JP2013050375A - Inspection method of ballast water - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inspection method of ballast water for accurately inspecting the population of L-size organisms and S-size organisms included in ballast water in a short time.SOLUTION: A method of inspecting microorganisms included in ballast water includes the steps of: inspecting the population of microorganisms with the minimum size of 50 μm or more on the basis of image data obtained by a scanner; and inspecting the population of microorganisms with the minimum size of 10 μm and more and less than 50 μm by a method other than that based on the image data obtained by a scanner.

Description

  The present invention relates to a method for testing ballast water, and more particularly to a method for testing microorganisms such as plankton contained in ballast water.

In order to stabilize the ship, the ship that is not loaded with cargo travels with ballast water and discharges the ballast water in the sea area where the luggage is loaded.
Ballast water is usually discharged into a sea area different from the sea area on which it is mounted. Therefore, problems such as the destruction of ecosystems by transporting microbes such as plankton and bacteria contained in the ballast water to sea areas other than the original habitat There is a risk of causing it.

  In order to deal with such problems, international rules regarding the regulation of ballast water have been formulated, and the “International Convention for the Regulation and Management of Ship Ballast Water and Sediment (Ballast Water Management Convention)” has been adopted. Yes.

The “Guideline for Ballast Water Sampling (G2)” related to the above Ballast Water Management Convention is the “Ballast Water Emission Standard (D-2)”. The allowable number of microorganisms contained in the ballast water discharged from the ship is For example, for microorganisms having a minimum size of 50 μm or more (hereinafter referred to as “L size organisms”), 10 microorganisms / m ³ or less, and for microorganisms having a minimum size of 10 μm or more but less than 50 μm (hereinafter referred to as “S size organisms”). Is defined as 10 cells / mL or less according to the minimum size of the microorganism.

In addition, the guideline (G2) defines that the “minimum size” of microorganisms in the discharge standard (D-2) means “the minimum dimension of a living body excluding stab, flagellum, or touch”. .
Therefore, in recent years, development of ballast water treatment apparatuses has been actively conducted so as to satisfy the above emission standard (D-2).

  By the way, conventionally, a method for inspecting microorganisms contained in ballast water using image data has been proposed (see Patent Document 1).

  The inspection method described in Patent Document 1 acquires a digital image of a microorganism using a fluorescence microscope, recognizes a connected region of pixels as one microorganism in a binary image obtained by binarizing the digital image, An area of the microorganism is calculated, and an elliptical minor axis having an area equal to the calculated area and an arbitrary ratio of minor axis and major axis of 1: 1.1-1: 8 is calculated. The recognized short diameter is regarded as the minimum size of the microorganism, and the recognized microorganism is counted separately for each minimum size of the microorganism in the ballast water discharge standard (D-2).

However, the inspection method described in Patent Document 1 assumes that the shape of the microorganism is an ellipse having the same area as that of the microorganism, and that the short diameter is the minimum size of the microorganism. The minor axis of the shape is clearly different from the definition of “minimum size” in the above guideline (G2).
In addition, although there are microorganisms having various shapes, the method described in Patent Document 1 assumes that the shape of the microorganism is simply an ellipse, has a large variation from actual measurement, and is reliable. It is scarce.

  Furthermore, since the fluorescence microscope used in the inspection method described in Patent Document 1 is expensive and complicated to operate, it is not suitable for daily simple inspection.

  Therefore, the present inventors have developed a method capable of accurately inspecting microorganisms contained in ballast water using image data acquired by a fluorescence scanner (see Patent Document 2).

  The inspection method described in Patent Document 2 inspects whether or not the minimum size of the microorganism is greater than or equal to a predetermined size depending on whether or not the two-dimensional shape of the microorganism passes through a slit having a predetermined length that is a two-dimensional space. Is.

According to the inspection method described in Patent Document 2, microorganisms contained in ballast water can be accurately divided and counted for each minimum size of microorganisms in the ballast water discharge standard (D-2).
Further, the inspection method described in Patent Document 2 uses image data acquired by a fluorescent scanner, and is suitable for daily simple inspection.

