Patent Application
20220283086
The present invention relates to an apparatus and method for inspecting ballast water, and in particular, to an inspection apparatus and method that are suitable for detecting live microorganisms, such as plankton, contained in ballast water, for example.
A vessel not loaded with cargo sails while being loaded with ballast water to make it stable, and discharges the ballast water in a sea area where cargo is loaded onto the vessel.
Ballast water is usually discharged in a sea area different from a sea area where the ballast water has been loaded. Therefore, microorganisms, such as plankton and bacteria, contained in the ballast water are carried to a sea area other than the native habitat of the microorganisms, and thus may damage the ecosystem.
To address such a problem, an international rule about the regulation of ballast water has been established. Specifically, the “International Convention for the Control and Management of Ships' Ballast Water and Sediments (Ballast Water Management Convention)” has been enacted and has entered into force.
In “Guidelines for ballast water sampling (G2)” related to the aforementioned Ballast Water Management Convention, “Ballast Water Performance Standard (D-2)” specifies the allowable limits for the number of live microorganisms contained in ballast water discharged from vessels based on the minimum size (i.e., minimum diameter) of the microorganisms. For example, the standard specifies that vessels shall discharge less than 10 organisms/m3 regarding organisms with a minimum size (i.e., minimum diameter) of 50 μm or greater (hereinafter referred to as “L-size organisms”) and less than 10 organisms/ml regarding microorganisms with a minimum size (i.e., minimum diameter) of 10 μm or greater and less than 50 μm (hereinafter referred to as “S-size organisms”).
As a method for confirming if the aforementioned discharge standard is met during the discharge of the ballast water, there is known a method of passing sea water, which has been pumped with a conveying pump, through a flow cell and performing measurement based on an image (for example, Patent Literature 1). As another means, there is also known a method of passing sea water, which has been pumped with a conveying pump, through a filter unit including filters with different apertures, and causing microorganisms on the filters to emit light, and then counting the number of the microorganisms (for example, Patent Literature 2). As further another means, there is also known a microorganism inspection apparatus for inspecting the number of microorganisms using a batch-type measurement cell (for example, Patent Literature 3).
A microorganism inspection apparatus described in Patent Literature 1 above includes a staining unit for staining organisms having live cells existing in a flowing liquid specimen, a concentration unit for increasing the concentration of the organisms in the flow of a stained specimen, an individual measuring unit for acquiring image information on the individuals containing the organisms in the concentrated specimen, and a control unit for measuring the organisms based on the image information on the individuals output from the individual measuring unit.
Accordingly, it is possible to perform flow cytometry inclusive of the step of staining organisms in a liquid specimen, the step of increasing the concentration of the organisms in the liquid, and the step of acquiring information on the organisms in the liquid, for example. Therefore, in comparison with a method of performing each step through a batch process, it is possible to significantly reduce the standby time for part of a specimen, which has undergone one step, to proceed to a next step, or set the standby time to zero, and thus acquire stable information about whether the organisms are alive or dead in the sense that degradation of the organisms in the stained state during the standby time can be prevented.
In addition, a microorganism inspection apparatus described in Patent Literature 2 above includes a step of passing sea water through a filter unit in which three types of filters with different apertures are arranged in series, a step of causing live microorganisms trapped by the filters to exhibit a color, emit light, or emit fluorescence, and a step of detecting one of the exhibited color, emitted light, and emitted fluorescence, and performing image analysis to count the number of microorganisms in the ballast water or the sea water.
Accordingly, it is possible to trap microorganisms of each size in stages, and thus quickly measure if the standard regulating the allowable residual microorganisms of each size is met.
Further, a microorganism inspection apparatus described in Patent Literature 3 above is adapted to add a sample and a fluorescent staining reagent into a batch-type sample container formed of a light-transmitting material, and stir and mix the sample solution, and then irradiate the sample solution with a light beam from a light source so as to calculate the number of microorganisms contained in a sample of the sample solution.
Accordingly, it is possible to easily detect and measure the number of microorganisms in ballast water in a short time with high accuracy.
