and the suction unit 13 are moved using the drive unit to remove the filter unit 3 from the filter container 2 and the suction unit 13, and then the position shown in FIG. 3C. After moving the filter unit 3 to B using the drive unit, the dyeing cap 14 and the dyeing unit 4 are attached to the filter unit 3.

Next, in step S160, the microorganisms captured on the membrane filter 23 are stained.

Next, in step S170, the dyeing cap 14 and the dyeing section 4 are moved by using the drive section, and then the filter unit 3 is moved by the drive section to the position C in FIG. 3B. The filter container 2 and the suction unit 13 are attached to the.

Next, in step S180, a predetermined amount of the washing liquid is poured into the filtration container 2 and suction filtered using the suction pump 24 to wash and remove the staining liquid.

Next, in step S190, the filter container 2, the filter unit 3, and the suction unit 13 are moved by using the drive unit to remove the filter unit 3 from the filter container 2 and the suction unit 13, and then the position shown in FIG. 3A. The filter unit 3 is moved to A by using a driving unit. In this way, it is possible to capture the microorganisms on the membrane filter 23 arranged in the filter unit 3 and stain the captured microorganisms.

The above-described processing is an example, and a part of the processing may be removed or another processing may be added. Further, in the above-described processing, the dyeing solution may be injected into the microorganisms captured on the membrane filter 23, and then the dyeing cap 14 may be attached to the filter unit 3.

[Setting of operating conditions: FIGS. 8A and 8B] Next,
An example of setting operating conditions in the above-described processing steps in the control unit 110 of the microorganism staining device 100 will be described with reference to FIGS. 8A and 8B. Note that the following description will be given by taking the case where the FISH method is used as the staining method as an example.

FIG. 8B shows an example of operating conditions in the process of capturing the microorganisms and the process of dyeing the captured microorganisms described in FIG. 7. The operating conditions in the capturing process of the microorganisms are Step 1 to Step 5, These are operation conditions Step 6 to Step 15 of the dyeing process.

That is, each condition will be briefly described. In order to start the process of capturing microorganisms in step 1, the test liquid (for example, 50 ml) is injected into the filtration container 2, and the step 2
Then, start the suction pump to start the process of capturing microorganisms,
In step 3, the test liquid is suction-filtered for a predetermined time (for example, 3 minutes) to capture the microorganisms on the membrane filter.
In Step 4, if necessary, a predetermined amount (for example, 25 ml) of the cleaning liquid is injected into the filtration container 2 to wash the filtration container 2, and then in Step 5, the cleaning liquid is supplied for a predetermined time (for example, 30 ml).
Sec) Suction filter to remove.

Next, in the dyeing process, in step 6, 1 ml of 50% ethanol was used as a pretreatment process for dyeing the captured microorganisms under a predetermined condition (for example, left at room temperature for 2 minutes) to dehydrate the microorganisms. Then, in step 7, the ethanol used for the dehydration treatment is removed by suction filtration. Although not shown in the figure for simplification of description, the dehydration treatment is performed by repeating the same treatments as in Steps 6 and 7 using 1 ml of ethanol of 75 and 99%, for example.

Next, in step 10, a predetermined dye solution (for example, containing the fluorescent substance shown in FIG. 6) is used in a predetermined amount (for example, 1).
ml) It is injected onto a membrane filter, and in step 11, the microorganism is dyed under predetermined conditions (for example, dyeing temperature 37 ° C., dyeing time 3 hours), and then the dyeing solution is suction filtered in step 11 to be used for dyeing. Remove excess stain that was not done.
Next, in step 13, a predetermined amount of cleaning liquid (for example, 10 ml)
After the injection, the washing liquid is removed by suction filtration in step 14, and the microorganism capturing process and the dyeing process are completed.

Although omitted in order to simplify the description in the above description, the control unit 110 is used to connect the filter container 3 and the suction unit 13 to the filter unit 3 using the drive unit described with reference to FIG. The setting of attachment and detachment, and attachment and detachment of the dyeing unit 4 and the dyeing cap 14 to and from the filter unit 3 are set in correspondence with the above steps.

