JP2007135582A - Method and apparatus for detecting microorganism in ballast water - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and an apparatus capable of effectively detecting residual microorganisms in ballast water after carrying out treatment for killing underwater microorganisms to ballast water poured into a vessel and to provide a method and an apparatus capable of effectively detecting the number of microorganisms in sea water before treatment in order to determine conditions for killing treatment of microorganisms to a ballast water. <P>SOLUTION: The method and the apparatus for detecting microorganisms in ballast water each comprises a step for passing sea water which is subjected to organism-killing treatment or sea water pouring into a vessel as ballast water through a filter unit in which three kinds of filters different in opening are arranged in series, a step for generating either one selected from coloring, luminescence and fluorescence by microorganisms collected on the filters and a step for detecting either one selected from coloring, luminescence and fluorescence and counting the number of microorganisms in ballast water or seawater by image analysis. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a method and an apparatus for detecting residual microorganisms in ballast water after treatment for killing the microorganisms (plankton, bacteria, etc.) in the water with respect to the ballast water injected into the ship.
The present invention also relates to a method and an apparatus for detecting the number of microorganisms in seawater before treatment in order to determine the killing treatment conditions for ballast water.

Ships sail with a large amount of ballast water injected into the ship, and this ballast water is dumped into the ocean when cargo is loaded. For this reason, the aquatic organisms of the ship leaving the sea where the ballast water was injected may diffuse into the sea area of the cargo loading area, and these may abnormally propagate and change the ecosystem or cause marine pollution.
In order to prevent this situation, the International Maritime Organization (IMO) has adopted a convention on the regulation and management of ballast water and sediment. In this Convention on the Regulation and Management of Ballast Water and Precipitate Discharge, the organisms in the ballast water shall be killed and it shall be confirmed that the following ballast water discharge standards are satisfied before ballast water discharge. Is requested.
(Ballast water discharge standard)
-Living organisms (50 µm or more) ····· 10 individuals / m ³ or less · Living organisms (10 µm to 50 µm or more) ··· 10 individuals / ml or less · E. coli (about 1 µm) ...・ ・ ・ ・ ・ ・ ・ ・ 250cfu / 100ml or less ・ Enterococci (about 1μm) ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ 100cfu / 100ml or less ・ V. cholerae (about 1μm) ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ 1cfu / 100ml or less

By the way, the following methods are known as methods for detecting microorganisms such as plankton and bacteria.
(1) Counting plankton Plankton is usually counted by microscopic observation after collecting and concentrating with plankton nets.
In addition, optical plankton counters and visual plankton recorders that analyze floating plankton images have been developed as automatic measurement devices, but it is difficult to distinguish suspended particles from plankton, and it is underestimated due to particle overlap. There is a problem of becoming overestimated by counting dead bodies.

(2) Microorganism detection Microorganism testing has been developed for the purpose of accurately and quickly detecting microorganisms in samples in clinical tests and food tests. The culture inspection method is generally performed in the microorganism inspection, but a highly sensitive rapid inspection method as described below has been developed for shortening and simplifying the time.
The specimen liquid containing microorganisms is filtered with a filter membrane that has been dipped in an ATP-degrading enzyme solution and then dried, the microorganisms contained in the specimen are captured on the filtration membrane, and the luminescent components in the microorganisms are extracted therefrom. A liquid extraction reagent and a luminescent reagent are sprayed in the form of a mist to emit light, and the number of light emission points is measured using a counting measurement device (see Patent Document 1).
A rapid microorganism detection apparatus comprising a substrate material to which a substance that specifically captures target microorganisms present in a sample is bound, and optically detecting target microorganisms captured on the substrate material (see Patent Document 2) ).
JP-A-11-137293 JP-A-2005-172680

In the ballast water microbiology test, the ballast water sample mixed with plankton and bacteria is divided into three levels of microbes: 50 μm or more, mainly zooplankton, 10-50 μm mainly phytoplankton, and 1 μm bacteria. On the other hand, different permissible remaining number criteria are applied.
In other words, in the case of plankton of 50 μm or more, the number of individuals per 1 m ³ is a problem, while in the plankton of 10 to 50 μm, the number of individuals per ml is the problem, and in bacteria, the number of cfu per 100 ml is a problem. The amount of water used as the standard is 10 ⁴ to 10 ⁶ times different.
In addition, in the case of performing a biological killing process when supplying ballast water, it is necessary to detect the number of microorganisms in seawater in a short time in order to determine the processing conditions.
As described above, in the microbiological examination of ballast water, as described above, different allowable remaining number standards are applied to a plurality of types of detection objects having different sizes, and rapid processing is required. There are two great specialities.
When the above-described conventional inspection method or apparatus is applied to the microbial inspection of ballast water having such special characteristics, there are the following problems.

The method of counting plankton collected by a plankton net, which is usually used for counting plankton, by microscopic observation requires a skillful technique and requires enormous time for relying on visual observation. Therefore, the bacteria cannot be counted and cannot be applied to a ballast water test that requires rapid processing.
In addition, the bacterial culture testing method commonly used for microbiological testing requires skill in testing and requires culture time, so it takes a lot of time to find the test results after obtaining the specimen. I need. Therefore, as with the plankton counting method, it cannot be applied to ballast water inspection.

In addition, although the microorganism rapid detection method disclosed in Patent Document 1 can detect bacteria, it is difficult to detect plankton due to differences in cell size and cell membrane structure.
Moreover, the object of the clinical test | inspection and the food test | inspection using the rapid detection apparatus of the microorganisms disclosed by patent document 2 is mainly pathogenic bacteria, and its application is difficult for the quick test | inspection of the plankton which is a ballast water regulation object.
As described above, any of the conventional methods cannot effectively detect organisms in the ballast water having the above-mentioned special characteristics.