The microorganism inspection apparatus described in Patent Literature 1 is adapted to sequentially pass sea water, which has been pumped with a conveying pump, through each step. Thus, the apparatus is large and the production cost may become high. In addition, although the sea water is sequentially passed through each step to shorten the standby time, it may take at least several hours to complete the measurement.
The microorganism inspection apparatus described in Patent Literature 2 is also adapted to sequentially pass sea water, which has been pumped with a conveying pump, through each step as with the technique described in Patent Literature 1. Thus, the apparatus is large and the production cost may become high.
In the microorganism inspection apparatus described in Patent Literature 3, a light-receiving unit is provided such that its light-receiving surface is arranged at an angle perpendicular to an excitation light beam. Thus, a number of optical components, such as a means for converting a light beam into a collimated light beam, a relay lens, a slit, and a condensing lens, are used. In addition, since a light source unit is provided on the lateral face side of the sample container, and the light-receiving unit is provided on the front face side or the rear face side of the sample container, the area for disposing the light source unit and the light-receiving unit as seen in plan view is large, which may result in a large body of the inspection apparatus.
It is a technical object of the present invention to provide a microorganism inspection apparatus with a reduced number of components and a reduced production cost, and also with a reduced size, and in which irradiation unevenness of an excitation light beam from a light source unit can be suppressed and thus inspection accuracy can be improved.
To solve the aforementioned problems, the present invention provides as a technical means a microorganism inspection apparatus for measuring the number of microorganisms in a sample solution, including stirring/mixing means for adding a sample and a fluorescent staining reagent into a batch-type sample container formed of a light-transmitting material, and stirring and mixing a sample solution; an excitation light source including a light source that irradiates an irradiation plane of the sample container with an excitation light beam in a vertical direction while the sample solution is stirred by the stirring/mixing means; light-receiving means arranged on a lateral face side of the sample container, the light-receiving means being configured to detect a fluorescence beam emitted in response to the excitation light beam from the excitation light source; and control means for converting the beam detected by the light-receiving means into an electric signal to detect the number of light emissions, and calculating the number of microorganisms contained in the sample in the sample container from the number of light emissions.
In addition, the excitation light source may be arranged above or below the sample container. Further, a reflective member may be provided in the sample container, the excitation light source may be arranged on a lateral side of the sample container, and an excitation light beam emitted from the excitation light source may irradiate the irradiation plane in the vertical direction by being reflected by the reflective member.
In addition, the microorganism inspection apparatus may further include an optical guide bar between the excitation light source and the irradiation plane.
Further, the microorganism inspection apparatus may further include a shielding plate on a side opposite to the excitation light source across the irradiation plane.
Accordingly, in comparison with an apparatus that performs measurement by leaving a sample solution to stand without stirring it, it is possible to allow microorganisms to emit bright light in an extremely short time and thus easily measure the number of microorganisms in ballast water in a short time. In addition, since the apparatus of the present invention uses a batch-type sample container, the size of the apparatus can be reduced, and the production cost can also be reduced. Further, since the number of optical members and the like can be reduced in comparison with that of the conventional art, the size of the apparatus can be further reduced, and the production cost can be further reduced. Furthermore, since excitation light beam irradiation is performed in a direction perpendicular to the water flow of the sample solution, the influence of the water flow can be reduced, and detection accuracy can be improved with suppressed irradiation unevenness.
In addition, since the excitation light source is arranged above or below the sample container, it is possible to reduce the number of members arranged around the sample container and the measurement unit as seen in plan view, and thus reduce the size of the apparatus and reduce the production cost.
Further, since the excitation light source is arranged on the lateral side of the sample container, the flexibility of the arrangement of the excitation light source can be increased. Furthermore, since excitation light beam irradiation is performed in a direction perpendicular to the water flow of the sample solution, the influence of the water flow can be reduced, and detection accuracy can be improved with suppressed irradiation unevenness.
In addition, since the optical guide bar is provided between the excitation light source and the irradiation plane, it is possible to allow an excitation light beam from the excitation light source to reliably irradiate the irradiation plane, and thus further improve detection accuracy.