Further, in step 15, when the microbial trapping process and the dyeing process are completed, the operation is signaled (for example, a buzzer, a lamp blinks), and the filter unit 3 is detached from the dyeing section 4 and the dyeing cap 14. The filter unit 3 is moved to a predetermined position.

Further, (a), (b), (c) of FIG. 8A,
(D) is an example of each screen for operating condition setting displayed on the display unit 13 of the control unit 110. This screen and the input unit 1
4 can be used to set the operating conditions of each step shown in FIG. 8B. That is, if the operating condition “A” is set by pressing the operating condition “A” on the screen of FIG. 8A, the screens of (b) to (d) are sequentially displayed. The conditions of each process of FIG. 8B can be set.

For example, in the screen of (b), steps 3 to
The operating conditions of step 6 are set, the operating conditions of steps 7 to 10 are set on the screen of (c), and in (d),
The operating conditions of steps 11 to 13 are set.

More specifically, for example, in step 3 of the screen of (b), "3" is set as "sample suction time".
If you enter "minutes", the default value "00
Since "0" is displayed, "+" of the hundred digit displayed is 1
By pressing the ten digit “+” eight times and the one digit “+” zero times, “1” “8” “0”, that is, “180” seconds (3 Minutes) can be entered. When it is desired to decrease each input numerical value, the "-" of each digit may be pressed.

When the input of steps 3 to 6 is completed on the screen of (b), the screen of (c) is displayed by pressing the "next page". Therefore, the same method as described above is used. Then, steps 7 to 10 may be sequentially input. When the input of the necessary steps is completed in this way, the setting of the operating condition can be completed by pressing the "setting end button" (not shown).

[Microbial Capture and Staining Process: FIG. 9A, 9
B] Next, with reference to FIGS. 9A and 9B, based on the operating conditions set in FIGS. 8A and 8B, the control unit 110 is displayed on the display unit 13 when performing the process of capturing and staining the microorganisms. An example of the driving situation will be described.

When the start of the process of capturing and staining the microorganisms is received in step S200 of FIG. 9A, the process proceeds to step S210, the operating condition setting screen shown in FIG. 9B (a) is displayed, and "AUTO" and "manual" are displayed. Encourage choices such as driving.

Next, in step S220, when it is received that the driver of the microorganism staining device 100 has impressed the "AUTO" button indicating the automatic operation in (a) of FIG. 9B, the process proceeds to step S230, and FIG. Display the operating condition setting screen shown in (a) of, and set the operating condition "A",
Prompt selection of operating conditions from "B", "C", and "D".

Next, in step S240, when it is detected that the driver has pressed the button of the operating condition "A", the process proceeds to step S250, in which the screen for driving preparation 1 shown in FIG. 9B (b) is displayed. Is displayed to prompt the driver to set the filter unit 3.

Next, in step S260, when it is detected that the driver has set the filter unit 3 to a predetermined position (position A in FIG. 3A) and has pressed the "go to step 1" button indicating the start of the operation. , Go to step S270,
The filter unit 3 is moved to a predetermined position to instruct the filter unit 3 to attach the filtration container 2 and the suction unit 13.

Next, in step S280, an instruction to inject the liquid to be inspected into the filtration container 2 is given, and then in step S290, a "start" button is pressed to notify that the injection of the liquid to be inspected is completed by the driver. When it is detected, the process proceeds to step S300, and a display screen (for example, FIG.
The screen of (d) of B is a screen for notifying the operation status of the process 3). Although omitted below, the screen of FIG. 9B (d) for each step is sequentially displayed.

Next, in step S310, a display screen (for example, (e) in FIG. 9B) for informing the operation status of each process during automatic operation is displayed. Next, when it is detected that the "end operation" button for ending the automatic operation is pressed, the process proceeds to step S320, and a display screen for notifying the end of the automatic operation (for example, the screen of (f) in FIG. 9B). Is displayed, and a series of processing ends.