The present invention has been made to solve such problems, and effectively detects living microorganisms in the ballast water after the treatment for killing the microorganisms in the water with respect to the ballast water poured into the ship. It is aimed to obtain a method and apparatus that can.
Another object of the present invention is to obtain a method and an apparatus capable of effectively detecting the number of microorganisms in seawater before treatment in order to determine the extinction treatment conditions for ballast water.

  (1) The method for detecting microorganisms in ballast water according to the present invention includes a filter unit in which three types of filters having different openings are arranged in series for ballast water that has undergone biological killing treatment or seawater that is poured into a ship as ballast water. A step of causing water to pass through, a step of generating any one of color development, luminescence, and fluorescence due to the activity of microorganisms collected and alive in the filter, and detecting any one of color development, luminescence, and fluorescence. And a step of counting the number of microorganisms in ballast water or seawater by analysis.

Since the microorganisms in the seawater are captured by passing water through a filter unit in which three types of filters with different mesh openings are arranged in series, it is possible to capture microorganisms in stages. It is possible to quickly measure whether or not the allowable residual standard regulated by the standard for each size is satisfied.
Further, since any one of color development, luminescence, and fluorescence generated by the activity of the microorganisms that are captured and survived by the filter is generated and detected, the microorganisms that are alive can be counted quickly.

The openings of the three types of filters having different openings are 50 μm, 10 μm, and 0.5 μm from the upstream side of the water flow, for example. By using these three types of filter openings, mainly zooplankton with a size of 50 μm or more, mainly phytoplankton with a size of 10 μm to 50 μm, and bacteria with a size of about 1 μm are collected. In addition, an appropriate amount of water in accordance with the allowable remaining number standard of ballast water can be passed through each filter.
In particular, generating any one of coloration, luminescence, and fluorescence by living microorganisms means that the biological component of the microorganism is dyed and colored, the biological component of the microorganism is dyed to generate fluorescence, the living organism of the microorganism Staining with a dye that generates fluorescence by reaction with components to generate fluorescence, staining with a dye that generates fluorescence by being decomposed by the electron reducing power possessed by the microorganism, and generating fluorescence, and ATP (adenosine possessed by the microorganism) Any method of adding a reagent that emits light with (triphosphate) and causing it to emit light may be selected, and a method suitable for the target microorganism is selected.

(2) The process of filtering and concentrating the ballast water that has been subjected to biological killing treatment or seawater injected as ballast water into a ship, and passing the concentrated ballast water or seawater through a filter having an opening of 25 to 100 μm And the ballast water or seawater filtered in the concentration step, or the ballast water that has been subjected to biocidal treatment or the seawater injected into the vessel as ballast water, is passed through two different filters with a mesh opening smaller than 50 μm. And a step of generating any one of color development, luminescence, and fluorescence generated by living microorganisms collected in two different filters having a mesh opening of 25 to 100 μm and a mesh opening smaller than 50 μm. And detecting any one of color development, luminescence, and fluorescence, and counting the number of microorganisms in the ballast water or seawater by image analysis.

The standard for the allowable number of remaining microorganisms in ballast water is that the number of allowable residual quantities for different amounts of water is determined for three-stage microorganisms: plankton of 50 μm or more, plankton of 10-50 μm, and bacteria of about 1 μm. In the case of plankton of 50 μm or more, the number of individuals per 1 m ³ is a problem, whereas in the case of plankton of 10 to 50 μm, the number of individuals per ml is the problem, and in bacteria, the number of cfu per 100 ml is a problem.
Ballast water that has been subjected to biocidal treatment or seawater injected into the ship as ballast water is filtered and concentrated, and the concentrated ballast water or seawater is passed through a filter having an opening of 25 to 100 μm, more preferably a filter of 50 μm. By capturing plankton of 50 μm or more in water, it is possible to efficiently detect plankton of 50 μm or more, which requires a large amount of water to flow, compared to detection of plankton of 10 to 50 μm or bacteria. When a filter having an opening larger than 50 μm is used, plankton of 50 μm or more having passed through the filter is captured by two different filters having an opening smaller than 50 μm.

(3) Further, the microorganism detecting apparatus for ballast water according to the present invention includes a filter unit in which three types of filters having different openings are arranged in series, and a filter unit having a large opening with respect to the filter unit. Means for flowing a fixed amount of biological killing water or seawater to be poured into a ship as ballast water, and taking out the filter when a predetermined amount of water determined for each filter is flowed to each filter Filter extraction mechanism, means for generating any one of color development, luminescence and fluorescence due to the activity of microorganisms collected and alive in the extracted filter, and detecting and detecting any one of color development, luminescence and fluorescence And means for counting the number of microorganisms in the seawater by analysis.

  The openings of the three types of filters having different openings are 50 μm, 10 μm, and 0.5 μm from the upstream side of the water flow, for example. By using these three types of filter openings, mainly zooplankton with a size of 50 μm or more, mainly phytoplankton with a size of 10 μm to 50 μm, and bacteria with a size of about 1 μm are collected. In addition, an appropriate amount of water in accordance with the allowable remaining number standard of ballast water can be passed through each filter.