However, in the inspection method, it is necessary to scan the filtered filter for each specimen of the L-size organism and the S-size organism with a fluorescent scanner to acquire image data, and the inspection takes time.
Further, for imaging of S-size organisms, a high resolution is required for the fluorescent scanner, and it takes time to acquire image data.
Furthermore, some phytoplanktons form a group of individuals, but the majority of these phytoplanktons are classified as S-size organisms, and there are many cases where the gap between individuals is 10 μm or less. It is difficult to recognize each individual phytoplankton that forms the colony from image data acquired by a fluorescent scanner.

On the other hand, at a meeting of the International Maritime Organization (IMO), a method for inspecting S-size organisms contained in ballast water using chlorophyll fluorescence measurement using pulse modulation has been proposed (see Non-Patent Document 1). .
This inspection method focuses on the fact that phytoplankton, which accounts for the majority of microorganisms classified as S-size organisms, has chlorophyll. Using a pulse-modulation fluorometer, the chlorophyll contained in a sample of ballast water is measured. The maximum quantum yield is measured, and the number of phytoplankton individuals included in the sample is indirectly examined based on the relationship between the measured value and the like and a preset number of phytoplankton individuals.

According to the inspection method, a commercially available pulse-modulated chlorophyll fluorometer (for example, a pulse-modulated chlorophyll fluorometer (Water-PAM) manufactured by Walz, Germany) is used, and S size that inhabits in a sample of ballast water. The number of organisms can be inspected accurately and easily.
However, this inspection method has a problem that it cannot cope with zooplankton, which accounts for the majority of microorganisms classified as L-size organisms.

JP 2010-63403 A Japanese Patent Application No. 2010-277543

BLG / 15/5/4 'DEVELOPMENT OF GUIDELINES AND OTHER DOCUMENTS FORUNIFORM IMPLEMENTATION OF THE 2004 BWM CONVENTION', Annex, Paragraph 9.14-9.16,9.25, [online]; INTERNATIONAL MARITIME ORGANIZATION, [retrieved on 21 December 2010] .Retrieved from the Internet: <URL: http://docs.imo.org/Category.aspx?cid=53>

  Therefore, the present invention accurately examines the number of individuals of L size organisms (microorganisms having a minimum size of 50 μm or more) and S size organisms (microorganisms having a minimum size of 10 μm or more and less than 50 μm) contained in ballast water in a short time. It aims at providing the inspection method of ballast water which can be performed.

  In order to achieve the above object, the ballast water inspection method of the present invention is an inspection method for microorganisms contained in ballast water, and for microorganisms having a minimum size of 50 μm or more, based on image data acquired by a scanner. The number is inspected, and the microorganism having a minimum size of 10 μm or more and less than 50 μm is characterized by inspecting the number of individuals by a method other than based on image data acquired by a scanner.

  Here, in the present invention, the “minimum size” of the microorganism follows the definition in the above “Guideline for Ballast Water Sampling (G2)”, which means “the minimum dimension of the living body excluding stab, flagella or tactile sensation”. Is.

  According to the ballast water inspection method of the present invention, for the microorganism having the minimum size of 50 μm or more, the image data of the microorganism is obtained by a scanner, the image data is binarized, and the binarized binary image is obtained. Based on the above, a connected region of pixels corresponding to the microorganism is specified, pixels constituting the contour are sequentially specified in the connected region of the pixels, and the contour is formed using one of the pixels constituting the contour as a starting point. When moving two points clockwise or counterclockwise along the pixel, the first point is moved in the first direction and the second point is moved in the second direction opposite to the first direction. Only when the linear distance to the first point is 50 μm or more, the first point is moved in the first direction, and the first point makes a round of the pixels constituting the contour and returns to the starting pixel. The contour is configured between If the first point and the second point do not coincide with each other when at least one of the first point and the second point passes through all the pixels to be processed, the minimum size of the microorganism is 50 μm or more It is preferable to check the number of individuals by counting the connected regions of pixels corresponding to the microorganisms whose minimum size is determined to be 50 μm or more.

  In the ballast water inspection method of the present invention, each time the second point moves the first point by one pixel in the first direction, the linear distance from the first point is less than 50 μm. It is preferable to move continuously in the second direction or continuously in the first direction until the linear distance to the first point is less than 50 μm.