[0138]

EXAMPLE Next, the above-described microorganism dyeing apparatus 100
An example in which a microorganism is captured from a liquid to be inspected on a membrane filter using the above, and the captured microorganism is dyed to prepare a sample for a fluorescence microscope will be described below.

<Embodiment 1: Evaluation of Microbial Capture> As a test liquid, 50 ml of beer forcibly inoculated with about 100 Pectinatus bacteria was used, and the dyeing apparatus 100 described above was used to capture the microorganisms. And dyeing treatment (FI
The SH method) was performed, and the quantitative analysis of microorganisms contained in the test liquid was analyzed with a fluorescence microscope.

First, the membrane filter 23 was set in the filter unit 3 (FIG. 5A), and the microorganism staining device 100 shown in FIG. 1A was used to capture and stain the microorganisms under the operating conditions shown in FIG. 8B.

That is, after the filtration of beer, 4 ml of 50%, 75%, and 99% ethanol was used, each of which was allowed to stand for 2 minutes and the filtration was intermittently repeated to perform a dehydration treatment as a pretreatment for the dyeing treatment. went. Subsequently, the dyeing process was performed in a sealed state (FIG. 5B) in which the dyeing cap 14 and the dyeing section 4 were attached to the filter unit 3.

For detection of Pectinatus, a staining solution containing 75 pmol of each of 24 fluorescent probes capable of detecting Pectinatus (hybridization buffer, 20 mM Tris [pH 7.6], 0.9M N 2
aCl, 5 mM EDTA, 0.02% SDS, 10%
Formamide) 1 ml and membrane filter 23
Dyeing was performed by bringing the dyeing solution into contact with the surface and holding it at 37 ° C. for 3 hours in the closed container of FIG. 5B that prevents evaporation of the dyeing solution.

After the dyeing treatment, the dyeing solution containing the excess fluorescent probe was removed by suction filtration, and the membrane filter 23 was washed with a washing solution and the washing solution was removed by suction filtration to prepare a fluorescent microscope sample.

ChemScan RDI (C
hemunex, France). The detection operation was performed according to the ChemScan work procedure manual. The setting of the optimum conditions for detecting Pectinatus was determined from the data obtained by conducting a model experiment in advance.

According to the observation result by the above fluorescence microscope observation, the fluorescence-detected bacteria were almost the same as the bacterial inoculation amount,
Almost all fluorescent objects were confirmed to be target bacteria by microscopic observation. Further, the number of Pectinatus bacteria detected by the above method and the number of colonies obtained as a result of subjecting the membrane filter 23 obtained by filtering beer containing an equal amount of Pectinatus bacteria to culture on a selective agar medium were almost the same. .

From this, it was confirmed that the use of the microorganism staining device 100 reliably captures the microorganisms contained in the test liquid and stains the captured microorganisms.

<Example 2: Evaluation of capture of microorganisms> About 100 Lactobacillus (Lact) was forcibly used as a test liquid.
obacillus) 50 ml of beer inoculated with the above-described dyeing device 100 is used to perform a microorganism capturing treatment and a dyeing treatment (esterase method), and a fluorescent microscope is used for quantitative analysis of the microorganisms contained in the test liquid. Analyzed.

First, the membrane filter 23 was set in the filter unit 3 (FIG. 5A), and the microorganism staining device 100 shown in FIG. 1A was used to carry out the microorganism capturing process and the staining process under the operating conditions shown in FIG. 8B.

That is, after the filtration of beer was completed, 800 μl of the esterase substrate solution was injected into the closed container shown in FIG. 5B so as to come into contact with the secondary side of the membrane filter 23, and the mixture was kept at 30 ° C. for 30 minutes to stain. The dyeing was performed while preventing the liquid from evaporating.

After the dyeing treatment, the dyeing solution containing excess fluorescent probe was removed by suction filtration, and the membrane filter 23 was washed with a wash solution and the wash solution was removed by suction filtration to prepare a sample for a fluorescence microscope.