(4) In addition, means for filtering and concentrating the ballast water that has been subjected to biological killing treatment or seawater injected into the ship as ballast water, and a filter having an opening of 25 to 100 μm for passing the concentrated ballast water or seawater And two kinds of filters having a mesh opening smaller than 50 μm and different from each other, in which the ballast water or seawater filtered by the concentrating means, the ballast water subjected to biological killing treatment, or seawater poured into the ship as ballast water is passed through, A filter take-out mechanism that takes out the filter when a predetermined amount of water determined for each filter is passed to each filter, and coloration, luminescence, and fluorescence by microorganisms that are collected and live in the taken-out filter In the ballast water or seawater by image analysis by detecting any one of color generation, luminescence and fluorescence And a means for counting the number of microorganisms.

(5) Further, in the above (3) or (4), there is provided a judging means for judging by comparing the counted number of microorganisms with a preset reference value.

In the present invention, since the microorganisms in the seawater are captured by passing the water through three types of filters having different openings, it is possible to capture microorganisms for each step size, and as a result, for each size. It is possible to quickly measure whether the acceptable residual standard regulated by the standard is satisfied.
Further, since any one of color development, luminescence, and fluorescence generated by the activity of the microorganisms that are captured and survived by the filter is generated and detected, the microorganisms that are alive can be counted quickly.

FIG. 1 is an explanatory diagram of a device for detecting microorganisms in ballast water according to an embodiment of the present invention. Ballast water after subjecting the ballast water to a biological killing process is used as a target sample.
The apparatus for detecting microorganisms in ballast water according to the present embodiment includes a filter unit 7 in which filters 1, 3, and 5 having openings of 50 μm, 10 μm, and 0.5 μm are arranged in series, and the filter unit 7. A water supply means 9 for supplying a predetermined amount of ballast water from the 50 μm filter side, and a filter take-out for taking out the filter when a predetermined amount of water determined for each filter is supplied to each filter 1, 3, 5 Mechanism 11, staining means 13 for staining the microorganisms to generate fluorescence due to the activity of the microorganisms collected and alive on the extracted filter, and irradiating light to generate fluorescence on the stained microorganisms The light irradiation means 15, the light detection / image analysis means 17 for detecting and analyzing the fluorescence emitted by the light irradiation, and the results analyzed by the light detection / image analysis means 17 are set in advance. It includes a determination unit 19 for determining whether the light of the allowable number of remaining reference meets the criteria, the. Hereinafter, each configuration will be described in more detail.

1. Filter unit The filter unit 7 is formed by arranging filters 1, 3 and 5 in series with openings of 50 μm, 10 μm and 0.5 μm. The filter 1 having an opening of 50 μm mainly captures zooplankton having a size of 50 μm or more, the filter 3 having an opening of 10 μm mainly captures phytoplankton having a size of 10 μm to 50 μm, and the filter 5 having an opening of 0.5 μm is large. It captures mainly bacteria with a length of about 1 μm.
Each filter is arranged from the upstream side in the flow direction of the ballast water in the order of 50 μm, 10 μm, and 0.5 μm, and is captured in order from the largest of the microorganisms contained in the ballast water. By arranging in this way, clogging of fine filters can be prevented, and the number of microorganisms regulated by the criteria for the number of remaining microorganisms per stage size of microorganisms by the convention on the regulation and management of discharge of ballast water and sediment. Capturing by size can be effectively realized.

As mentioned above, in the Convention on the Control and Management of Ballast Water and Sediment, microorganisms of 50 μm or more are based on the number of individuals per 1 m ³ , and microorganisms of 10 μm to 50 μm are based on the number of individuals per 1 ml. As a standard, about 1 μm of bacteria is based on cfu per 100 ml.
In this way, since the amount of water used as the allowable remaining number standard is different for the microorganisms captured by each filter, the amount of water passing through the filter becomes smaller toward the downstream side, such as 50 μm, 10 μm, and 0.5 μm. It is desirable to adjust so that it may decrease.
By adjusting the amount of water passing through the filter in this way, it becomes unnecessary to take out a filter with a small amount of passing water extremely quickly.

  Each filter only needs to be able to capture microorganisms, such as a hydrophilic filtration membrane (for example, hydrophilic polytetrafluoroethylene), a hydrophobic filtration membrane (for example, PVDF (polyvinylidene fluoride), PTFE (polytetrafluoroethylene), PC (polycarbonate), PP (polypropylene), etc.). What fixed the filtration membrane to the frame, and what was fixed with the reinforcement net | network can also be used.

2. Water supply means The water supply means 9 pumps up the biocidal ballast water stored in the ballast tank 8, and supplies the filter unit 7 with a predetermined flow rate of ballast water. In this example, the water supply means 9 is realized by a water supply pump. Yes. By providing a flow meter for measuring the flow rate of water supplied to the filter unit 7 and an adjustment valve for adjusting the flow rate of water supply, the flow rate of water supplied to the filter unit 7 can be adjusted and set.
Further, before the water is poured into the ballast tank, or the ballast water that has been subjected to the biological killing process is sent to the filter unit 7 from the downstream side of the biological killing apparatus that pumps the ballast water from the ballast tank and performs the biological killing process. Good.

3. Filter take-out mechanism The filter take-out mechanism 11 is used when a predetermined amount of ballast water determined for each filter is sent to each of the filters 1, 3 and 5 having an opening of the filter unit 7 of 50 μm, 10 μm and 0.5 μm. The filters 1, 3 and 5 are taken out from the filter unit 7 and moved to a section for staining and a section for detecting fluorescence by microorganisms. The filter take-out mechanism 11 may be either manual or automatic as long as it is a mechanism having the above function.