  In the method for examining ballast water according to the present invention, it is preferable to use phytoplankton as a microorganism having the minimum size of 10 μm or more and less than 50 μm.

  In the method for inspecting ballast water of the present invention, it is preferable to inspect the phytoplankton population by chlorophyll fluorescence measurement using pulse modulation.

  In the ballast water inspection method of the present invention, the ballast water sample is irradiated with pulse-modulated measurement light, the ballast water sample irradiated with the measurement light is irradiated with pulse light, and the measurement light is irradiated with the measurement light. Measure the minimum fluorescence intensity Fo released by chlorophyll contained in the sample when irradiated, measure the maximum fluorescence intensity Fm emitted by chlorophyll contained in the sample when irradiated with the pulsed light, The maximum quantum yield Fv / Fm = (Fm−Fo) / Fm of the chlorophyll contained in the sample is calculated from the measured values of the minimum fluorescence intensity Fo and the maximum fluorescence intensity Fm, and the minimum fluorescence intensity is calculated. Based on the relationship between Fo and the maximum quantum yield Fv / Fm and a preset number of phytoplankton individuals, the plant plan contained in the ballast water sample It is preferable to indirectly inspect the population of tons.

  In the ballast water inspection method of the present invention, it is preferable that the scanner is a fluorescent scanner.

  In the method for inspecting ballast water in the present invention, for microorganisms having a minimum size of 50 μm or more (L size organisms), the number of individuals is inspected based on image data acquired by a scanner, and microorganisms having a minimum size of 10 μm or more and less than 50 μm ( For S size organisms, the number of individuals is inspected by a method other than based on image data acquired by a scanner. Therefore, for L size organisms, inspection is performed by a simple method using a scanner. For S size organisms, Since the inspection can be performed in parallel with the inspection of the L-size organism by another appropriate method, the number of L-size organisms and S-size organisms contained in the ballast water can be inspected with high accuracy in a short time. .

  According to the ballast water inspection method of the present invention, for the microorganism having the minimum size of 50 μm or more, the image data of the microorganism is obtained by a scanner, the image data is binarized, and the binarized binary image is obtained. Based on the above, a connected region of pixels corresponding to the microorganism is specified, pixels constituting the contour are sequentially specified in the connected region of the pixels, and the contour is formed using one of the pixels constituting the contour as a starting point. When moving two points clockwise or counterclockwise along the pixel, the first point is moved in the first direction and the second point is moved in the second direction opposite to the first direction. Only when the linear distance to the first point is 50 μm or more, the first point is moved in the first direction, and the first point makes a round of the pixels constituting the contour and returns to the starting pixel. Until the said contour If the first point and the second point do not coincide with each other when at least one of the first point and the second point passes through all the pixels constituting the minimum size of the microorganism, If the number of connected individuals of the pixels corresponding to the microorganisms determined to be 50 μm or more and the minimum size is determined to be 50 μm or more is to be examined, the number of individuals of L size organisms is image-processed. The technology enables easy and accurate inspection.

  In the ballast water testing method according to the present invention, if the phytoplankton is a microorganism having the minimum size of 10 μm or more and less than 50 μm, the number of individuals of S size organisms can be easily tested.

  In the ballast water inspection method according to the present invention, if the number of phytoplankton individuals is to be inspected by chlorophyll fluorescence measurement using pulse modulation, a commercially available pulse-modulated chlorophyll fluorescence measuring instrument is used. The number of S-size organisms that inhabit can be inspected accurately and easily.