ChemScan RDI (C
hemunex, France). The detection operation was performed according to the ChemScan work procedure manual. The optimum conditions for detecting Lactobacillus were set based on the data obtained by conducting a model experiment in advance.

According to the observation result by the above fluorescence microscope observation, the fluorescence-detected bacteria were almost the same as the bacterial inoculation amount,
Almost all fluorescent objects were confirmed to be target bacteria by microscopic observation. Further, the number of Lactobacillus detected by the above method and the number of colonies obtained as a result of subjecting the membrane filter 23 obtained by filtering beer containing an equal amount of Lactobacillus to culture on a selective agar medium were almost the same. .

From this, it was confirmed that the use of the microorganism staining device 100 reliably captures the microorganisms contained in the test liquid and stains the captured microorganisms.

<Embodiment 3: Evaluation of prevention of drying of dyeing solution in dyeing treatment> The membrane filter 23 is set in the filter unit 3 and the same microorganism trapping treatment and dyeing as in Embodiment 1 are performed using the microorganism dyeing device 100. The treatment was carried out. However, in the third embodiment, the cap 14 for dyeing is provided in the middle of the dyeing treatment (dehydration, injection of the dyeing liquid) subsequent to the treatment for capturing the microorganisms (filtration and washing of beer) in the first embodiment.
The sealed filter unit 3 (FIG. 5B) to which the dyeing part 4 and the dyeing part 4 were attached was taken out at appropriate time, and the amount of the dyeing liquid remaining on the primary side of the membrane filter was measured for the amount of residual liquid when the dyeing liquid was injected.

After the measurement, the filter unit 3 is immediately returned to the microorganism staining device 100, and the staining process is continued.
After the lapse of a predetermined time, the amount of the staining liquid remaining on the primary side of the membrane filter was measured. This work is performed by the dyeing processing time (4.
It carried out 4 times (every 1 hour) during 5 hours.

[Change in Remaining Amount of Staining Solution with Time: FIG. 10] □ (closed) in FIG. 10 shows the change with time in the remaining amount of the staining solution measured by the above method. FIG. 10 shows a relative display of the amount of the remaining staining liquid after 1, 2, 3, 4 hours, based on the amount of the staining liquid remaining immediately after the injection of the staining liquid. From the figure, it can be seen that the dye solution near the time immediately after the injection of the dye solution remains in the sealed filter unit 3 even after 4 hours. Further, the excess dyeing solution remaining after the dyeing treatment can be easily removed by suction filtration or by suction filtration using a washing solution without remaining on the surface of the microorganisms and the membrane filter.

[0157] The solid squares (open) in Fig. 10 are the results of the comparative test for the squares (closed). That is, in the comparative test, in order to compare with the above condition (closed state in which the dyeing cap is attached to the filter unit 3), the filter unit 3 to the dyeing cap 1 shown in FIG.
4-1 is removed, the filter unit 3 is moved into a closed container (volume: 1000 cm ³ ) kept at 37 ° C., and the filter unit 3 is kept for the same time as the above condition and the membrane filter 2
The remaining staining solution in 3 was measured.

It can be seen from (1) (open) in FIG. 10 that the residual amount of the dyeing solution sharply decreases when the dyeing cap 14-1 is removed from the filter unit 3. Further, the dyeing liquid remaining after this dyeing treatment adhered to the surfaces of the microorganisms and the membrane filter and could not be removed from the surfaces of the microorganisms and the membrane filter even by suction filtration using a washing liquid. Further, when the microorganisms and the stains adhered to the surface of the membrane filter remain in this manner, it is impossible to measure the microorganisms accurately under the influence of the adhered stains in the fluorescence microscope observation performed in Example 1. all right.

Therefore, in the microbial dyeing process using the microbial dyeing apparatus 100, the dyeing cap 14-1 as shown in FIG. 5B is attached to the filter unit 3 to remove the dyeing liquid from the surface of the membrane filter 23. It was found that it is important to prevent the evaporation and remove the stain solution remaining after the staining process of the microorganisms from the surface of the microorganisms and the membrane filter by suction filtration in order to detect the microorganisms accurately.