4). Dyeing means The dyeing means 13 dyes microorganisms with a staining solution in order to generate any one of color development, luminescence and fluorescence due to the activity of the living microorganisms in the ballast water trapped in the filter.
As a method for staining microorganisms with a staining solution, the staining solution may be dropped on the filter, the staining solution may be sprayed on the filter with an ultrasonic sprayer, or the filter may be immersed in the staining solution. Alternatively, a filter may be placed on a filter paper or agar soaked with a staining solution.

  Generating any one of color development, luminescence, and fluorescence due to the activity of living microorganisms can be described in detail by staining and coloring the biological component of the microorganism, generating fluorescence by staining the biological component of the microorganism, microorganism Staining with a dye that generates fluorescence by reaction with biological components of the substance to generate fluorescence, staining with a dye that generates fluorescence by being decomposed by the electron reducing power possessed by the microorganism, and generating fluorescence, and ATP possessed by the microorganism Either of adding a reagent that emits light with (adenosine triphosphate) and causing it to emit light is performed. Select a method suitable for the target microorganism and use the corresponding staining solution. Hereinafter, each method and the staining solution will be described.

1) Dyeing of biological components of microorganisms
(1) Staining with a dye detected with visible light For example, dyeing with a dye such as amide black or Giemsa and detecting the stained microorganism with visible light.
(2) Staining with a fluorescent dye For example, a fluorescent dye such as acridine orange, DAPI, or rhodamine is used to detect fluorescence emitted by laser irradiation.
At this time, by staining with a fluorescent dye with excellent cell membrane permeability (SYTO9, etc.), only the viable cells with strong cell membranes are colored, and at the same time, stained with a fluorescent dye (PI, etc.) with poor cell membrane permeability. By doing so, live cells and dead cells can be stained and separated.

2) Fluorescent or luminescent by the reaction of living (active) microorganism biological components
(1) The microorganism is stained with a dye (such as FDA (Fluorescein diacetate)) that is decomposed by esterase contained in the cell and emits fluorescence. Only living cells having esterase activity develop green fluorescence upon laser irradiation.
(2) Staining with a dye (such as CTC (Cyanoditoyl tetrazolium chloride)) that is broken down by the electron-reducing power of the cells and emits fluorescence. Only living cells having respiratory reduction ability develop red fluorescence by laser irradiation.
(3) Bioluminescence is caused by adding a luminescent reagent (for example, luciferin and luciferase) to ATP (adenosine triphosphate) contained in the cells of the microorganism. Only living organisms can be bioluminescent.

  In addition, detection sensitivity can also be improved by extracting the biological component of microorganisms using a suitable extractant before reaction with the microorganisms capture | acquired by the filter, and a dyeing | staining liquid. As the extractant, ethanol, surfactant, trichloroacetic acid and the like can be used.

As described above, various dyes for living microorganisms in ballast water can be considered. However, as described above, the ballast water discharge standard is for zooplankton, phytoplankton, and bacteria, and because the size and ecology are greatly different, the staining method that has been applied to bacteria cannot be applied as it is. . For example, detection of a living zooplankton having a strong membrane structure and low membrane permeability requires a staining method different from phytoplankton and bacteria.
However, it is complicated to use a complex combination of staining methods corresponding to each organism for organisms of different sizes and ecology.

Therefore, the inventor has conducted extensive research and found that staining using esterase activity is the most suitable for determining the ballast water discharge standard for organisms having greatly different sizes and ecology. The reason is as follows.
A stain that reacts with an enzyme esterase produced by a living organism is decomposed into a fluorescent substance by esterase when it is taken into living cells. Since the fluorescent substance has a strong membrane structure and low permeability in living cells, it is retained in the cell without passing through the cell membrane and flowing out, and fluorescence can be detected by laser irradiation.
More specific description will be given below.

FDA (Fluorescein) is a staining agent that serves as a substrate for esterase activity.
diacetate). In order to incorporate this stain into the cells of a living zooplankton having a strong membrane structure and low membrane permeability, the membrane permeability may be improved by esterification of the carboxyl group of the stain.
The staining agent with improved membrane permeability penetrates not only into bacterial cells but also into zooplankton and phytoplankton having living cells with a strong membrane structure. Therefore, it penetrates into zooplankton, phytoplankton, and bacterial cells regardless of whether the organism is alive or dead.
When the staining agent penetrates into living cells, it is decomposed into fluorescent substance Fluorescein by esterase in the cells. Viable cells have a low membrane permeability due to their strong membrane structure, and the resulting fluorescent substance Fluorescein does not flow out of the cell, but accumulates in living cells due to the polarity of Fluorescein.
On the other hand, although FDA permeates through dead cells, dead cells that have lost esterase activity are not degraded to fluorescein, which is a fluorescent substance.
Therefore, when FDA staining is applied to a living organism and excited with blue light with a wavelength of 488 nm, live cells emit green fluorescence with a wavelength of 530 nm derived from Fluorescein, and dead cells do not produce a fluorescent substance, and the staining agent FDA Since it is non-fluorescent itself, it does not show fluorescent coloration, so it can be distinguished between life and death.
Further, as described above, in the living cells, the fluorescent substance Fluorescein generated without permeability due to the strong membrane structure is accumulated in the living cells, so that the accuracy of fluorescence detection is increased.
As the FDA staining condition, a 10 μM concentration solution and a treatment for 2 minutes at room temperature are sufficient, and the concentration can be further lowered.

Staining agents that measure esterase activity other than FDA include calcein AM, Carboxyfluoresceindiacetate (CFDA), BCECF / AM,
Examples include CFSE and CytoRed. The fluorescence characteristics of calcein AM, CFDA, BCECF / AM, CFSE, and CytoRed are excitation wavelengths / fluorescence wavelengths of 490/515, 495/520, 590/526, 496/516, and 535/590 nm, respectively.