An example of plankton images with a fluorescence microscope. The flowchart of the inspection method of L size organisms. The image figure of a binary image. Explanatory drawing of a raster scan. Explanatory drawing of a contour tracking method. The figure which attached the outline number in order of outline tracking in FIG. The flowchart of L size biological judgment. Explanatory drawing 1 about the determination example 1 of L size living thing. Explanatory drawing 2 about the example 1 of judgment of L size living thing. FIG. 3 is an explanatory diagram for a determination example 1 of an L-size organism. Explanatory drawing 4 about the example 1 of judgment of L size living thing. Explanatory drawing 5 about the example 1 of judgment of L size living thing. The image figure of a binary image. Explanatory drawing 1 about the example 2 of judgment of L size living thing. Explanatory drawing 2 about the example 2 of judgment of L size living thing. FIG. 3 is an explanatory diagram for a determination example 2 of an L-size organism. FIG. 4 is a diagram for explaining a determination example 2 of an L size organism. Explanatory drawing 5 about the example 2 of judgment of L size living thing. Explanatory drawing which shows the principle of the chlorophyll fluorescence measurement using pulse modulation. The graph which shows the relationship between the light irradiated to chlorophyll, and fluorescence intensity.

Embodiments of the present invention will be described with reference to the drawings.
FIG. 1 shows an example of a fluorescence microscope image of a microorganism to be examined in the embodiment of the present invention.
Fig. 1 (a) shows barnacle larvae (zooplankton), (b) shows copepods (zooplankton), and (c) and (d) show diatoms (phytoplankton).
In each image, a portion indicated by an arrow corresponds to the “minimum size” of the microorganism defined in the guideline (G2) of the Ballast Water Management Convention.

<Inspection of L-size organisms (microorganisms with a minimum size of 50 μm or more)>
In the inspection of the L-size organism in the present embodiment, it is assumed that the “minimum size” of the microorganism is the minimum size of the slit length through which the planar shape of the microorganism in the image can pass.

  And the inspection method of the L size living thing in this Embodiment is that the minimum size of the said microorganisms is 50 micrometers or more because the connection area | region of the pixel corresponding to the microorganisms in an image cannot pass the slit of length 50 micrometers. The number of connected L-sized organisms is examined by counting the connected regions of the pixels corresponding to the microorganisms that are determined to have a minimum size of 50 μm or more.

FIG. 2 shows a flow of an inspection method for L-size organisms.
In the present embodiment, the inspection of the L-size organism includes the imaging step (S1), the binarization processing step (S2), the labeling step (S3), the contour tracking step (S4), the size determining step (S5), and the counting step. It is performed through each step of (S6).

(1) Imaging process: S1
In this step, a specimen of ballast water is filtered with a filter, and an image of microorganisms on the filter is acquired with a scanner.
Here, if microorganisms in the ballast water are previously stained with a fluorescent stain, preferably calcein AM, and an image of the microorganisms on the filter is obtained with a fluorescence scanner, microorganisms having living cells can be easily identified.

(2) Binarization process: S2
In this step, the image data acquired in the imaging step (S1) is binarized to obtain a binary image.
Here, FIG. 3 shows an image of a binary image acquired by the binarization processing. A plurality of white or black squares in FIG. 3 each indicate a pixel. In FIG. 3, pixels corresponding to microorganisms are shown in white.

(3) Labeling process: S3
In this step, the pixel region corresponding to the microorganism is identified from the binary image acquired in the binarization processing step (S2) by a technique such as raster scan so that each pixel region can be distinguished. Perform labeling.
Here, the pixel region is a single pixel region in which one pixel having a pixel value 1 is independent or a connected region in which a plurality of pixels having a pixel value 1 are connected.
The example shown in FIG. 3 shows a connected region of pixels corresponding to microorganisms.

(4) Outline tracking step: S4
In this step, the connected region of the pixels specified in the labeling step (S3) starts from one of the pixels (contour pixel) constituting the contour, and the contour of the connected region is tracked in units of pixels. Here, the outline in the present embodiment refers to the outer outline of the connected area of the pixels.
At this time, the first contour pixel as the starting point can be specified by, for example, raster scanning. Further, the tracking of the contour can be performed by a known method such as 4-neighbor tracking or 8-neighbor tracking for obtaining a boundary of connected pixels.

FIG. 4 shows an example of searching for the first contour pixel by raster scanning in the binary image shown in FIG. Raster scanning is a scanning method in which the upper left corner of an image is the starting point, the pixels are examined from the left end to the right, and after reaching the right end, the rows are moved down one row and the pixels are examined from the left end to the right.
FIG. 5A shows a method for tracking an outline by the 4-neighbor tracking, and FIG. 5B shows a method for tracking an outline by the 8-neighbor tracking. Each neighborhood tracking shown in FIGS. 5A and 5B is a method of searching for the next contour pixel in the numerical order shown in the clockwise direction from the tracking direction around the searched contour pixel O.