Further, even when a lid and the like are not provided, it is possible to perform measurement without the operator feeling dazzling. Furthermore, the measurement range of microorganisms, including its vicinity, can be reliably irradiated with an excitation light beam.
According to the present invention, it is possible to reduce the number of components and reduce the production cost, and also reduce the size of an inspection apparatus, and further suppress irradiation unevenness of an excitation light beam from a light source unit, and thus improve inspection accuracy.
To describe the characteristics of the invention of the present application, a summary of conventional art described in Patent Literature 3 will be described first.
An inspection apparatus of the conventional art includes a batch-type sample container 105 formed of a light-transmitting, transparent material (for example, glass, quartz, or acrylic resin), and a measurement unit 106 that optically counts the number of microorganisms in the sample container 105. The sample container 105 includes a rotor 107 to stir a sample solution S in the sample container 105. The rotor 107 is configured to be rotationally driven by a stirrer provided in a body (not illustrated).
The inspection apparatus illustrated in
In addition, the batch-type sample container 105 formed of a light-transmitting, transparent material is formed in the shape of a prism having a bottom face with a size of 50 mm×50 mm and a height of 60 mm, and has an internal volume set to 100 ml (milliliter) when the water level in the container is 40 mm. The shape of the sample container 105 is not limited to such a prism shape, and may be a cylindrical shape or a cubic shape as long as an internal volume of about 100 ml (milliliter) can be secured.
The measurement unit 106 includes a sample container housing unit 109 that houses and holds the sample container 105, a light source unit 113 that emits an excitation light beam toward the sample container 105, and a light-receiving unit 119 for observing microorganisms stained by a luminescence reagent and floating in the sample container 105 with the excitation light beam emitted from the light source unit 113. The light-receiving unit 119 is electrically connected to a CPU (not illustrated) that counts the number of microorganisms in the sample solution S and performs an information processing operation or a statistical processing operation on the measurement results, for example.
The sample container housing unit 109 is formed with holding plates 108a and 108b surrounding at least two faces of the sample container 105, and houses and holds the sample container 105 so as not to block a light beam emitted from the light source unit 113.
In addition, as illustrated in
The means 111 for converting a light beam into a collimated light beam illustrated in
The light-receiving unit 119 is provided such that its light-receiving surface F is arranged at an angle perpendicular to an excitation light beam in the direction of the normal P from the light source unit 113. The light-receiving unit 119 includes a detector 114 arranged and configured to receive fluorescence along an optical axis perpendicular to an excitation light beam emitted from the LED light source 110 toward the sample container 105, a light-receiving filter 115 arranged on the front face side of the detector 114, a condensing lens 116 arranged on the front face side of the light-receiving filter 115, a slit 117 arranged on the front face side of the condensing lens 116, and a relay lens 118 arranged in the gap between the slit 117 and the sample container 105 and adapted to excite a fluorescent material contained in microorganisms and collect the resulting fluorescence emission to form an image.
In such an inspection apparatus, since the rotor 107 rotates on the bottom face of the sample container 105 as illustrated in
In view of the foregoing, an embodiment of the present invention will be described hereinafter.
An inspection apparatus 1 of the present embodiment includes a body unit 2 that incorporates a control mechanism, such as a CPU board, and performs an operation of processing measurement results, for example, an operation unit 3 that is arranged next to the body unit 2 and includes operation buttons, for example, and a display unit 4 for displaying the measurement results, for example. The display unit 4 includes a liquid crystal panel, for example. The operation unit 3 includes a power button 3a, a measurement start button 3b, an external output button 3c, and a setting button 3d. Pressing the power button 3a can control the on/off switching, and pressing the measurement start button 3b can start measurement. In addition, pressing the external output button 3c can transfer data to an external printer or personal computer, and pressing the setting button 3d can switch the type of measurement, such as the setting of the size of microorganisms as a measurement target, change the setting of a threshold, and change the setting of the measurement time.