[0160]

As described above, according to the microorganism inspection apparatus of the present invention, for example, the microorganisms contained in the liquid to be inspected such as beer are reliably captured on the membrane filter, and the captured microorganisms are fluorescently stained. By removing the remaining staining solution from the surface of the microorganism and the membrane filter at the time of performing, it is possible to prepare a sample in which the microorganism can be detected accurately with a fluorescence microscope.

[Brief description of drawings]

FIG. 1A is an external view of a microorganism dyeing device according to an embodiment of the present invention as viewed from the front.

FIG. 1B is an external view of the microorganism dyeing apparatus according to the embodiment of the present invention as seen from above.

FIG. 2 is a diagram showing a control unit of a microorganism dyeing device.

FIG. 3A is a diagram illustrating the positional relationship of each part in the process of capturing a microorganism and the process of staining.

FIG. 3B is a diagram showing a state in which a filter container and a suction unit are attached to the filter unit.

FIG. 3C is a diagram showing a state in which a dyeing cap and a dyeing unit are attached to the filter unit.

FIG. 4A is a diagram showing details of a state where a filter container and a suction unit are attached to a filter unit.

FIG. 4B is a diagram illustrating supply of a solution and suction filtration of the solution in a process for capturing a microorganism.

FIG. 5A is a diagram showing details of a state in which a dyeing cap and a dyeing unit are attached to the filter unit.

FIG. 5B is a diagram illustrating the supply of a staining solution in a microorganism staining process.

FIG. 6 is a diagram showing the relationship between the fluorescent labeling substance used in the present embodiment and its excitation light and fluorescence.

FIG. 7 is a flowchart illustrating a process of capturing a microorganism contained in a liquid to be inspected and a staining process of the microorganism.

FIG. 8A is a diagram illustrating an example of setting the operating conditions of the processing steps of the microorganism inspection device according to the embodiment of the present invention.

FIG. 8B is a diagram illustrating an example of processing steps of the microorganism inspection device according to the embodiment of the present invention.

FIG. 9A is a flowchart illustrating an automatic process.

FIG. 9B is a diagram illustrating an example of an operation condition setting screen in automatic processing and a screen during automatic operation.

FIG. 10 is a diagram showing the relationship between the remaining amount of dyeing solution and the dyeing time.

[Explanation of symbols]

100 Microorganism dyeing equipment 110 control unit 1 injection part 2 filtration containers 3 filter units 4 dyeing section 5 Filter container holder 6 Filter unit holder 7 Staining section holding section 8 Solution holder 9 Liquid feed pump 10 boxes 11 Temperature controller 12 Temperature controller 13 Display 14 Input section

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] November 5, 2001 (2001.11.
5)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 19

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 33

[Correction method] Change

[Correction content]

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 1/30 G01N 1/30 33/48 33/48 P (72) Inventor Yasuro Motoyama Moriya, Kitasoma-gun, Ibaraki 1-1-1, Midori 21 Asahi Breweries Ltd. Liquor Research Institute (72) Inventor Go Koyamauchi 4074 Miyadera Miyajima, Iruma City, Saitama Prefecture Inside Lifetech Co., Ltd. (72) Kazuo Yamaguchi 4074 Miyadera Iruma City, Saitama Prefecture Lifetech Co., Ltd. Inner F-term (reference) 2G043 AA03 BA17 CA09 DA01 DA06 DA08 EA01 FA02 GA28 GB07 GB09 GB16 GB21 GB28 KA02 KA05 MA16 2G045 AA28 BB05 BB24 CB21 FA11 FA16 2G052 AA26 AA36 AB16 AB17 AB18 AB20 AD09 AD32 GA09 GA03 AD08 GA32 AD52 AD03 AD08 AD32 AD52 AD03 AD32 AD52 EA03 BB02 CC03 FA02 4B063 QA01 QA18 QQ06 QR32 QR35 QR56 QR66 QS34 QS36 QX02 QX10