In the present embodiment, in the pixel connection region, the first contour pixel is specified by raster scanning, and the contour pixel is sequentially searched from the first contour pixel in the clockwise direction by tracking eight neighbors. The tracking of the contour ends when the searched pixel is the first contour pixel and the pixel to be searched next is a searched pixel.
6 shows that the first contour pixel in the binary image shown in FIG. (1), and contour numbers (contour No. (2) to No. (16)) are assigned in the order of contour pixel tracking.

(5) Size determination step: S5
In this step, it is determined whether or not the minimum size of the microorganism corresponding to the connected region is 50 μm or more for the connected region in which the contour pixel is specified in the contour tracking step (S4).
In the present embodiment, in the binary image shown in FIG. (1) is a starting pixel A, and two points B and C from the starting pixel A are moved along the contour pixels in the order of the contour numbers.

For the first point B, the contour No. (1) → No. (2) → No. (3) Move in the first direction in the clockwise direction. For the second point C, the contour No. (1) → No. (16) → No. (15) → ... and move it counterclockwise in the second direction.
Here, for the second point C, every time the first point B is moved to a pixel of the next contour number, the contour number is within a range where the distance from the first point B is less than 50 μm. In order, it is continuously moved in the second direction. When the distance to the first point B is equal to or greater than 50 μm, the distance is continuously moved in the first direction in the order of the contour numbers until the distance is less than 50 μm.

  Then, the first point B goes around the contour pixels in the order of the contour numbers, and the contour No. When the first point B and the second point C intersect with pixels having the same contour number before returning to the starting pixel A in (1), that is, all of the contour pixels are When the first point B and the second point C coincide with each other when at least one of the point B and the second point C has passed, the connecting region can pass through a 50 μm slit, It is determined that the minimum size of the microorganism corresponding to the connection region is less than 50 μm.

  On the other hand, the first point B goes around the contour pixel without matching with the pixel having the same contour number as the second point C. Returning to the starting pixel A in (1), it is determined that the minimum size of the microorganism corresponding to the connection region is 50 μm or more, assuming that the connection region cannot pass through a slit having a length of 50 μm.

(6) Counting step: S6
In this step, the connected regions of the pixels that have been determined in the size determining step (S5) to have a minimum microorganism size of 50 μm or more are sequentially counted. The number of connected regions of the pixels counted in this step is the number of individuals of L size organisms to be examined in the present embodiment.

  Hereinafter, determination of the “minimum size” of microorganisms in the present embodiment will be described based on an example.

(Example in which the minimum size of microorganisms is determined to be less than 50 μm)
FIG. 7 shows a flowchart for determining the minimum size of microorganisms using image processing. 8 to 12 show examples in which the flowchart shown in FIG. 7 is applied to the binary image shown in FIG. 8A to 12B, (a) shows an image of a binary image, and (b) shows a contour list created based on the contour numbers shown in FIG.
In this example, the distance between point B and point C (distance between BC points) is the distance between the pixel centers where the points are located. In this example, 50 μm is 2.5 pixels.

  First, as shown in FIG. (1) is determined as the starting pixel A. At this time, since both the points B and C are located on the starting pixel A, the distance between the BC points is 0.0 pixels as shown in the outline list of FIG. 8B.

  Next, the points B and C are moved in the order of the contour numbers. First, as shown in FIG. (1) → No. (2) Move one pixel clockwise in the first direction. In this case, as shown in the outline list of FIG. 9B, since the distance between the BC points is 1.0 pixel, it is determined that the distance is less than 50 μm. (1) → No. (16) and move one pixel counterclockwise in the second direction. In this case, as shown in the outline list of FIG. 10B, since the distance between the BC points is 1.4 pixels, it is determined that the distance is less than 50 μm, and the C point is subsequently set to the outline No, (16) → No, (15) and move one pixel in the second direction.