  The conventional stains used for bacterial life and death have the problem that the stains do not permeate the organisms with thick cell membranes, such as zooplankton. By selecting, it is possible to determine whether the zooplankton is alive or dead. Furthermore, by using a sample obtained by collecting zooplankton with a filter and concentrating it, it is possible to use a small amount of a high-concentration dyeing agent to enhance the dyeability, and to make life / death discrimination more accurate. Moreover, it can permeate | transmit and dye | stain, without making the usage-amount of a coloring agent excessive.

Next, regarding the staining of bacteria, the fact that it was difficult to stain by the conventional method will be described.
What should be noted in the staining of microorganisms is that the dyeing method differs in each stage of the bacterial life cycle. The normal life cycle of bacteria is in the logarithmic growth phase, stationary phase, and death phase. Bacteria in the logarithmic growth phase and stationary phase are likely to be stained, and they have fluorescence due to their esterase activity. Not dyed. Therefore, such normal bacteria are relatively easy to distinguish between life and death.

  On the other hand, the life cycle of some bacteria is divided into a logarithmic growth phase, stationary phase, spore formation phase, and death phase. Vegetative cells in the logarithmic growth phase and stationary phase are strongly stained, as are bacteria that do not form spores. Cells at death are not stained because they have lost esterase activity. Therefore, for some of these bacteria, life / death discrimination is relatively easy when they are in the logarithmic growth phase, stationary phase, or death phase. The problem is bacteria in the spore formation stage.

A spore is a highly durable cell structure formed by some bacteria. When bacteria having the ability to form spores are placed in a poor environment such as nutrients and temperature, or when they come into contact with a compound that is toxic to the bacteria, dormant spores are formed inside the bacterial cells. The germinating spore has esterase activity and emits green fluorescence by FDA staining. However, in the dormant spore, the stain is difficult to penetrate into the dormant spore, and the fluorescence is weak.
For this reason, with the conventional method, it was difficult to determine whether a dormant spore is viable or dead.

  Therefore, even for such dormant spores, the use of the above-described staining agent with excellent membrane permeability makes it possible to determine whether or not the animal is alive or dead as in the case of the zooplankton described above. In addition, instead of using a stain with excellent membrane permeability, the membrane permeability is increased by pretreatment, the cell membrane structure is loosened by cavitation in ballast water sterilization treatment, etc. Life-and-death discrimination staining is also possible for dormant spore cells. However, such treatment may be performed in addition to using a staining agent having excellent membrane permeability.

  As described above, by using a staining agent that reacts with esterase activity with improved membrane permeability, it is possible to stain and detect living cells of both zooplankton, phytoplankton, and bacteria. In the conventional method, when various microorganisms exist at different concentrations, such as seawater, it is difficult to detect living organisms with one staining method. By selecting an excellent stain, life / death discrimination can be detected by staining with a single stain.

5. Light Irradiation Unit The light irradiation unit 15 irradiates light, for example, laser light, in order to generate fluorescence on the filter coated with the staining solution. Moreover, the light for generating fluorescence may be irradiated by irradiating excitation light.
However, in the case of the method of adding a luminescent reagent (for example, luciferin and luciferase) to ATP (adenosine triphosphate) described in 2) and 3) in the description of the staining means 13, in order to generate fluorescence for bioluminescence. No light irradiation is required.

6). Photodetection / image analysis means The photodetection / image analysis means 17 captures color development, luminescence, or fluorescence caused by microorganisms on the filter with an imager 18 such as a CCD image sensor, and optically detects it as image information to perform image analysis. And count the number of microorganisms.
If impurities exist, the size and shape of the microorganism are identified in the image analysis process, and impurities having other shapes are excluded.
The image analysis means 17 can use an existing bioluminescence image analysis system.

7). Determination means The determination means 19 compares the number of viable microorganisms analyzed by the light detection / image analysis means 17 with a preset allowable residual number reference value, and passes if the number of viable microorganisms is less than the reference value. When the number of surviving microorganisms is larger than the reference value, it is determined as rejected. And when it passes or fails, it is displayed on the display means which is not illustrated. As a display means, it may be displayed on the screen of the personal computer with characters of pass or fail, or a green lamp is turned on when it passes, and a red lamp is turned on when it fails. May be.

The operation of the present embodiment configured as described above will be described.
First, the microorganisms are killed with respect to the seawater poured into the ballast tank 8. The killing treatment method may be a method of adding a bactericidal agent, a method of combining filter filtration, bactericidal agent addition and cavitation treatment.

The ballast water stored in the ballast tank after performing the killing process of the microorganisms is supplied to the filter unit 7 by operating the water supply pump 9. As described above, the filter unit 7 is provided with three types of filters having openings of 50 μm, 10 μm, and 0.5 μm, and each filter is taken out when a predetermined amount of water is passed through them.
Filter removal is performed by the filter removal mechanism 11.
When the filter removal mechanism 11 is manual, the amount of water supplied to the filter unit 7 is confirmed, and each filter is removed manually.
When the filter take-out mechanism 11 is automatic, each filter is automatically taken out when a predetermined amount of water is passed through each filter.

  The dyed liquid is sprayed on the taken out filter by a staining means 13 such as an ultrasonic sprayer. Then, the filter sprayed with the staining solution is installed in the field of view of the image pickup device 18 of the light detection / image analysis means 17, and the laser beam is irradiated to generate fluorescence by the activity of the microorganism. And the number of microorganisms is counted by image analysis.