The body unit 2 includes a measurement unit 6. The measurement unit 6 houses a batch-type sample container 5 and optically counts the number of microorganisms in a sample solution S in the sample container 5. The sample container 5 is formed of a transparent material, such as glass, quartz, or acrylic resin, for example, and thus is configured to transmit light. The sample container 5 houses a rotor 7, and the rotor 7 stirs the sample solution S. The rotor 7 is housed in the sample container 5 together with the sample solution S and a luminescence reagent, and is closed with a lid 30. In addition, the rotor 7 is configured to be, when the sample container 5 is housed in the measurement unit 6, rotationally driven by a stirrer 27 incorporated in the measurement unit 6.
The inspection apparatus 1 illustrated in
The sample container 5 is formed in the shape of a prism having a bottom face with a size of 50.5 mm×51.5 mm and a height of 67 mm, and has a volume set to about 100 ml when the water level in the container is 43 mm. Although the sample container 5 is formed in the shape of a prism in the present embodiment, the sample container 5 may have other shapes, such as a cylindrical shape. In such a case, however, the sample container 5 is desirably dimensioned to be able to secure a volume of about 100 ml to facilitate measurement.
Further, the electrical control configuration of the inspection apparatus 1 of the present embodiment will be described based on
The CPU board 23 has electrically connected thereto the detector 14, the LED light source 10, a RAM 25 as a read/write memory portion, and a ROM 26 as a read-only memory portion.
Besides, the CPU board 23 has connected thereto the stirrer 27, which rotates the rotor 7 with a magnetic force, the display unit 4 formed with a liquid crystal panel, for example, a cooling fan 28 for control devices, such as the CPU board 23, and an external output terminal 29, such as RS-232C.
Next, the configuration of the measurement unit 6 of the present embodiment will be described based on
The rotor 7 is arranged on the bottom face of the sample container 5. Thus, when the rotor 7 is rotated by the stirrer 27, the flow of water is stirred mainly in the horizontal direction along the bottom face. Therefore, although excitation light beam irradiation is performed in a direction parallel with the flow of water in the conventional art, in the present embodiment, excitation light beam irradiation is performed in a direction perpendicular to the flow of water. Therefore, the influence of the flow of water is reduced, and detection of the number of microorganisms can be performed with higher accuracy and with suppressed irradiation unevenness.
An excitation light beam excited by the LED light source 10 passes through the excitation filter 12 so that unwanted wavelengths are filtered out, and then, the beam shape is adjusted by the diaphragm 11. Accordingly, it is possible to reduce variation in the amount of fluorescence emitted by microorganisms depending on the portion through which an excitation light beam passes.
In the present embodiment, the number of optical members on the side of the light source unit 13 and the side of the light-receiving unit 19 can be reduced in comparison with that of the conventional art, and thus, the optical configuration can be simplified.
Specifically, the number of the LED light sources 10 can be reduced from 6 to 1, and the number of the excitation filters 12 can be reduced from 2 to 1. The diameter of the LED light source 10 can also be reduced to half, specifically, from 25 mm to 12.5 mm. In addition, the number of pieces of cover glass provided on the excitation filter 12 can also be reduced from 2 to 1, and the size of the cover glass can also be reduced.
Regarding the light source unit 13, although the LED light source 10 is used as a light source, it is possible to use not only the LED light source 10 but also a collimated-light-beam LED light source capable of emitting a collimated light beam, a laser light source, or a light bulb as long as a fluorescent material contained in microorganisms can be excited.
Regarding the optical components on the side of the light-receiving unit 19, there is no need to use a relay lens, a slit, or a cylindrical lens provided in the conventional art. In addition, regarding the light-receiving filter 15 and the detector 14, a single light-receiving filter 15 and a single detector 14 may be provided.
In this manner, reducing the number of optical components on the side of the light source unit 13 and the side of the light-receiving unit 19 to simplify the configuration can reduce the overall size of the apparatus.
Although the sample container 5 in the present embodiment is formed in the shape of a prism, the sample container 5 may be formed in a cylindrical shape. Further, the sample container 5 may also be formed in the shape of a polygonal prism, such as a triangular prism, a pentagonal prism, or a hexagonal prism.