  After that, until it is determined that the distance between the BC points is 50 μm or more, the C point is designated as the contour No. (15) → No. (14) → No. (13) →... And continuously moved in the second direction. Then, as shown in FIG. When moved to (12), as shown in the outline list of FIG. 10 (b), if the distance between the BC points is 3.2 pixels and is determined to be 50 μm or more, then the C point is Outline No. (12) → No. In contrast to (13), one pixel is moved clockwise in the first direction. Then, as shown in the outline list of FIG. 11B, since the distance between the BC points is 2.2 pixels, it is determined that the distance is less than 50 μm. (2) → No. (3) and move one pixel in the first direction.

  The movement of point B and point C is repeated as described above, and as shown in FIG. (7) → No. (8) and move one pixel in the first direction. In this case, as shown in the contour list of FIG. 12B, the point C is contour No. until the distance between the BC points is determined to be 50 μm or more. (12) → No. (11) → No. (10) → No. (9) → No. (8) and move continuously in the second direction.

  Thus, in this example, the points B and C are contour Nos. Matches at pixel (8). Therefore, in this example, it is determined that the minimum size of plankton corresponding to the connected region of the binary image shown in FIG. 3 is less than 50 μm.

(Example in which the minimum size of microorganisms is determined to be 50 μm or more)
FIG. 13 shows an image of a binary image used in this example. In the binary image shown in FIG. 13, the first contour pixel is added to the contour pixel searched in the contour tracking step (S4). As (1), contour numbers (contour Nos. (2) to No. (16)) are given in the order of tracking.
14 to 18 show an example in which the flow shown in FIG. 7 is applied to the binary image shown in FIG. In each of FIGS. 14 to 18, (a) shows an image of a binary image, and (b) shows a contour list created based on the contour number shown in FIG. 13.
In this example as well, the distance between BC points is the distance between the pixel centers where each point is located. In this example, 50 μm is 2.5 pixels.

  First, as shown in FIG. (1) is determined as the starting pixel A. At this time, since both the points B and C are located on the starting pixel A, the distance between the BC points is 0.0 pixels as shown in the outline list of FIG.

  Next, the points B and C are moved in the order of the contour numbers. First, as shown in FIG. (1) → No. (2) Move one pixel clockwise in the first direction. In this case, as shown in the outline list of FIG. 15B, since the distance between the BC points is 1.0 pixel, it is determined that the distance is less than 50 μm. (1) → No. (16) and move one pixel counterclockwise in the second direction. In this case, as shown in the outline list of FIG. 15B, the distance between the BC points is 1.4 pixels, so it is determined that the distance is less than 50 μm. (16) → No. (15) and move one pixel in the second direction.

  After that, until it is determined that the distance between the BC points is 50 μm or more, the C point is designated as the contour No. (15) → No. (14) → No. (13) →... And continuously moved in the second direction. Then, as shown in FIG. When moved to (12), as shown in the outline list of FIG. 15B, if it is determined that the distance between the BC points is 3.2 pixels and is not less than 50 μm, the C point is then set to the outline No. (12) → No. In contrast to (13), one pixel is moved clockwise in the first direction. Then, since the distance between the BC points is 2.2 pixels, it is determined that the distance is less than 50 μm. (2) → No. (3) and move one pixel in the first direction.

  The movement of point B and point C is repeated as described above, and as shown in FIG. (4) → No. (5) and move one pixel in the first direction. In this case, as shown in the outline list of FIG. Since the distance between the BC points is 2.8 pixels and the distance between the BC points is determined to be 50 μm or more, and the distance between the BC points is determined to be less than 50 μm, the contour point No. (13) → No. (14) → No. (15) → No. (16) → No. (1) → No. (2) → No. (3) and move continuously in the first direction.

  Further, the movement of points B and C is repeated as described above, and as shown in FIG. (15) → No. (16) and move one pixel in the first direction. In this case, as shown in the contour list of FIG. (7) → No. (8) → No. (9) → No. (10) → No. (11) → No. (12) → No. (13) and move continuously in the first direction.

  Then, as shown in FIG. (16) → No. (1) and move one pixel in the first direction.