As described above, in the present embodiment, since the filter unit 7 in which the filters having openings of 50 μm, 10 μm, and 0.5 μm are arranged in series is used, the discharge regulation and management of ballast water and sediment are related. Capable of capturing each size of microorganisms regulated by the acceptable number of remaining standards for each size in the treaty, and as a result, can quickly measure whether the acceptable number of remaining standards regulated by the above conventions are met .
Further, since color development, luminescence, or fluorescence from the microorganisms captured by the filter is optically detected and analyzed by a CCD image sensor or the like, the living microorganisms can be quickly counted.

  In the above-described embodiment, an example in which the remaining microorganisms in the ballast water are detected using the ballast water that has been subjected to the killing process of the microorganisms as a sample is shown, but the present invention is not limited to this, and the microorganisms It can also be used in the case where microorganisms that survive in seawater are measured for each size using seawater before killing as a sample. In this case, it is possible to select the optimum method and conditions for the microorganism killing process in accordance with the number of surviving microorganisms living in the seawater for each size.

  Collected seawater and killed seawater are passed through a filter unit with three types of filters with openings of 50, 10, 0.5 μm, and FDA solution is added to the microorganisms collected on each filter. The sample was stained at 30 ° C. for 30 minutes, irradiated with light having a wavelength of 460 nm as excitation light and 530 nm for fluorescence. The generated fluorescence was captured as image information by a CCD image sensor, and the number of microorganisms was counted. The results are shown in Table 1.

  As shown in Table 1, it was confirmed that microorganisms in seawater before treatment and in seawater after death treatment could be detected in a short time. And it has confirmed that it was killed by the killing process to such an extent that the ballast water discharge standard is satisfied.

  FIG. 2 is an explanatory view of an example embodying a filter unit and a filter take-out mechanism. FIG. 2 (a) is an explanatory view of the entire configuration, and FIG. FIG. In addition, the same code | symbol is attached | subjected to a part of part which has the same function as what was shown in FIG. 1, and description is abbreviate | omitted.

In the example shown in FIG. 2, a filter take-out mechanism 71 is provided for each filter. In FIG. 2, only the filter 1 having an opening of 50 μm is shown, but the other filters 3 and 5 (FIG. 1) are shown. The same mechanism is applied to the reference).
The filter take-out mechanism 71 has a rotating shaft 73 at the center, a disk 75 that is rotatably installed around the rotating shaft 73, and a motor 79 that rotationally drives the disk 75 via a gear 77. The disk 75 is provided with an installation part in which the filter 1 can be detachably installed.

In this example, the ballast water in the ballast tank 8 (see FIG. 1) is not sent directly to the filter 1 but a predetermined amount of ballast water is stored in the ballast water auxiliary tank 81 and the ballast water in the ballast water auxiliary tank 81 is stored. Water is sent to the filter 1.
The water supply line on the downstream side of the ballast water auxiliary tank 81 will be described. A water supply pump 85 is installed in the main water supply pipe 83 connected to the ballast water auxiliary tank 81, and an inverted U-shaped bypass pipe is provided on the downstream side of the water pump 85. 87 is connected so that the filter 1 can be attached to and detached from the bypass pipe 87. That is, a part of the bypass pipe 87 joined to the filter 1 installed on the disk 75 is a movable pipe 89 that can move up and down, and the filter 1 and the bypass pipe 87 are moved up and down. Can be connected and disconnected.
An inlet side valve 91 and an outlet side valve 93 are provided at both ends of the bypass pipe 87, and a valve 95 is also provided between the two connecting parts of the main water supply pipe 83 to the bypass pipe 87.

  In the apparatus configured as described above, when water is supplied to the bypass pipe 87, the moving pipe 89 is pushed up to close the gap between the filter 1 and the bypass pipe 87, and the inlet side valve 91 and the outlet side valve 91 are closed. 93 is opened and water is passed through the filter 1. When detecting a living organism after passing a predetermined amount of ballast water through the filter 1, the water supply pump 85 is stopped and the inlet valve 91 is closed. In this state, the moving pipe 89 is lowered to disconnect the bypass pipe 87 from the filter 1. Then, the motor 79 is driven to move to the staining reagent injection position (see FIG. 2B). Here, the above-described staining is performed, and then the rotating plate 73 is further rotated, and fluorescence detection and image analysis are performed at the imaging position. Thereafter, the rotating plate is further rotated to the filter replacement position to replace the filter 1, and the rotating plate 73 is further rotated to collect organisms as necessary at the filtering position.

[Embodiment 2]
FIG. 3 is an explanatory diagram of another embodiment of the present invention. In the present embodiment, a concentrating device 21 that filters and concentrates seawater is installed in a filter 1 having an opening of 50 μm in a line that feeds seawater, and the water is fed to the filter 1 after the seawater is concentrated by the concentrating device 21. It is what you do.
The two filters 3 and 5 are installed in the filter unit 8 so that the seawater that has passed through the concentrating device 21 is passed through.