Claims (36)

[Claims] 1. A dyeing device for dyeing microorganisms contained in a liquid to be inspected, comprising: capturing means for filtering the microorganisms contained in the liquid to be inspected and capturing them on a filter; Microorganisms comprising: a dyeing means for introducing a dyeing solution, and a dyeing means for dyeing a microorganism with the dyeing solution; and a sealing means for shielding a periphery of the filter into which the dyeing solution is introduced to form a sealed space. Dyeing equipment. 2. The sealing means prevents the dyeing liquid from evaporating to a high concentration during dyeing and sticking to the surface of the microorganisms and the filter so that they cannot be removed. Microorganism dyeing equipment. 3. The microorganism staining apparatus according to claim 1, wherein the sealed space formed by the sealing means has a volume of 350 cm ³ or less. 4. The capturing means has a filtration container including a membrane filter, wherein the test liquid is filtered by the filtration container to capture microorganisms contained in the test liquid on the membrane filter. The dyeing apparatus for microorganisms according to any one of claims 1 to 3, which is characterized by the above-mentioned. 5. The microbial dyeing apparatus according to claim 1, wherein the capturing unit has an accelerating unit that accelerates the filtration speed. 6. The sealing means holds the dyeing solution so that a predetermined amount of the dyeing solution remains in the sealed space during the dyeing period. The dyeing apparatus for microorganisms according to 1. 7. The apparatus for dyeing microorganisms according to claim 1, wherein the sealing means is formed of a light blocking material that does not transmit light. 8. The transport means for transporting the capturing means to preset first and second positions, and the transport control means for controlling the transport means, wherein the capturing means includes the transport means. 2. The method according to claim 1, wherein the microorganisms are transported to the first position by capturing the microorganisms, and then the transportation means transports the microorganisms to the second position to stain the microorganisms by the staining means. Item 7. A device for staining a microorganism according to any one of items 7. 9. The dyeing means is a solution containing the dyeing agent from a primary side of the filter, which is a side for injecting the test liquid into the filter, or a secondary side of the filter facing the primary side. Is injected.
9. The apparatus for dyeing microorganisms according to claim 8. 10. The dyeing means has constant temperature means for keeping a closed space closed by the closing means at a predetermined temperature and dyeing time control means for controlling the dyeing time to be a predetermined time. The staining apparatus for microorganisms according to any one of claims 1 to 9. 11. After dyeing a microorganism with the dyeing solution,
The microorganism staining apparatus according to any one of claims 1 to 10, further comprising a removing unit that removes the staining solution. 12. The method according to claim 1, further comprising a supply unit that supplies the inspection target liquid, a pretreatment liquid for dyeing, a sterilizing liquid, and a cleaning liquid to the capturing unit. An apparatus for dyeing microorganisms according to the item. 13. The sterilizing solution and / or cleaning solution is
The microorganism staining apparatus according to claim 12, wherein the microorganism is supplied after being stained with the stain. 14. The microorganism staining apparatus according to claim 12, wherein the pretreatment liquid and / or the cleaning liquid is supplied after the inspection target liquid is passed through the filter. 15. The capturing means has a filtration container holding the inspection target liquid, the supply means has a rotary nozzle for spraying the cleaning liquid, and a nozzle driving unit, and the supply means has the filtration container. The dyeing apparatus for microorganisms according to claim 12, wherein the liquid to be inspected attached to and remaining on the inner wall of the microorganism is removed by using the rotary nozzle. 16. The filter according to claim 1, wherein the filter has a predetermined filter diameter and is processed into a shape that can be installed in the capturing means.
An apparatus for dyeing microorganisms according to the item. 17. The filter is a flat membrane filter, and the smoothness of the flat surface has a maximum error of 100 μm.