  In this way, in this example, the point B goes around the contour pixel without crossing the point B and the point C with the pixel having the same contour number, and the contour No. Return to the starting pixel A in (1). Therefore, in this example, it is determined that the minimum size of the microorganism corresponding to the connected region of the binary image shown in FIG. 13 is 50 μm or more.

  In each of the above examples, it is assumed that the contour of the connection region of the image is a line connecting the centers of the contour pixels, and the distance between BC points is the distance between the pixel centers where the points are located. Assuming that the contour of the region is a line connecting the vertices in the outermost contour of the contour pixel, and the distance between the BC points is the distance using the vertices of the four corners of the pixel where each point is located, the microorganism can be more accurately It is possible to determine the minimum size of.

  In the above-described embodiment of the present invention, since the planar shape of a microorganism cannot pass through a slit having a length of 50 μm that is a two-dimensional space, the minimum size of the microorganism is determined to be 50 μm or more. If the three-dimensional shape can be identified, the three-dimensional shape cannot pass through a circle having a diameter of 50 μm, which is a two-dimensional space, so that the minimum size of the microorganism can be determined with higher accuracy if it is 50 μm or more.

  Note that the inspection of L-size organisms is not limited to the above embodiment, and as long as an image of microorganisms acquired by a scanner is used, the size of microorganisms can be determined by other image processing techniques. Good.

<Inspection of S-size organisms (microorganisms having a minimum size of 10 μm to less than 50 μm)>
In the inspection of S-size organisms in the present embodiment, a feature that the majority of phytoplankton is classified as S-size organisms, and the majority of zooplankton is classified as L-size organisms, and S-size organisms are phytoplankton. Assume that there is.
And the inspection method of the S size living organism | raw_food in this Embodiment test | inspects the number of individuals of the phytoplankton contained in the sample of ballast water by the chlorophyll fluorescence measurement using pulse modulation.

  Chlorophyll fluorescence measurement using pulse modulation is a well-known technique. For example, by using a pulse modulation chlorophyll fluorescence measurement device (Water-PAM) manufactured by Walz, Germany, individual phytoplankton contained in the sample of ballast water You can easily check the number.

Here, the chlorophyll fluorescence is fluorescence emitted from the chlorophyll when the measurement light is irradiated to chlorophyll (chlorophyll a bonded to the photosystem II complex).
In addition, chlorophyll fluorescence measurement using pulse modulation is the measurement of the fluorescence emitted by chlorophyll when chlorophyll (chlorophyll a bonded to the photosystem II complex) is irradiated with pulse-modulated measuring light (Measuring beam). Among them, the fluorescence emitted based on the measurement light can be measured separately from the fluorescence emitted based on other light.

FIG. 19 shows the principle of chlorophyll fluorescence measurement using pulse modulation. FIG. 20 shows the relationship between the light applied to chlorophyll and the fluorescence intensity.
In the inspection of the S-size organism in the present embodiment, first, as shown in FIG. 19A, a sample of ballast water is irradiated with pulse-modulated measurement light, and is included in the sample by irradiation of the measurement light. The minimum fluorescence intensity Fo emitted by chlorophyll is measured.

  Next, as shown in FIG. 19 (b), the ballast water sample irradiated with the measurement light is irradiated with a strong pulsed light (Flash), and the pulsed light gives energy used by the chlorophyll for photosynthesis. By saturating, the maximum fluorescence intensity Fm emitted by the chlorophyll upon irradiation with the measurement light is measured.

  Then, the maximum quantum yield Fv / Fm = (Fm−Fo) / Fm of chlorophyll contained in the sample is calculated from the measured values of the minimum fluorescence intensity Fo and the maximum fluorescence intensity Fm, and the minimum Based on the relationship between the fluorescence intensity Fo and the maximum quantum yield Fv / Fm and the number of phytoplankton individuals contained in the preset ballast water sample, the individual phytoplankton contained in the ballast water sample Check the number indirectly.

  Here, the relationship between the minimum fluorescence intensity Fo and the maximum quantum yield Fv / Fm and the number of phytoplankton individuals contained in the sample of the ballast water can be determined in advance by a detailed experiment. .