Various forms of the concentrating device 21 can be applied.
FIG. 4 is an explanatory diagram of a single cylinder type concentration device 22 as an example of the concentration device 21. The single cylinder type concentrator 22 shown in this example includes a cylindrical main body container 23 and a plankton net 25 having an opening of 50 μm that divides the inside of the main body container into two chambers. The inner side of the container body divided by the plankton net 25 is a concentrated water chamber 27, and the outer side is a filtered water chamber 29.
One end side of the main body container 23 is connected to an inflow pipe 31 for allowing the organism extinction ballast water or seawater (hereinafter simply referred to as “detection target water”) to be detected to flow into the concentrated water chamber 27. The inflow pipe 31 is provided with an inflow valve (V1).
Further, a cleaning water pipe 33 for allowing the cleaning water to flow into the concentration chamber 27 is connected to one end side of the main body container 23 adjacent to the inflow pipe 31, and the cleaning water pipe 33 is provided with a cleaning valve (V5). .
The other end side of the main body container 23 is connected to a concentrated water take-out pipe 35 for taking out the concentrated water in the concentrating chamber 27, and the concentrated water take-out pipe 35 is provided with a concentrated water take-out valve (V3). The concentrated water take-out pipe 35 is connected to the filter 1 side.
Further, a drainage pipe 37 for discharging filtered water flowing from the inflow pipe 31 and filtered by the plankton net 25 is connected to the lower part of the side surface of the main body container 23. The drainage pipe 37 is provided with a drain valve (V2). It has been.
Further, a washing water pipe 39 for allowing washing water for back washing the plankton net 25 to flow in is connected to the upper part of the side surface of the main body container 23, and the washing water pipe 39 is provided with a washing valve (V4).
The cleaning water pipe 33 and the cleaning water pipe 39 can be attached with a device for increasing the cleaning efficiency, for example, a spray nozzle. In addition, the washing water pipe 39 can be connected to a ring-shaped pipe in the main body container 23 so that the entire surface of the plankton net 25 is back-washed.

FIG. 5 is an explanatory diagram for explaining the operation of the single-tube concentrator 22 shown in FIG. As shown in FIG. 5, the inflow pipe 31 is connected to an auxiliary tank 96 having a capacity of 1 m ³ , and the ballast water from the ballast tank 8 can flow into the auxiliary tank 96. Also. A cleaning water tank 97 for cleaning is connected to the cleaning water pipe 33. Further, a branch pipe 98 is provided on the downstream side of the drain pipe 37, and a filter 3 having an opening of 10 μm and a filter 5 having an opening of 0.5 μm are installed in series on the branch pipe 98. The branch pipe 98 is provided with an on-off valve (V6) in front of the filter 3, an on-off valve (V7) is provided in front of the filter 5, and is further opened and closed downstream of the junction of the drain pipe 37 with the branch pipe 98. A valve (V8) is provided. By adjusting the opening degree of these on-off valves (V6), (V7) and (V8), the amount of water passing through the filters 3 and 5 can be adjusted.

  Hereinafter, the operation of the single cylinder type concentrator 22 will be described with reference to FIG. First, the inflow valve (V1) is opened, and the detection target water stored in the auxiliary tank 96 is caused to flow into the concentrated water chamber 27 inside the plankton net having an opening of 50 μm. At this time, the drain valve (V2) is set to “open”, and the washing valves (V4 and V5) and the concentrated water take-off valve (V3) are set to “closed”. The on-off valves (V6), (V7), and (V8) are adjusted to an opening degree that allows a proper amount of water to pass through the filters 3 and 5.

The detection target water supplied to the inside of the plankton net is that microorganisms and water of 50 μm or less move to the filtered water chamber 29 outside the plankton net and flow into the drainage pipe 37, part of which flows into the branch pipe 98, and the others Returned to the ballast tank 8.
After supplying a predetermined amount of detection target water, the cleaning valves (V4, V5) are set to “open”, cleaning water is supplied from the cleaning water pipes 33, 39, the plankton net 25 is cleaned, and the microorganisms attached to the plankton net 25 Wash off. As a result, concentrated water in which microorganisms of 50 μm or more are concentrated remains in the concentration chamber 27. Thereafter, the concentrated water take-off valve (V3) is opened, the concentrated water is discharged to the filter 1 side, and the filter 1 collects microorganisms of 50 μm or more. On the other hand, the filter 3 collects 10 to 50 μm microorganisms, and the filter 5 collects 0.5 to 10 μm microorganisms.
Subsequent counting of microorganisms and the like are performed in the same manner as in the first embodiment.

FIG. 6 is an explanatory diagram of a two-cylinder concentrator 41 as another example of the concentrator 21. The two-cylinder concentrator 41 shown in this example includes a cylindrical first container 45 that forms the concentrated water chamber 43 and a cylindrical second container 49 that forms the filtered water chamber 47. The first container 45 and the second container 49 communicate with each other at the side thereof, and a plankton net 51 having an opening of 50 μm is installed in the communication part.
A detection target water inflow pipe 53a for allowing the detection target water to flow into the first container 45 is connected to the upper end of the first container 45, and the detection target water inflow pipe 53a is provided with an inflow valve (V1-a). Yes.
Further, a concentrated water take-out pipe 55a for taking out the concentrated water in the first container 45 is connected to a lower end portion of the first container 45, and a concentrated water take-out valve (V4-a) is provided in the concentrated water take-out pipe 55a. It has been.
Furthermore, one end of a drain pipe 56a is connected to the upper side of the first container 45, and a drain valve top (V2-a) is installed in the drain pipe 56a. A drainage pump 57 is provided on the other end side of the drainage pipe 56a and is connected to the filter unit 8 side.
Further, one end of the first container 45 is connected to the drain pipe 59a at the lower side of the side surface, and the other end of the drain pipe 59a is connected to the drain pipe 56a. A drain valve (V3-a) is provided in the drain pipe 59a.
Further, a cleaning water pipe 61a for supplying cleaning water is connected to the center of the side surface of the first container 45, and the cleaning water pipe 61a is provided with a cleaning valve (V5-a).

  Further, the second container 49 is provided with the same piping and valves as those provided in the first container 45. In FIG. 6, these piping and valves are provided on the first container 45 side. The code | symbol (code | symbol which attached | subjected the subscript b with the same number) corresponding to piping and valves is attached | subjected.