Although an example in which a photomultiplier (PMT) is used as a light-receiving sensor of the light-receiving unit 19 has been illustrated, the present invention is not limited thereto, and it is possible to use various photodetectors, such as a silicon photodiode (SiPD) and an avalanche photodiode (APD), that can detect light emission of a fluorescent material contained in microorganisms as with a photomultiplier (PMT).
(Step 1) An operator collects 100 ml (milliliter) of a sample from ballast water at a temperature of about 20° C., using a pipette, for example, and then puts the sample into the sample container 5.
(Step 2) A fluorescent staining reagent is added into the sample container 5. As the fluorescent staining reagent, it is possible to use commonly known calcein AM (Calcein-AM manufactured by Promocell GMBH in Germany) or FDA (Fluorescein Diacetate manufactured by Tokyo Chemical Industry Co., Ltd.), for example. Calcein AM tends to stain phytoplankton more easily, while FDA tends to stain zooplancton more easily. Therefore, when staining with a staining reagent is performed with a reagent containing a mixture of calcein AM and FDA, it is possible to shorten the reagent staining time and specifically reduce the time required for staining to half that of the conventional art.
(Step 3) The operator puts the rotor 7 into the sample container 5 and places the sample container 5 in the measurement unit 6 of the inspection apparatus 1, and then puts the lid 30 of the measurement unit 6, thereby completing the preparation for measurement. Then, the operator presses the power button 3a to cause the rotor 7 to rotate with the drive of the magnetic stirrer 27 incorporated in the measurement unit 6, and thus stir the sample solution S.
(Step 4) The operator presses the measurement start button 3b of the operation unit. Then, after a predetermined time has elapsed, the LED light source 10 is lighted so that the sample container 5 is irradiated with a light beam transmitted through the bandpass filter 12 for an excitation light beam. At this time, for example, the sample container 5 is irradiated with a light beam with a wavelength of 450 nm to 490 nm as the wavelength characteristics so that a specimen (i.e., microorganisms) in the sample container 5 emits fluorescence.
(Step 5) The detector 14 of the light-receiving unit 19 detects the fluorescence.
(Step 6) The detector 14 detects the emission of fluorescence by converting the light energy into electrical energy utilizing the photoelectric effect, and also with a current amplification function added thereto. The detected electric signal is sent to the CPU board 23 so that received-light waveforms that are greater than or equal to a given threshold are counted.
(Step 7) Further, the CPU board 23 estimates the number of microorganisms existing in 100 ml (milliliter) of water in the sample container 5 from the counted value of the received-light waveforms, and then displays on the display unit 4 information about whether the estimated number of microorganisms satisfies the effluent standard.
The configuration of the measurement unit 6 according to another embodiment will be described based on
The configuration of the measurement unit 6 according to further another embodiment will be described based on
The configuration of the measurement unit 6 according to still another embodiment will be described based on
The slit 17 is used to narrow the observation plane in a slit form. Using the slit 17 can narrow the light-receiving area of the light-receiving surface and can also narrow the area of the background fluorescence emission that would become noise. This improves the ratio of a signal of the fluorescence emission of microorganisms to the background fluorescence emission, and thus improves detection accuracy for the fluorescence emission of microorganisms.
The configuration of the measurement unit 6 according to yet another embodiment will be described based on
The configuration of the measurement unit 6 according to yet another embodiment will be described based on
Although the aforementioned embodiments have illustrated examples in which a single LED light source is provided, it is also possible to arrange a plurality of LED light sources, such as three LED light sources. In such a case, if the LED light sources are arranged on the bottom face side of the sample container 5, for example, the LED light sources are preferably arranged in the horizontal direction along the direction of the flow of the sample solution S based on the rotor 7. In addition, in such embodiments, although the rotor 7 is arranged on the bottom face and excitation light beam irradiation is performed in the vertical direction with respect to the rotor 7, it is also possible to dispose the rotor 7 on the lateral face and perform excitation light beam irradiation in the horizontal direction perpendicular to the rotor 7.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-152722 | Aug 2019 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/028165 | 7/20/2020 | WO |