  In this regard, according to the document of International Maritime Organization (IMO) (see Non-Patent Document 1), when the condition that the fluorescence intensity Fo is 20 or more and the maximum quantum yield Fv / Fm is 0.3 or more is satisfied, ballast The number of S-size organisms contained in the water sample is related to 20 or more.

  In addition, the inspection of S-size organisms is not limited to the above embodiment, and the individual can be obtained by other methods for inspecting the number of phytoplankton individuals, or by other appropriate methods other than using microorganism images acquired by a scanner. The number may be inspected.

  The ballast water inspection method according to the present embodiment is such that, for L-size organisms, the number of individuals is examined by a simple method using a scanner, and for S-size organisms, for example, chlorophyll fluorescence measurement using pulse modulation, etc. Since the L size organism and the S size organism can be inspected in parallel by the appropriate method, the number of the L size organism and the S size organism contained in the ballast water can be inspected with high accuracy in a short time. .

  It goes without saying that the present invention is not limited to the above-described embodiment, and the configuration thereof can be changed as appropriate without departing from the scope of the invention.

  The test method for ballast water of the present invention can test the number of L-size organisms and S-size organisms contained in ballast water with high accuracy in a short time, and is extremely practical.

Claims (7)

A method for testing microorganisms contained in ballast water,
For microorganisms with a minimum size of 50 μm or more, the number of individuals is inspected based on image data acquired by a scanner,
A method for examining ballast water, characterized in that, for microorganisms having a minimum size of 10 μm or more and less than 50 μm, the number of individuals is examined by a method other than based on image data acquired by a scanner. For microorganisms with a minimum size of 50 μm or more,
Obtain image data of microorganisms with a scanner,
Binarizing the image data;
Based on the binarized binary image, specify a connected region of pixels corresponding to the microorganism,
Sequentially identifying pixels constituting the contour in the connected region of the pixels,
When moving two points clockwise or counterclockwise along the pixels constituting the contour, starting from one of the pixels constituting the contour, the first point is moved in the first direction, The second point is moved in the second direction opposite to the first direction and is moved in the first direction only when the linear distance to the first point is 50 μm or more. The stage in which at least one of the first point and the second point has passed through all the pixels constituting the contour until the point goes around the pixels constituting the contour and returns to the starting pixel. If the first point and the second point do not match, the minimum size of the microorganism is determined to be 50 μm or more,
The ballast water test method according to claim 1, wherein the number of individuals is tested by counting connected regions of pixels corresponding to the microorganisms whose minimum size is determined to be 50 μm or more.   Each time the second point is moved one pixel in the first direction, the second point is continuously moved in the second direction within a range where the linear distance from the first point is less than 50 μm. The ballast water inspection method according to claim 2, wherein the ballast water is continuously moved in the first direction until the linear distance to the first point is less than 50 μm.   The method for examining ballast water according to any one of claims 1 to 3, wherein the minimum size is a microorganism having a minimum size of 10 µm or more and less than 50 µm with phytoplankton.   The method for inspecting ballast water according to claim 4, wherein the number of individuals of the phytoplankton is inspected by chlorophyll fluorescence measurement using pulse modulation. Irradiating the sample of ballast water with pulse-modulated measurement light;
Irradiating the sample of ballast water irradiated with the measurement light with pulsed light,
Measure the minimum fluorescence intensity Fo released by chlorophyll contained in the sample when irradiated with the measurement light,
Measure the maximum fluorescence intensity Fm emitted by the chlorophyll contained in the sample when irradiated with the pulsed light,
The maximum quantum yield Fv / Fm = (Fm−Fo) / Fm of chlorophyll contained in the sample is calculated from the measured values of the minimum fluorescence intensity Fo and the maximum fluorescence intensity Fm,
Based on the relationship between the minimum fluorescence intensity Fo and the maximum quantum yield Fv / Fm and a preset number of phytoplankton individuals, the number of phytoplankton individuals contained in the ballast water sample is indirectly determined. The method for inspecting ballast water according to claim 4 or 5, wherein the ballast water is inspected.   The method for inspecting ballast water according to claim 1, wherein the scanner is a fluorescent scanner.

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