The operation of the two-tube concentrator 41 configured as described above will be described.
First, the inflow valve (V1-a) on the first container 45 side and the drain valve (V2-b) on the second container 49 side are opened, and the other valves are closed.
In this state, the detection target water is supplied from the detection target water inflow pipe 53a to the first container 45 side. In the detection target water supplied to the concentrated water chamber 43 of the first container 45, microorganisms and water of 50 μm or less move to the filtered water chamber 47 through the plankton net 51 and are discharged from the drain pipe 56b. On the other hand, microorganisms of 50 μm or more remain in the concentrated water chamber 43.
If necessary, the washing water valves (V5-a and V5-b) of the concentrated water chamber 43 and the filtration water chamber 47 are opened to wash the microorganisms adhering to the plankton net 51.
After supplying the detection target water at a predetermined flow rate to the concentrated water chamber 43, the drain valve (V3-b) of the filtered water chamber 47 is opened to discharge the water. As a result, water enriched with microorganisms of 50 μm or more remains in the concentrated water chamber 43.
The concentrated water is taken out by opening the concentrated water take-off valve (V4-a) and supplied to the filter 1 side.
Subsequent counting of microorganisms and the like are performed in the same manner as in the first embodiment.

  As described above, in the present embodiment, the concentration device 21 that filters and concentrates seawater is installed in the filter 1 having an opening of 50 μm in the line that feeds the seawater, and the concentration device 21 concentrates the seawater. By sending water from the filter to the filter 1, it is possible to efficiently detect plankton of 50 μm or more, which has to pass a large amount of water, compared to detection of 10 to 50 μm plankton and bacteria. It is possible to prevent damage to the filter 1 due to a large amount of seawater.

  In the description of the second embodiment, the example in which the detection target water that has passed through the concentration device 21 is passed through the filter unit 8 having two types of filters having an opening of 10, 0.5 μm is shown. The water to be passed to the filter unit 8 does not necessarily pass through the concentrating device 21, and the detection target water may be passed directly to the filter unit 8 separately.

It is explanatory drawing of the microorganisms detection apparatus in the ballast water which concerns on one embodiment of this invention. It is explanatory drawing of an example which actualized the filter unit and filter extraction mechanism which were shown in FIG. It is explanatory drawing of the microorganisms detection apparatus in the ballast water which concerns on other embodiment of this invention. It is explanatory drawing of one aspect | mode of the concentration apparatus in other embodiment of this invention. It is operation | movement explanatory drawing of the concentration apparatus shown in FIG. It is explanatory drawing of the other aspect of the concentration apparatus in other embodiment of this invention.

Explanation of symbols

    1 50 μm filter, 3 10 μm filter, 5 0.5 μm filter, 7 filter unit, 9 water supply means, 11 filter take-out mechanism, 13 dyeing means, 15 light irradiation means, 17 light detection / image analysis means, 19 judgment means, 21 concentrator .

Claims (5)

Passing the ballast water subjected to the biological killing process or seawater injected into the ship as ballast water through a filter unit in which three kinds of filters having different openings are arranged in series;
A step of generating any one of coloration, luminescence and fluorescence by microorganisms collected and alive in the filter;
A method for detecting microorganisms in ballast water, the method comprising: detecting any one of color development, luminescence, and fluorescence and counting the number of microorganisms in ballast water or seawater by image analysis. Filtering and concentrating the ballast water that has been subjected to biocidal treatment or the seawater injected into the ship as ballast water; and
Passing the concentrated ballast water or seawater through a filter having an aperture of 25 to 100 μm;
Passing the ballast water or seawater filtered in the concentration step, or the ballast water subjected to biological killing treatment or seawater poured into the vessel as ballast water through two different filters having an opening smaller than 50 μm; and
A step of generating any one of coloring, luminescence and fluorescence by the living microorganisms collected in two different filters having a mesh opening of 25 to 100 μm and a mesh opening smaller than 50 μm;
A method for detecting microorganisms in ballast water, the method comprising: detecting any one of color development, luminescence, and fluorescence and counting the number of microorganisms in ballast water or seawater by image analysis. A filter unit in which three kinds of filters having different openings are arranged in series, and ballast water in which a predetermined amount of organism killing processing has been performed on the filter unit from the filter side having a large opening or water injection as ballast water to a ship Means for flowing sea water, a filter take-out mechanism for taking out the filter when a predetermined amount of water determined for each filter is flowed to each filter, and being collected and alive by the taken-out filter Means for generating any one of coloration, luminescence and fluorescence by the microorganism, and means for detecting any one of the coloration, luminescence and fluorescence and counting the number of microorganisms in the ballast water or seawater by image analysis A device for detecting microorganisms in ballast water. Means for filtering and concentrating the ballast water that has been subjected to biological killing treatment or seawater poured into the ship as ballast water, a filter having an opening of 25 to 100 μm for passing the concentrated ballast water or seawater, and the concentration means The two types of filters that have a mesh opening smaller than 50 μm and different from each other, and for each of the filters. A filter take-out mechanism that takes out the filter when a predetermined amount of water determined for each filter flows, and any one of color development, luminescence, and fluorescence by microorganisms that are collected and alive in the taken-out filter The number of microorganisms in ballast water or seawater is detected by image analysis after detecting any of the means to generate and color development, luminescence and fluorescence. A device for detecting microorganisms in the ballast water. The apparatus for detecting a microorganism in ballast water according to claim 3 or 4, further comprising determination means for comparing the counted number of microorganisms with a preset reference value.

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