The dyeing device for microorganisms according to claim 16, wherein the dyeing device has a diameter of m or less. 18. The microorganism dyeing device according to claim 17, wherein the pore size of the filter is smaller than 50 μm. 19. A dyeing method for dyeing microorganisms contained in a liquid to be inspected, comprising a capturing step of filtering the microorganisms contained in the liquid to be inspected and capturing them on a filter, wherein the dyeing liquid is introduced. A sealing step of shielding the periphery of the filter to form a closed space, and introducing the staining solution into the filter that has captured microorganisms,
And a dyeing step of dyeing a microorganism with the dyeing solution. 20. The sealing step prevents the dyeing solution from being evaporated during dyeing to have a high concentration and being fixed to the surfaces of the microorganisms and the filter so that they cannot be removed. Method for dyeing microorganisms. 21. The method for staining a microorganism according to claim 19 or 20, wherein the closed space formed by the closing step has a volume of 350 cm ³ or less. 22. The capturing step is characterized in that the inspection target liquid is filtered with a filtration container including a membrane filter, and the microorganisms contained in the inspection target liquid are captured on the membrane filter. 22. A method for dyeing a microorganism according to claim 21. 23. The pressurizing or depressurizing pump is used to accelerate the speed of the filtration in the capturing step, according to any one of claims 19 to 22.
The method for dyeing a microorganism according to the item. 24. The method according to claim 19, wherein in the sealing step, a predetermined amount of the staining liquid is retained in the sealed space during the dyeing period.
The method for dyeing a microorganism according to any one of 1. 25. The method for staining a microorganism according to claim 19, wherein the closed space is closed by a light blocking material that does not transmit light. 26. The method further comprises a carrying step of carrying the filter to preset first and second positions, and a carrying control step of controlling the carrying step, wherein the filter is configured to carry out the first step by the carrying step. That the microorganisms are captured on the filter by the capturing step after being transported to the first position, the filter is transported to the second position by the transporting step, and the microorganisms are stained by the staining step. Claims 19 to 25, characterized in that
The method for dyeing a microorganism according to any one of 1. 27. In the dyeing step, the dye solution is injected from a primary side of the filter, which is a side into which the test liquid is injected into the filter, or a secondary side of the filter facing the primary side. 27. The method for staining a microorganism according to any one of claims 19 to 26, wherein: 28. In the dyeing step, a temperature controller that keeps the sealed small space at a predetermined temperature and a dyeing time controller that controls the dyeing time to be a predetermined time are used. The method for dyeing a microorganism according to any one of claims 19 to 27. 29. After staining a microorganism with the staining solution,
The method for staining a microorganism according to any one of claims 19 to 28, further comprising a removal step of removing the staining liquid. 30. The method according to claim 19, further comprising a supplying step of supplying the inspection target liquid, the pretreatment liquid for dyeing, the sterilizing liquid, and the cleaning liquid to the filter. The method for dyeing a microorganism according to 1. 31. The sterilizing solution and / or the cleaning solution is
The method for dyeing a microorganism according to claim 30, wherein the microorganism is supplied after being dyed with the dye. 32. The method for staining a microorganism according to claim 30, wherein the pretreatment liquid and / or the cleaning liquid is supplied after the inspection target liquid is passed through the filter. 33. In the capturing step, a filtration container holding the inspection target liquid is used, and in the supplying step, the cleaning liquid is rotated with respect to the inspection target liquid remaining on the inner wall of the filtration container. 31. The method for dyeing a microorganism dyeing device according to claim 30, wherein the method is a method for dyeing with a microorganism. 34. The filter according to any one of claims 19 to 33, wherein the filter has a predetermined filter diameter and is processed into a shape that can be installed in a filtration container including the filter. The method for dyeing a microorganism according to 1. 35. The filter is a flat membrane filter, and the smoothness of the flat surface has a maximum error of 100 μm.
35. The method for dyeing a microorganism according to claim 34, wherein the method is less than or equal to m. 36. The method for staining a microorganism according to claim 35, wherein the pore size of the filter is smaller than 50 μm.